Dr. Fredric Kraemer is a Professor in the Division of Endocrinology, Gerontology and Metabolism. He is a board certified endocrinologist for over 40 years who specializes in the treatment diabetes mellitus, particularly type 1 diabetes, and the treatment of lipid disorders, such as elevated cholesterol or triglycerides.

Clinical Focus

  • Endocrinology / Diabetes
  • Lipid Metabolism Disorders
  • Endocrinology
  • Diabetes and Metabolism

Administrative Appointments

  • Chief, Division of Endocrinology, Gerontology and Metabolism, Stanford University (2002 - 2022)
  • Associate Chief of Staff for Research and Development, VA Palo Alto Health Care System (2002 - 2007)

Honors & Awards

  • Gerald M. Reaven, M.D. Professor in Endocrinology, Stanford University (2018)
  • Stanford University Professorship in Endocrinology, Stanford University (2002-2018)
  • SmithKline Beecham Junior Faculty Award In Diabetes, SmithKline Beecham (1998)
  • Hume Faculty Scholar, Stanford University (1984-1988)
  • Mellon Foundation Fellow, Stanford University (1983-1984)
  • Special Emphasis Research Career Award, NIH (1982-1987)

Professional Education

  • Fellowship: Stanford University School of Medicine (1982) CA
  • Medical Education: New York University School of Medicine (1974) NY
  • Board Certification: American Board of Internal Medicine, Endocrinology, Diabetes and Metabolism (1981)
  • Residency: Kings County Hospital (1978) NY
  • Residency: Kings County Hospital (1977) NY
  • Board Certification: American Board of Internal Medicine, Internal Medicine (1977)
  • Internship: Kings County Hospital (1977) NY
  • BA, Emory University, Chemistry (1970)
  • MD, New York University, Medicine (1974)

Current Research and Scholarly Interests

Our research interests are in the general area of cellular lipid and lipoprotein metabolism. The work is aimed primarily at understanding the mechanisms regulating cholesterol and triglyceride accumulation in cells. We utilize a variety of techniques from cell biology, biochemistry, and molecular biology. Current research projects focus on the regulation of hormone sensitive lipase (HSL). HSL is an intracellular enzyme responsible for the cytosolic hydrolysis of intracellular triglyceride and cholesteryl esters. Thus, it has an important role in fat mobilization, but also in cholesterol metabolism. We are examining all aspects of the biology of HSL, including its mechanisms of action and its structure-function relationships in regard to adipose cell function and steroid hormone synthesis.

Clinical Trials

  • Treatment of Psychotic Major Depression With Mifepristone Not Recruiting

    The purpose of this research study is to see how certain hormones cause changes in mood and thinking in some depressed patients and to determine the effectiveness of mifepristone in treating some forms of depression. This study is conducted in conjunction with an observational study "Clinical and Biological Characteristics of Psychotic Depression".

    Stanford is currently not accepting patients for this trial. For more information, please contact Lakshika Tennakoon, (650) 723 - 3305.

    View full details

2022-23 Courses

Graduate and Fellowship Programs

All Publications

  • Early enforcement of cell identity by a functional component of the terminally differentiated state. PLoS biology Bahrami-Nejad, Z., Zhang, Z. B., Tholen, S., Sharma, S., Rabiee, A., Zhao, M. L., Kraemer, F. B., Teruel, M. N. 2022; 20 (12): e3001900


    How progenitor cells can attain a distinct differentiated cell identity is a challenging problem given the fluctuating signaling environment in which cells exist and that critical transcription factors are often not unique to a differentiation process. Here, we test the hypothesis that a unique differentiated cell identity can result from a core component of the differentiated state doubling up as a signaling protein that also drives differentiation. Using live single-cell imaging in the adipocyte differentiation system, we show that progenitor fat cells (preadipocytes) can only commit to terminally differentiate after up-regulating FABP4, a lipid buffer that is highly enriched in mature adipocytes. Upon induction of adipogenesis in mouse preadipocyte cells, we show that after a long delay, cells first abruptly start to engage a positive feedback between CEBPA and PPARG before then engaging, after a second delay, a positive feedback between FABP4 and PPARG. These sequential positive feedbacks both need to engage in order to drive PPARG levels past the threshold for irreversible differentiation. In the last step before commitment, PPARG transcriptionally increases FABP4 expression while fatty acid-loaded FABP4 increases PPARG activity. Together, our study suggests a control principle for robust cell identity whereby a core component of the differentiated state also promotes differentiation from its own progenitor state.

    View details for DOI 10.1371/journal.pbio.3001900

    View details for PubMedID 36469503

  • Mice Deficient in ER Protein Seipin Have Reduced Adrenal Cholesteryl Ester Lipid Droplet Formation and Utilization. Journal of lipid research Shen, W. J., Cortez, Y., Singh, A., Chen, W., Azhar, S., Kraemer, F. B. 2022: 100309


    Cholesteryl ester-rich lipid droplets accumulate in steroidogenic tissues under physiological conditions and constitute an important source of cholesterol as the precursor for the synthesis of all steroid hormones. The mechanisms specifically involved in cholesteryl ester-rich lipid droplet formation have not been directly studied and are assumed by most to occur in a fashion analogous to triacylglycerol-rich lipid droplets. Seipin is an ER protein that forms oligomeric complexes at ER-lipid droplet contact sites, and seipin deficiency results in severe alterations in lipid droplet maturation and morphology as seen in Berardinelli-Seip congenital lipodystrophy type 2. While seipin is critical for triacylglycerol-rich lipid droplet formation, no studies have directly addressed whether seipin is important for cholesteryl ester-rich lipid droplet biogenesis. To address this issue, mice with deficient expression of seipin specifically in adrenal, testis and ovary, steroidogenic tissues that accumulate cholesteryl ester-rich lipid droplets under normal physiological conditions were generated. We found that the steroidogenic-specific seipin deficient mice displayed a marked reduction in lipid droplet and cholesterol/cholesteryl ester accumulation in the adrenals, demonstrating the pivotal role of seipin in cholesteryl ester-rich lipid droplet accumulation/formation. Moreover, the reduction in cholesteryl ester-rich lipid droplets was associated with significant defects in adrenal and gonadal steroid hormone production that could not be completely reversed by addition of exogenous lipoprotein cholesterol. We conclude that seipin has a heretofore unappreciated role in intracellular cholesterol trafficking.

    View details for DOI 10.1016/j.jlr.2022.100309

    View details for PubMedID 36332685

  • Corrigendum to "Hormone sensitive lipase ablation promotes bone regeneration" [Biochim. Biophys. Acta Mol. Basis Dis. Volume 1868, Issue 9, 1 September 2022, 166449]. Biochimica et biophysica acta. Molecular basis of disease Shen, W. J., Still, C., Han, L., Yang, P., Chen, J., Wosczyna, M., Rando, T. A., Salmon, B. J., Perez, K. C., Li, J., Cuevas, P. L., Liu, B., Azhar, S., Helms, J., Qi, L. S., Kraemer, F. B. 2022; 1868 (11): 166506

    View details for DOI 10.1016/j.bbadis.2022.166506

    View details for PubMedID 35926435

  • Flattening of circadian glucocorticoid oscillations drives acute hyperinsulinemia and adipocyte hypertrophy. Cell reports Tholen, S., Patel, R., Agas, A., Kovary, K. M., Rabiee, A., Nicholls, H. T., Bielczyk-Maczyńska, E., Yang, W., Kraemer, F. B., Teruel, M. N. 2022; 39 (13): 111018


    Disruption of circadian glucocorticoid oscillations in Cushing's disease and chronic stress results in obesity and adipocyte hypertrophy, which is believed to be a main source of the harmful effects of obesity. Here, we recapitulate stress due to jet lag or work-life imbalances by flattening glucocorticoid oscillations in mice. Within 3 days, mice achieve a metabolic state with persistently high insulin, but surprisingly low glucose and fatty acids in the bloodstream, that precedes a more than 2-fold increase in brown and white adipose tissue mass within 3 weeks. Transcriptomic and Cd36-knockout mouse analyses show that hyperinsulinemia-mediated de novo fatty acid synthesis and Cd36-mediated fatty acid uptake drive fat mass increases. Intriguingly, this mechanism by which glucocorticoid flattening causes acute hyperinsulinemia and adipocyte hypertrophy is unexpectedly beneficial in preventing high levels of circulating fatty acids and glucose for weeks, thus serving as a protective response to preserve metabolic health during chronic stress.

    View details for DOI 10.1016/j.celrep.2022.111018

    View details for PubMedID 35767959

  • Hormone sensitive lipase ablation promotes bone regeneration. Biochimica et biophysica acta. Molecular basis of disease Shen, W. J., Chris Still, I. I., Han, L., Yang, P., Chen, J., Wosczyna, M., Salmon, B. J., Perez, K. C., Li, J., Cuevas, P. L., Liu, B., Azhar, S., Helms, J., Qi, L. S., Kraemer, F. B. 2022: 166449


    There is an inverse relationship between the differentiation of mesenchymal stem cells (MSCs) along either an adipocyte or osteoblast lineage, with lineage differentiation known to be mediated by transcription factors PPARγ and Runx2, respectively. Endogenous ligands for PPARγ are generated during the hydrolysis of triacylglycerols to fatty acids through the actions of lipases such as hormone sensitive lipase (HSL). To examine whether reduced production of endogenous PPARγ ligands would influence bone regeneration, we examined the effects of HSL knockout on fracture repair in mice using a tibial mono-cortical defect as a model. We found an improved rate of fracture repair in HSL-ko mice documented by serial μCT and bone histomorphometry compared to wild-type (WT) mice. Similarly, accelerated rates of bone regeneration were observed with a calvarial model where implantation of bone grafts from HSL-ko mice accelerated bone regeneration at the injury site. Further analysis revealed improved MSC differentiation down osteoblast and chondrocyte lineage with inhibition of HSL. MSC recruitment to the injury site was greater in HSL-ko mice than WT. Finally, we used single cell RNAseq to understand the osteoimmunological differences between WT and HSL-ko mice and found changes in the pre-osteoclast population. Our study shows HSL-ko mice as an interesting model to study improvements to bone injury repair. Furthermore, our study highlights the potential importance of pre-osteoclasts and osteoclasts in bone repair.

    View details for DOI 10.1016/j.bbadis.2022.166449

    View details for PubMedID 35618183

  • Hormone-sensitive lipase protects adipose triglyceride lipase-deficient mice from lethal lipotoxic cardiomyopathy. Journal of lipid research Yamada, M., Suzuki, J., Sato, S., Zenimaru, Y., Saito, R., Konoshita, T., Kraemer, F. B., Ishizuka, T. 2022: 100194


    Lipid droplets (LDs) are multifunctional organelles that regulate energy storage and cellular homeostasis. The first step of triacylglycerol (TAG) hydrolysis in LDs is catalyzed by adipose triglyceride lipase (ATGL), deficiency of which results in lethal cardiac steatosis. Although hormone-sensitive lipase (HSL) functions as a diacylglycerol (DAG) lipase in the heart, we hypothesized that activation of HSL might compensate for ATGL deficiency. To test this hypothesis, we crossed ATGL-knockout (KO) mice and cardiac-specific HSL-overexpressing mice (cHSL) to establish homozygous ATGL-KO (AKO) mice and AKO mice with cardiac-specific HSL overexpression (AKO+cHSL). We found that cardiac TAG content was 160-fold higher in AKO relative to wild type (Wt) mice, while that of AKO+cHSL mice was comparable to the latter. In addition, AKO cardiac tissues exhibited reduced mRNA expression of PPARalpha-regulated genes, and upregulation of genes involved in inflammation, fibrosis, and cardiac stress. In contrast, AKO+cHSL cardiac tissues exhibited expression levels similar to those observed in Wt mice. AKO cardiac tissues also exhibited macrophage infiltration, apoptosis, interstitial fibrosis, impaired systolic function, and marked increases in ceramide and DAG contents, while no such pathological alterations were observed in AKO+cHSL tissues. Furthermore, electron microscopy revealed considerable LDs, damaged mitochondria, and disrupted intercalated discs in AKO cardiomyocytes, none of which were noted in AKO+cHSL cardiomyocytes. Importantly, the lifespan of AKO+cHSL mice was comparable to that of Wt mice. We conclude cardiac HSL overexpression normalizes lipotoxic cardiomyopathy in AKO mice. These findings highlight the applicability of cardiac HSL activation as a therapeutic strategy for ATGL deficiency-associated lipotoxic cardiomyopathies.

    View details for DOI 10.1016/j.jlr.2022.100194

    View details for PubMedID 35283217

  • SNAP25 mutation disrupts metabolic homeostasis, steroid hormone production and central neurobehavior. Biochimica et biophysica acta. Molecular basis of disease Hao, X., Zhu, B., Yang, P., Dong, D., Sahbaie, P., Oliver, P. L., Shen, W., Azhar, S., Kraemer, F. B. 2021: 166304


    OBJECTIVE: SNAP-25 is one of the key proteins involved in formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes that are at the core of hormonal secretion and synaptic transmission. Altered expression or function of SNAP-25 can contribute to the development of neuropsychiatric and metabolic disease. A dominant negative (DN) I67T missense mutation in the b-isoform of SNAP-25 (DN-SNAP25mut) mice leads to abnormal interactions within the SNARE complex and impaired exocytotic vesicle recycling, yet the significance of this mutation to any association between the central nervous system and metabolic homeostasis is unknown.METHODS: Here we explored aspects of metabolism, steroid hormone production and neurobehavior of DN-SNAP25mut mice.RESULTS: DN-SNAP25mut mice displayed enhanced insulin function through increased Akt phosphorylation, alongside increased adrenal and gonadal hormone production. In addition, increased anxiety behavior and beigeing of white adipose tissue with increased energy expenditure were observed in mutants.CONCLUSIONS: Our results show that SNAP25 plays an important role in bridging central neurological systems with peripheral metabolic homeostasis, and provide potential insights between metabolic disease and neuropsychiatric disorders in humans.

    View details for DOI 10.1016/j.bbadis.2021.166304

    View details for PubMedID 34826585

  • Hormone-sensitive lipase deficiency affects the expression of SR-BI, LDLr, and ABCA1 receptors/transporters involved in cellular cholesterol uptake and efflux and disturbs fertility in mouse testis. Biochimica et biophysica acta. Molecular and cell biology of lipids Casado, M. E., Huerta, L., Marcos-Diaz, A., Ortiz, A. I., Kraemer, F. B., Lasuncion, M. A., Busto, R., Martin-Hidalgo, A. 2021: 159043


    Hormone-sensitive lipase (HSL) hydrolyse acylglycerols, cholesteryl and retinyl esters. HSL is a key lipase in mice testis, as HSL deficiency results in male sterility. The present work study the effects of the deficiency and lack of HSL on the localization and expression of SR-BI, LDLr, and ABCA1 receptors/transporters involved in uptake and efflux of cholesterol in mice testis, to determine the impact of HSL gene dosage on testis morphology, lipid homeostasis and fertility. The results of this work show that the lack of HSL in mice alters testis morphology and spermatogenesis, decreasing sperm counts, sperm motility and increasing the amount of Leydig cells and lipid droplets. They also show that there are differences in the localization of HSL, SR-BI, LDLr and ABCA1 in HSL+/+, HSL+/- and HSL-/- mice. The deficiency or lack of HSL has effects on protein and mRNA expression of genes involved in lipid metabolisms in mouse testis. HSL-/- testis have augmented expression of SR-BI, LDLr, ABCA1 and LXRbeta, a critical sterol sensor that regulate multiple genes involved in lipid metabolism; whereas LDLr expression decreased in HSL+/- mice. Plin2, Abca1 and Ldlr mRNA levels increased; and LXRalpha (Nr1h3) and LXRbeta (Nr1h2) decreased in testis from HSL-/- compared with HSL+/+; with no differences in Scarb1. Together these data suggest that HSL deficiency or lack in mice testis induces lipid homeostasis alterations that affect the cellular localization and expression of key receptors/transporter involved in cellular cholesterol uptake and efflux (SR-BI, LDRr, ABCA1); alters normal cellular function and impact fertility.

    View details for DOI 10.1016/j.bbalip.2021.159043

    View details for PubMedID 34461308

  • Chemerin regulates formation and function of brown adipose tissue: Ablation results in increased insulin resistance with high fat challenge and aging. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Zhang, Y., Shen, W., Qiu, S., Yang, P., Dempsey, G., Zhao, L., Zhou, Q., Hao, X., Dong, D., Stahl, A., Kraemer, F. B., Leung, L. L., Morser, J. 2021; 35 (7): e21687


    Apart from its role in inflammation and immunity, chemerin is also involved in white adipocyte biology. To study the role of chemerin in adipocyte metabolism, we examined the function of chemerin in brown adipose tissue. Brown and white adipocyte precursors were differentiated into adipocytes in the presence of Chemerin siRNA. Chemerin-deficient (Chem-/- ) mice were compared to wild-type mice when fed a high-fat diet. Chemerin is expressed during brown adipocyte differentiation and knock down of chemerin mRNA results in decreased brown adipocyte differentiation with reduced fatty acid uptake in brown adipocytes. Chem-/- mice are leaner than wild-type mice but gain more weight when challenged with high-fat diet feeding, resulting in a larger increase in fat deposition. Chem-/- mice develop insulin resistance when on a high-fat diet or due to age. Brown adipose depots in Chem-/- mice weigh more than in wild-type mice, but with decreased mitochondrial content and function. Compared to wild-type mice, male Chem-/- mice have decreased oxygen consumption, CO2 production, energy expenditure, and a lower respiratory exchange ratio. Additionally, body temperature of Chem-/- mice is lower than that of wild-type mice. These results revealed that chemerin is expressed during brown adipocyte differentiation and has a pivotal role in energy metabolism through brown adipose tissue thermogenesis.

    View details for DOI 10.1096/fj.202100156R

    View details for PubMedID 34089273

  • FXR activation promotes intestinal cholesterol excretion and attenuates hyperlipidemia in SR-B1-deficient mice fed a high-fat and high-cholesterol diet. Physiological reports Singh, A. B., Dong, B., Kraemer, F. B., Liu, J. 2020; 8 (5): e14387


    Obeticholic acid (OCA) activates the farnesoid X receptor (FXR) to lower circulating total cholesterol (TC) and high density lipoprotein-cholesterol (HDL-C) concentrations and to stimulate fecal cholesterol excretion in mice by increasing hepatic SR-B1 expression. Here we show that hepatic SR-B1 depletion by an adenovirus expressing Sr-b1 shRNA (Ad-shSR-B1) attenuated these beneficial effects of OCA in mice on a chow diet. The mRNA levels of ABC cholesterol transporter genes (Abca1, Abcg1, Abcg5, and Abcg8) were unchanged in the liver of hepatic SR-B1-depleted mice regardless of OCA treatment; however, a modest increase in Abca1, Abcg5, and Abcg8 mRNA levels was observed in the ileum of vehicle-treated control mice and Abca1 and Abcg8 mRNA levels were increased more by OCA administration. OCA treatment of Sr-b1 knock out (KO) mice (Sr-b1-/-) fed a normal chow diet (NCD) displayed a similar lack of transhepatic cholesterol movement, as well as a modest increase in the levels of ileum cholesterol transporter expression. However, OCA treatment of Sr-b1 KO mice fed a cholesterol-enriched diet reduced circulating cholesterol and increased fecal cholesterol output to comparable degrees to that of wild-type (WT) mice, and these effects were accompanied by substantial elevations of mRNA levels of Abca1, Abcg1, Abcg5, and Abcg8 in the ileum of Sr-b1 KO mice. Our studies suggest that FXR activation stimulates intestinal cholesterol excretion and reduces diet-induced hyperlipidemia through increased expression of ileal cholesterol transporters when hepatic SR-B1-mediated cholesterol movement is absent.

    View details for DOI 10.14814/phy2.14387

    View details for PubMedID 32170842

  • Molecular changes in hepatic metabolism in ZDSD rats-A new polygenic rodent model of obesity, metabolic syndrome, and diabetes. Biochimica et biophysica acta. Molecular basis of disease Han, L. n., Bittner, S. n., Dong, D. n., Cortez, Y. n., Bittner, A. n., Chan, J. n., Umar, M. n., Shen, W. J., Peterson, R. G., Kraemer, F. B., Azhar, S. n. 2020: 165688


    In recent years, the prevalence of obesity, metabolic syndrome and type 2 diabetes is increasing dramatically. They share pathophysiological mechanisms and often lead to cardiovascular diseases. The ZDSD rat was suggested as a new animal model to study diabetes and the metabolic syndrome. In the current study, we have further characterized metabolic and hepatic gene expression changes in ZDSD rats. Immuno-histochemical staining of insulin and glucagon on pancreas sections of ZDSD and control SD rats revealed that ZDSD rats have severe damage to their islet structures as early as 15 weeks of age. Animals were followed till they were 26 weeks old, where they exhibited obesity, hypertension, hyperglycemia, dyslipidemia, insulin resistance and diabetes. We found that gene expressions involved in glucose metabolism, lipid metabolism and amino acid metabolism were changed significantly in ZDSD rats. Elevated levels of ER stress markers correlated with the dysregulation of hepatic lipid metabolism in ZDSD rats. Key proteins participating in unfolded protein response pathways were also upregulated and likely contribute to the pathogenesis of dyslipidemia and insulin resistance. Based on its intact leptin system, its insulin deficiency, as well as its timeline of disease development without diet manipulation, this insulin resistant, dyslipidemic, hypertensive, and diabetic rat represents an additional, unique polygenic animal model that could be very useful to study human diabetes.

    View details for DOI 10.1016/j.bbadis.2020.165688

    View details for PubMedID 31987840

  • Slc43a3 is a regulator of free fatty acid flux. Journal of lipid research Hasbargen, K. B., Shen, W. J., Zhang, Y. n., Hou, X. n., Wang, W. n., Shuo, Q. n., Bernlohr, D. A., Azhar, S. n., Kraemer, F. B. 2020


    Adipocytes take up long chain fatty acids through diffusion and protein mediated transport, whereas fatty acid efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating fatty acid flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated fatty acid efflux, while also increasing fatty acid uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased fatty acid uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate fatty acid flux in adipocytes, functioning as a positive regulator of fatty acid efflux and as a negative regulator of fatty acid uptake.

    View details for DOI 10.1194/jlr.RA119000294

    View details for PubMedID 32217606

  • Identification of p115 as a novel ACSL4 interacting protein and its role in regulating ACSL4 degradation. Journal of proteomics Sen, P. n., Kan, C. F., Singh, A. B., Rius, M. n., Kraemer, F. B., Sztul, E. n., Liu, J. n. 2020: 103926


    Long-chain acyl-CoA synthetase 4 (ACSL4) is an ACSL family member that exhibits unique substrate preference for arachidonic acid. ACSL4 has a functional role in hepatic lipid metabolism, and is dysregulated in non-alcoholic fatty liver disease. Our previous studies demonstrated AA-induced ACSL4 degradation via the ubiquitin-proteasomal pathway (UPP). To characterize this unique mechanism, we applied proteomic approaches coupled with LC-MS/MS and identified the intracellular general vesicular trafficking protein p115 as the prominent ACSL4 interacting protein in HepG2 cells. Importantly, we found that AA greatly enhanced p115-ACSL4 association. Combined AA treatment with p115 knockdown suggested an additive role for p115 in AA-driven ACSL4 degradation. Furthermore, in vivo studies revealed a significant upregulation of p115 protein in the liver of mice fed a high fat diet that has been previously reported to induce downregulation of ACSL4 protein expression. This new finding has revealed a novel inverse correlation between ACSL4 and p115 proteins in the liver. p115 is crucial for ER-Golgi trafficking and Golgi biogenesis. Thus far, p115 has not been reported to interact with UPP proteins nor with FA metabolism enzymes. Overall, our current study provides a novel insight into the connection between ER-Golgi trafficking and UPP machinery with p115 as a critical mediator. SIGNIFICANCE: ACSL4 is uniquely regulated by its own substrate AA, and in this study, we have found that AA leads to an enhanced interaction of ACSL4 with a novel interacting partner, the intracellular vesicle trafficking protein p115. The latter is crucial for Golgi biogenesis and ER-Golgi transport and is not known to be associated with the ubiquitin-proteasome machinery or protein stability regulation until now. This study is the first report of a possible coordination of the protein secretion pathway and the UPP in regulating a key metabolic enzyme. Our study lays the foundation to this unique crosstalk between the two major cellular pathways- secretion and protein degradation and opens up a new avenue to explore this partnership in controlling hepatic lipid metabolism. Overall, the complete elucidation of the AA-mediated ACSL4 regulation will help identify key targets in participating pathways that can be further studied for the development of therapeutics against diseases such as NAFLD, NASH and hepatocarcinoma, which are associated with dysregulated ACSL4 function.

    View details for DOI 10.1016/j.jprot.2020.103926

    View details for PubMedID 32736139

  • SOD2 deficiency-induced oxidative stress attenuates steroidogenesis in mouse ovarian granulosa cells. Molecular and cellular endocrinology Zaidi, S. K., Shen, W. J., Cortez, Y. n., Bittner, S. n., Bittner, A. n., Arshad, S. n., Huang, T. T., Kraemer, F. B., Azhar, S. n. 2020: 110888


    This study investigated the effects of SOD2 (MnSOD)-deficiency-induced excessive oxidative stress on ovarian steroidogenesis in vivo and isolated and cultured granulosa cells using WT and Sod2 ± mice. Basal and 48 h eCG-stimulated plasma progesterone levels were decreased ∼50% in female Sod2 ± mice, whereas plasma progesterone levels were decreased ∼70% in Sod2 ± mice after sequential stimulation with eCG followed by hCG. Sod2 ± deficiency caused about 50% reduction in SOD2 activity in granulosa cells. SOD2-deficiency also caused a marked reduction in progestins and estradiol in isolated granulosa cells. qRT-PCR measurements indicated that the mRNA expression levels of StAR protein and steroidogenic enzymes are decreased in the ovaries of Sod2 ± mice. Further studies showed a defect in the movement of mobilized cytosolic cholesterol to mitochondria. The ovarian membrane from Sod2 ± mice showed higher susceptibility to lipid peroxidation. These data indicates that SOD2-deficiency induced oxidative stress inhibits ovarian granulosa cell steroidogenesis primarily by interfering with cholesterol transport to mitochondria and attenuating the expression of Star protein gene and key steroidogenic enzyme genes.

    View details for DOI 10.1016/j.mce.2020.110888

    View details for PubMedID 32717420

  • Scavenger receptor class B, type 1 facilitates cellular fatty acid uptake. Biochimica et biophysica acta. Molecular and cell biology of lipids Wang, W., Yan, Z., Hu, J., Shen, W., Azhar, S., Kraemer, F. B. 2019: 158554


    SR-B1 belongs to the class B scavenger receptor, or CD36 super family. SR-B1 and CD36 share an affinity for a wide array of ligands. Although they exhibit similar ligand binding specificity, SR-B1 and CD36 have some very specific lipid transport functions. Whereas SR-B1 primarily facilitates the selective delivery of cholesteryl esters (CEs) and cholesterol from HDL particles to the liver and non-placental steroidogenic tissues, as well as participating in cholesterol efflux from cells, CD36 primarily mediates the uptake of long-chain fatty acids in high fatty acid-requiring organs such as the heart, skeletal muscle and adipose tissue. However, CD36 also mediates cholesterol efflux and facilitates selective lipoprotein-CE delivery, although less efficiently than SR-B1. Interestingly, the ability or efficiency of SR-B1 to mediate fatty acid uptake has not been reported. In this paper, using overexpression and siRNA-mediated knockdown of SR-B1, we show that SR-B1 possesses the ability to facilitate fatty acid uptake. Moreover, this function is not blocked by BLT-1, a specific chemical inhibitor of HDL-CE uptake activity of SR-B1, nor by sulfo-N-succinimidyl oleate, which inhibits fatty acid uptake by CD36. Attenuated fatty acid uptake was also observed in primary adipocytes isolated from SR-B1 knockout mice. In conclusion, facilitation of fatty acid uptake is an additional function that is mediated by SR-B1.

    View details for DOI 10.1016/j.bbalip.2019.158554

    View details for PubMedID 31678516

  • Cardiac overexpression of perilipin 2 induces atrial steatosis, connexin 43 remodeling, and atrial fibrillation in aged mice. American journal of physiology. Endocrinology and metabolism Sato, S., Suzuki, J., Hirose, M., Yamada, M., Zenimaru, Y., Nakaya, T., Ichikawa, M., Imagawa, M., Takahashi, S., Ikuyama, S., Konoshita, T., Kraemer, F. B., Ishizuka, T. 2019


    Atrial fibrillation (AF) is prevalent in patients with obesity and diabetes, and such patients often exhibit cardiac steatosis. Since the role of cardiac steatosis per se in the induction of AF has not been elucidated, the present study was designed to explore the relation between cardiac steatosis and AF. Transgenic (Tg) mice with cardiac-specific overexpression of perilipin 2 (PLIN2) were housed in the laboratory for more than 12 months before the study. Electron microscopy of the atrium of PLIN2-Tg mice showed accumulation of small lipid droplets around mitochondrial chains, and 5- to 9-fold greater atrial triacylglycerol (TAG) content compared to wildtype (Wt) mice. Electrocardiography showed significantly longer RR intervals in PLIN2-Tg mice than Wt mice. Transesophageal electrical burst pacing resulted in significantly higher prevalence of sustained (>5 min) AF (69%) in PLIN2-Tg mice than Wt mice (24%), although it was comparable in younger (4-month-old) mice. Connexin 43 (Cx43), a gap junction protein, was localized at the intercalated discs in Wt atria, but was heterogeneously distributed on the lateral side of cardiomyocytes in PLIN2-Tg atria. Langendorff-perfused hearts using the optical mapping technique showed slower and heterogeneous impulse propagation in PLIN2-Tg atria compared with Wt atria. Cardiac overexpression of hormone-sensitive lipase in PLIN2-Tg mice resulted in atrial TAG depletion and amelioration of AF susceptibility. The results suggest that PLIN2-induced steatosis is associated with Cx43 remodeling, impaired conduction propagation, and higher incidence of AF in aged mice. Therapies targeting cardiac steatosis could be potentially beneficial against AF in patients with obesity or diabetes.

    View details for DOI 10.1152/ajpendo.00227.2019

    View details for PubMedID 31661297

  • Tissue Specific Ablation of ACSL4 Results in Disturbed Steroidogenesis. Endocrinology Wang, W., Hao, X., Han, L., Yan, Z., Shen, W., Dong, D., Hasbargen, K., Bittner, S., Cortez, Y., Greenberg, A. S., Azhar, S., Kraemer, F. B. 2019


    ACSL4 is a member of the ACSL family that catalyzes the conversion of long chain fatty acids to acyl CoAs, which are essential for fatty acid incorporation and utilization into diverse metabolic pathways including cholesteryl ester synthesis. Cholesteryl esters in steroidogenic tissues such as the adrenal gland are particularly enriched in cholesteryl esters of long chain polyunsaturated fatty acids and constitute an important pool supplying cholesterol for steroid synthesis. The current studies addressed whether ACSL4 is required for normal steroidogenesis. CYP11A1 promoter-mediated Cre was used to generate steroid-tissue specific ACSL4 KO mice. Results demonstrate that ACSL4 plays an important role in adrenal cholesteryl ester formation, as well as determining the fatty acyl composition of adrenal cholesteryl esters, with ACSL4 deficiency leading to reductions in cholesteryl ester storage and alterations in cholesteryl ester composition. Statistically significant reductions in corticosterone and testosterone, but not progesterone, production were observed in vivo, and these deficits were accentuated in ex vivo and in vitro studies of isolated steroid tissues and cells from ACSL4 deficient mice. However, these effects on steroid production appear due to reductions in cholesteryl ester stores rather than disturbances of signaling pathways. We conclude that ACSL4 is dispensable for normal steroidogenesis.

    View details for DOI 10.1210/en.2019-00464

    View details for PubMedID 31504388

  • Liver-specific knockdown of long-chain acyl-CoA synthetase 4 reveals its key role in VLDL-TG metabolism and phospholipid synthesis in mice fed a high-fat diet AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Singh, A. B., Kan, C. K., Kraemer, F. B., Sobel, R. A., Liu, J. 2019; 316 (5): E880–E894
  • Anti-hyperlipidaemic effects of synthetic analogues of nordihydroguaiaretic acid in dyslipidaemic rats BRITISH JOURNAL OF PHARMACOLOGY Singh, M., Bittner, S., Li, Y., Bittner, A., Han, L., Cortez, Y., Inayathullah, M., Arif, Z., Parthasarathi, R., Rajadas, J., Shen, W., Nicolls, M. R., Kraemer, F. B., Azhar, S. 2019; 176 (3): 369–85

    View details for DOI 10.1111/bph.14528

    View details for Web of Science ID 000455517100003

  • Novel ABCA1 peptide agonists with antidiabetic action MOLECULAR AND CELLULAR ENDOCRINOLOGY Azhar, S., Bittner, S., Hu, J., Shen, W., Cortez, Y., Hao, X., Han, L., Lagerstedt, J. O., Kraemer, F. B., Johansson, J. O. 2019; 480: 1–11
  • Creosote bush-derived NDGA attenuates molecular and pathological changes in a novel mouse model of non-alcoholic steatohepatitis (NASH). Molecular and cellular endocrinology Han, L. n., Bittner, S. n., Dong, D. n., Cortez, Y. n., Dulay, H. n., Arshad, S. n., Shen, W. J., Kraemer, F. B., Azhar, S. n. 2019: 110538


    Creosote bush (Larrea tridentata)-derived nordihydroguaiaretic acid (NDGA) was shown to have profound effects on the core components of metabolic syndrome. This study investigated the in vivo potential of NDGA for prevention or attenuation of the pathophysiologic abnormalities of NASH. A novel dietary NASH model with feeding C57BL/6J mice with a high trans-fat, high cholesterol and high fructose (HTF) diet, was used. The HTF diet fed mice exhibited obesity, insulin resistance, hepatic steatosis, fibrosis, inflammation, ER stress, oxidative stress, and liver injury. NDGA attenuated these metabolic abnormalities as well as hepatic steatosis and fibrosis together with attenuated expression of genes encoding fibrosis, progenitor and macrophage markers with no effect on the levels of mRNAs for lipogenic enzymes. NDGA increased expression of fatty acid oxidation genes. In conclusion, NDGA exerts anti-NASH/anti-fibrotic actions and raises the therapeutic potential of NDGA for treatment of NASH patients with fibrosis and other associated complications.

    View details for DOI 10.1016/j.mce.2019.110538

    View details for PubMedID 31415794

  • The role of miRNAs in regulating adrenal/gonadal steroidogenesis. Journal of molecular endocrinology Azhar, S. n., Dong, D. n., Shen, W. J., Hu, Z. n., Kraemer, F. B. 2019


    MicroRNAs (miRNAs) are endogenous noncoding single-stranded small RNAs of ~22 nucleotides in length that post-transcriptionally repress the expression of their various target genes. They contribute to the regulation of a variety of physiologic processes including embryonic development, differentiation and proliferation, apoptosis, metabolism, hemostasis and inflammation. In addition, aberrant miRNA expression is implicated in the pathogenesis of numerous diseases including cancer, hepatitis, cardiovascular diseases and metabolic diseases. Steroid hormones regulate virtually every aspect of metabolism and acute and chronic steroid hormone biosynthesis is primarily regulated by tissue-specific trophic hormones involving transcriptional and translational events. In addition, it is becoming increasingly clear that steroidogenic pathways are also subject to post-transcriptional and post-translational regulations including processes such as phosphorylation/dephosphorylation, and protein‒protein interactions and regulation by specific miRNAs, although the latter is in its infancy state. Here, we summarize the recent advances in miRNA-mediated regulation of steroidogenesis with emphasis on adrenal and gonadal steroidogenesis.

    View details for DOI 10.1530/JME-19-0105

    View details for PubMedID 31671401

  • Novel ABCA1 peptide agonists with antidiabetic action. Molecular and cellular endocrinology Azhar, S., Bittner, S., Hu, J., Shen, W., Cortez, Y., Hao, X., Lu, H., Lagerstedt, J. O., Kraemer, F. B., Johansson, J. O. 2018


    Previously, apoE-derived ABCA1 agonist peptides have been shown to possess anti-atherosclerotic and possibly antidiabetic properties. Here we assessed the in vitro and in vivo actions of a second generation of ABCA1 peptide agonists, CS6253 and T6991-2, on glucose homeostasis. The results show that these two peptides improve glucose tolerance in a prediabetic diet-induced obesity mouse model by enhancing insulin secretion. It was further demonstrated that T6991-2 also improved glucose tolerance in leptin-deficient (ob/ob) mice. CS6253 increased insulin secretion both under basal conditions and in response to high glucose stimulation in pancreatic INS-1 beta-cells rendered leptin receptor deficient with specific siRNA. Additional in vitro cell studies suggest that the CS6253 agonist attenuates hepatic gluconeogenesis and glucose transport. It also potentiates insulin-stimulated glucose uptake and utilization. These observed anti-diabetic actions suggest additional benefits of the CS6253 and T6991-2 ABCA1 peptide agonists about cardiovascular disease beyond their direct anti-atherosclerosis properties previously described.

    View details for PubMedID 30290217

  • Farnesoid X Receptor Activation by Obeticholic Acid Elevates Liver Low-Density Lipoprotein Receptor Expression by mRNA Stabilization and Reduces Plasma Low-Density Lipoprotein Cholesterol in Mice. Arteriosclerosis, thrombosis, and vascular biology Singh, A. B., Dong, B., Kraemer, F. B., Xu, Y., Zhang, Y., Liu, J. 2018; 38 (10): 2448-2459


    Objective- The objective of this study was to determine whether and how activation of farnesoid X receptor (FXR) by obeticholic acid (OCA), a clinical FXR agonist, modulates liver low-density lipoprotein receptor (LDLR) expression under normolipidemic conditions. Approach and Results- Administration of OCA to chow-fed mice increased mRNA and protein levels of LDLR in the liver without affecting the sterol-regulatory element binding protein pathway. Profiling of known LDLR mRNA-binding proteins demonstrated that OCA treatment did not affect expressions of mRNA degradation factors hnRNPD (heterogeneous nuclear ribonucleoprotein D) or ZFP36L1 but increased the expression of Hu antigen R (HuR) an mRNA-stabilizing factor. Furthermore, inducing effects of OCA on LDLR and HuR expression were ablated in Fxr-/- mice. To confirm the post-transcriptional mechanism, we used transgenic mice (albumin-luciferase-untranslated region) that express a human LDLR mRNA 3' untranslated region luciferase reporter gene in the liver. OCA treatment led to significant rises in hepatic bioluminescence signals, Luc-untranslated region chimeric mRNA levels, and endogenous LDLR protein abundance, which were accompanied by elevations of hepatic HuR mRNA and protein levels in OCA-treated transgenic mice. In vitro studies conducted in human primary hepatocytes and HepG2 cells demonstrated that FXR activation by OCA and other agonists elicited the same inducing effect on LDLR expression as in the liver of normolipidemic mice. Furthermore, depletion of HuR in HepG2 cells by short interfering RNA transfection abolished the inducing effect of OCA on LDLR expression. Conclusions- Our study is the first to demonstrate that FXR activation increases LDLR expression in liver tissue by a post-transcriptional regulatory mechanism involving LDLR mRNA-stabilizing factor HuR.

    View details for DOI 10.1161/ATVBAHA.118.311122

    View details for PubMedID 30354208

    View details for PubMedCentralID PMC6206879

  • Plasma membrane cholesterol trafficking in steroidogenesis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Deng, B., Shen, W., Dong, D., Azhar, S., Kraemer, F. B. 2018: fj201800697RRR


    Cholesterol is an important component of plasma membranes (PMs) and the precursor of all steroid hormones. In steroidogenic tissues, upon hormone stimulation, there is a rapid transfer of cholesterol to the mitochondria, which is the site of the initial step in steroidogenesis. In the current study, we examined PM cholesterol trafficking for steroidogenesis. In a mitochondrial reconstitution assay, adrenal PMs supported steroidogenesis in the absence of additional transport proteins. Depletion of cholesterol in PMs by 50% eliminated the membranes' ability to support steroidogenesis in vitro and reduced steroid production in intact Y1 adrenocortical cells. Syntaxin (STX)-5 and alpha-soluble N-ethylmaleimide-sensitive factor attachment protein (alpha-SNAP) are enriched in adrenal PMs, and adrenocorticotropic hormone treatment of rats recruited STX5 and alpha-SNAP to adrenal PMs and mitochondria. Immunodepletion of STX5 and alpha-SNAP from PMs decreased steroidogenesis supported by PMs in vitro. Protease digestion of PMs decreased, whereas recombinant STX5 or alpha-SNAP restored, the PMs' ability to support steroidogenesis. Knockdown of either STX5 or alpha-SNAP in Y1 cells decreased stimulated steroidogenesis. These results indicate that STX5 and alpha-SNAP facilitate cholesterol trafficking from PMs to mitochondria for adrenal steroid synthesis and underscore the importance of vesicular trafficking of PM cholesterol for steroidogenesis.-Deng, B., Shen, W.-J., Dong, D., Azhar, S., Kraemer, F. B. Plasma membrane cholesterol trafficking in steroidogenesis.

    View details for PubMedID 30133326

  • Scavenger receptor B type 1 : Expression, Molecular Regulation, and Cholesterol Transport Function. Journal of lipid research Shen, W., Asthana, S., Kraemer, F. B., Azhar, S. 2018


    Cholesterol is required for maintenance of plasma membrane fluidity and integrity and for many cellular functions. Cellular cholesterol can be obtained from lipoproteins in a selective pathway of HDL-cholesteryl ester (CE) uptake without parallel apolipoprotein uptake. Scavenger receptor B type 1 (SR-B1) is a cell surface HDL receptor that mediates HDL-CE uptake. It is most abundantly expressed in liver, where it provides cholesterol for bile acid synthesis, and in steroidogenic tissues, where it delivers cholesterol needed for storage or steroidogenesis in rodents. SR-B1 transcription is regulated by trophic hormones in the adrenal gland, ovary, and testis; in the liver and elsewhere, SR-B1 is subject to post-transcriptional and post-translational regulation. SR-B1 operates in several metabolic processes and contributes to pathogenesis of atherosclerosis, inflammation, HCV infection, and other conditions. Here, we summarize characteristics of the selective uptake pathway and involvement of microvillar channels as facilitators of selective HDL-CE uptake. We also present the potential mechanisms of SR-B1-mediated selective cholesterol transport; the transcriptional, post-transcriptional, and post-translational regulation of SR-B1; and the impact of gene variants on expression and function of human SR-B1. A better understanding of this unique pathway and the role of SR-B1 may yield improved therapies for a wide variety of conditions.

    View details for PubMedID 29720388

  • SR-B1: A Unique Multifunctional Receptor for Cholesterol Influx and Efflux ANNUAL REVIEW OF PHYSIOLOGY, VOL 80 Shen, W., Azhar, S., Kraemer, F. B., Julius, D. 2018; 80: 95–116


    The scavenger receptor, class B type 1 (SR-B1), is a multiligand membrane receptor protein that functions as a physiologically relevant high-density lipoprotein (HDL) receptor whose primary role is to mediate selective uptake or influx of HDL-derived cholesteryl esters into cells and tissues. SR-B1 also facilitates the efflux of cholesterol from peripheral tissues, including macrophages, back to liver. As a regulator of plasma membrane cholesterol content, SR-B1 promotes the uptake of lipid soluble vitamins as well as viral entry into host cells. These collective functions of SR-B1 ultimately affect programmed cell death, female fertility, platelet function, vasculature inflammation, and diet-induced atherosclerosis and myocardial infarction. SR-B1 has also been identified as a potential marker for cancer diagnosis and prognosis. Finally, the SR-B1-linked selective HDL-cholesteryl ester uptake pathway is now being evaluated as a gateway for the delivery of therapeutic and diagnostic agents. In this review, we focus on the regulation and functional significance of SR-B1 in mediating cholesterol movement into and out of cells.

    View details for PubMedID 29125794

  • Nordihydroguaiaretic acid, a lignan from Larrea tridentata (Creosote bush) protects against ALIOS diet-induced metabolic dysfunction in mice. The Journal of pharmacology and experimental therapeutics Chan, J. K., Bittner, S. n., Bittner, A. n., Atwal, S. n., Shen, W. J., Inayathullah, M. n., Rajada, J. n., Nicolls, M. R., Kraemer, F. B., Azhar, S. n. 2018


    To determine the effects of NDGA on metabolic and molecular changes in response to feeding mice typical American fast food or Western diet, mice were fed with ALIOS diet and subjected to metabolic analysis. Male C57BL/6J mice were randomly assigned to: ALIOS, ALIOS + NDGA, or control diet and maintained on the specific diet for 8 weeks. Mice fed ALIOS diet, showed increased body, liver and epididymal fat pad weight, plasma ALT and AST levels (a measure of liver injury), and liver triglyceride (TG) content. Co-administration of NDGA normalized body and epididymal fat pad weight, ALT and AST levels, and liver TG. NDGA treatment also improved insulin sensitivity but not glucose intolerance in ALIOS diet fed mice. In ALIOS diet fed mice, NDGA supplementation induced PPARα (the master regulator of fatty acid oxidation) and mRNA levels of Cpt1c and Cpt2, key genes involved in fatty acid oxidation as compared to ALIOS diet. NDGA significantly reduced liver ER stress response CHOP protein, as compared to chow or ALIOS diet and also ameliorated ALIOS diet-induced elevation of apoptosis signaling protein, CASP3. Likewise, NDGA downregulated the ALIOS-diet induced mRNA levels of Pparg, Fasn, and Dgat2. NDGA treatment of ALIOS fed mice upregulated the hepatic expression of antioxidant enzymes, GPX4 and PRDX3 proteins. In conclusion, we provide evidence that NDGA improves metabolic dysregulation by simultaneously modulating PPARα transcription factor and key genes involved in fatty acid oxidation, key antioxidant and lipogenic enzymes, and apoptosis and ER stress signaling pathways.

    View details for PubMedID 29472517

  • Cardiac overexpression of perilipin 2 induces dynamic steatosis: prevention by hormone-sensitive lipase AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Ueno, M., Suzuki, J., Hirose, M., Sato, S., Imagawa, M., Zenimaru, Y., Takahashi, S., Ikuyama, S., Koizumi, T., Konoshita, T., Kraemer, F. B., Ishizuka, T. 2017; 313 (6): E699–E709


    Cardiac intracellular lipid accumulation (steatosis) is a pathophysiological phenomenon observed in starvation and diabetes mellitus. Perilipin 2 (PLIN2) is a lipid droplet (LD)-associated protein expressed in nonadipose tissues, including the heart. To explore the pathophysiological function of myocardial PLIN2, we generated transgenic (Tg) mice by cardiac-specific overexpression of PLIN2. Tg hearts showed accumulation of numerous small LDs associated with mitochondrial chains and high cardiac triacylglycerol (TAG) content [8-fold greater than wild-type (WT) mice]. Despite massive steatosis, cardiac uptake of glucose, fatty acids and VLDL, systolic function, and expression of metabolic genes were comparable in the two genotypes, and no morphological changes were observed by electron microscopy in the Tg hearts. Twenty-four hours of fasting markedly reduced steatosis in Tg hearts, whereas WT mice showed accumulation of LDs. Although activity of adipose triglyceride lipase in heart homogenate was comparable between WT and Tg mice, activity of hormone-sensitive lipase (HSL) was 40-50% less in Tg than WT mice under both feeding and fasting conditions, suggesting interference of PLIN2 with HSL. Mice generated through crossing of PLIN2-Tg mice and HSL-Tg mice showed cardiac-specific HSL overexpression and complete lack of steatosis. The results suggest that cardiac PLIN2 plays an important pathophysiological role in the development of dynamic steatosis and that the latter was prevented by upregulation of intracellular lipases, including HSL.

    View details for PubMedID 28851734

  • WNT-activated bone grafts repair osteonecrotic lesions in aged animals SCIENTIFIC REPORTS Salmon, B., Liu, B., Shen, E., Chen, T., Li, J., Gillette, M., Ransom, R. C., Ezran, M., Johnson, C. A., Castillo, A. B., Shen, W. J., Kraemer, F. B., Smith, A. A., Helms, J. A. 2017; 7: 14254


    The Wnt pathway is a new target in bone therapeutic space. WNT proteins are potent stem cell activators and pro-osteogenic agents. Here, we gained insights into the molecular and cellular mechanisms responsible for liposome-reconstituted recombinant human WNT3A protein (L-WNT3A) efficacy to treat osteonecrotic defects. Skeletal injuries were coupled with cryoablation to create non-healing osteonecrotic defects in the diaphysis of the murine long bones. To replicate clinical therapy, osteonecrotic defects were treated with autologous bone graft, which were simulated by using bone graft material from syngeneic ACTB-eGFP-expressing mice. Control osteonecrotic defects received autografts alone; test sites received autografts treated ex vivo with L-WNT3A. In vivo µCT monitored healing over time and immunohistochemistry were used to track the fate of donor cells and assess their capacity to repair osteonecrotic defects according to age and WNT activation status. Collectively, analyses demonstrated that cells from the autograft directly contributed to repair of an osteonecrotic lesion, but this contribution diminished as the age of the donor increased. Pre-treating autografts from aged animals with L-WNT3A restored osteogenic capacity to autografts back to levels observed in autografts from young animals. A WNT therapeutic approach may therefore have utility in the treatment of osteonecrosis, especially in aged patients.

    View details for PubMedID 29079746

  • Regulation of adrenal and ovarian steroidogenesis by miR-132 JOURNAL OF MOLECULAR ENDOCRINOLOGY Hu, Z., Shen, W., Kraemer, F. B., Azhar, S. 2017; 59 (3): 269–83


    miR-132 is hormonally regulated in steroidogenic cells of the adrenal gland, ovary and testis. Here, we examined the potential role of miR-132 in the control of steroidogenesis. Transfection of Y1 adrenal cells with miR-132 increased mRNAs of 3β-HSD and 20α-HSD enzymes, which catalyze the sequential conversion of pregnenolone to progesterone to biologically inactive 20α-hydroxyprogesterone (20α-OHP). Overexpression of miR-132 reduced MeCP2 and StAR protein expression, basal progestin (progesterone and 20α-OHP) production, but enhanced their production in response to cAMP stimulation. Use of [3H] pregnenolone and free-diffusible 22(R)-hydroxycholesterol further confirmed that miR-132 promotes the production of 20α-OHP by upregulating 3β-HSD and 20α-HSD. Evidence is also presented that StAR is a direct target of miR-132. Transient transfection of Y1 cells with miR-132 demonstrated that miR-132 induction of 3β-HSD and 20α-HSD was accompanied by significant suppression of one of its target gene products, MeCP2. In contrast, co-expression of miR-132 plus MeCP2 protein partially blocked the ability of miR-132 to upregulate the expression and function of 3β-HSD and 20α-HSD. Moreover, suppression of MeCP2 protein with siRNA resulted in increased expression of 3β-HSD and 20α-HSD, further demonstrating that miR-132 induces the expression of these two enzymes via inhibition of MeCP2. Likewise, overexpression of miR-132 increased 20α-OHP production with and without HDL loading, while knockdown of miR-132 resulted in a significant decrease of 20α-OHP production by granulosa cells. In conclusion, our data suggest that miR-132 attenuates steroidogenesis by repressing StAR expression and inducing 20α-HSD via inhibition of MeCP2 to generate a biologically inactive 20α-OHP.

    View details for PubMedID 28729436

  • PPARs: regulators of metabolism and as therapeutic targets in cardiovascular disease. Part I: PPAR-a. Future cardiology Han, L., Shen, W., Bittner, S., Kraemer, F. B., Azhar, S. 2017


    This article provides a comprehensive review about the molecular and metabolic actions of PPAR-α. It describes its structural features, ligand specificity, gene transcription mechanisms, functional characteristics and target genes. In addition, recent progress with the use of loss of function and gain of function mouse models in the discovery of diverse biological functions of PPAR-α, particularly in the vascular system and the status of the development of new single, dual, pan and partial PPAR agonists (PPAR modulators) in the clinical management of metabolic diseases are presented. This review also summarizes the clinical outcomes from a large number of clinical trials aimed at evaluating the atheroprotective actions of current clinically used PPAR-α agonists, fibrates and statin-fibrate combination therapy.

    View details for DOI 10.2217/fca-2016-0059

    View details for PubMedID 28581332

  • PPARs: regulators of metabolism and as therapeutic targets in cardiovascular disease. Part II: PPAR-ß/d and PPAR-?. Future cardiology Han, L., Shen, W., Bittner, S., Kraemer, F. B., Azhar, S. 2017


    The PPARs are a subfamily of three ligand-inducible transcription factors, which belong to the superfamily of nuclear hormone receptors. In mammals, the PPAR subfamily consists of three members: PPAR-α, PPAR-β/δ and PPAR-γ. PPARs control the expression of a large number of genes involved in metabolic homeostasis, lipid, glucose and energy metabolism, adipogenesis and inflammation. PPARs regulate a large number of metabolic pathways that are implicated in the pathogenesis of metabolic diseases such as metabolic syndrome, Type 2 diabetes mellitus, nonalcoholic fatty liver disease and cardiovascular disease. The aim of this review is to provide up-to-date information about the biochemical and metabolic actions of PPAR-β/δ and PPAR-γ, the therapeutic potential of their agonists currently under clinical development and the cardiovascular disease outcome of clinical trials of PPAR-γ agonists, pioglitazone and rosiglitazone.

    View details for DOI 10.2217/fca-2017-0019

    View details for PubMedID 28581362

  • SNAREs and cholesterol movement for steroidogenesis MOLECULAR AND CELLULAR ENDOCRINOLOGY Kraemer, F. B., Shen, W., Azhar, S. 2017; 441 (C): 17-21


    Steroidogenesis is a complex process through which cholesterol traffics to mitochondria and is converted via a series of enzymatic steps to steroid hormones. Although the rate-limiting step in this process is the movement of cholesterol from the outer to the inner mitochondrial membrane via the actions of StAR, a continuous supply of cholesterol must be delivered to the outer mitochondrial membrane during active steroidogenesis and this is derived from multiple sources, including lipoprotein uptake, endogenous cholesterol synthesis and release from stores within cytoplasmic lipid droplets. A number of mechanisms have been suggested to contribute to cholesterol trafficking to mitochondria; however, there is no definitive consensus and this is particularly so in regards to trafficking from cytoplasmic lipid droplets. In this paper we review experiments in which we have surveyed the expression of SNARE proteins in steroidogenic tissue and cells and examined the role of SNAREs in mediating cholesterol movement from lipid droplets to the mitochondria based on multiple studies that identified SNAREs as components of cytoplasmic lipid droplets. We established and characterized an in vitro mitochondria reconstitution assay system that enabled us to examine the impact of adding recombinant SNARE proteins specifically on the movement of cholesterol from model lipid droplets to the outer mitochondrial membrane. Using this reconstitution assay system in combination with siRNA knockdown experiments in rat primary granulosa cells or in steroidogenic cell lines, we showed that several SNARE proteins are important components in the trafficking of cholesterol from lipid droplets to the mitochondria for steroidogenesis.

    View details for DOI 10.1016/j.mce.2016.07.034

    View details for Web of Science ID 000393247500004

  • Microarray analysis of gene expression in liver, adipose tissue and skeletal muscle in response to chronic dietary administration of NDGA to high-fructose fed dyslipidemic rats NUTRITION & METABOLISM Zhang, H., Shen, W., Li, Y., Bittner, A., Bittner, S., Tabassum, J., Cortez, Y. F., Kraemer, F. B., Azhar, S. 2016; 13


    Nordihydroguaiaretic acid (NDGA), the main metabolite of Creosote Bush, has been shown to have profound effects on the core components of metabolic syndrome, including lowering of blood glucose, free fatty acids and triglyceride levels, attenuating elevated blood pressure in several rodent models of dyslipidemia, and improving body weight, insulin resistance, diabetes and hypertension. In the present study, a high-fructose diet fed rat model of hypertriglyceridemia, dyslipidemia, insulin resistance and hepatic steatosis was employed to investigate the global transcriptional changes in the lipid metabolizing pathways in three insulin sensitive tissues: liver, skeletal muscle and adipose tissue in response to chronic dietary administration of NDGA. Sprague-Dawley male rats (SD) were fed a chow (control) diet, high-fructose diet (HFrD) or HFrD supplemented with NDGA (2.5 g/kg diet) for eight weeks. Dietary administration of NDGA decreased plasma levels of TG, glucose, and insulin, and attenuated hepatic TG accumulation. DNA microarray expression profiling indicated that dietary administration of NDGA upregulated the expression of certain genes involved in fatty acid oxidation and their transcription regulator, PPARα, decreased the expression of a number of lipogenic genes and relevant transcription factors, and differentially impacted the genes of fatty acid transporters, acetyl CoA synthetases, elongases, fatty acid desaturases and lipid clearance proteins in liver, skeletal muscle and adipose tissues. These findings suggest that NDGA ameliorates hypertriglyceridemia and steatosis primarily by inhibiting lipogenesis and enhancing fatty acid catabolism in three major insulin responsive tissues by altering the expression of key enzyme genes and transcription factors involved in de novo lipogenesis and fatty acid oxidation.

    View details for DOI 10.1186/s12986-016-0121-y

    View details for Web of Science ID 000384584200001

    View details for PubMedID 27708683

    View details for PubMedCentralID PMC5041401

  • Hormone-sensitive lipase deficiency disturbs lipid composition of plasma membrane microdomains from mouse testis. Biochimica et biophysica acta Casado, M. E., Pastor, O., García-Seisdedos, D., Huerta, L., Kraemer, F. B., Lasunción, M. A., Martín-Hidalgo, A., Busto, R. 2016; 1861 (9): 1142-1150

    View details for DOI 10.1016/j.bbalip.2016.06.018

    View details for PubMedID 27355565

  • SNAREs and cholesterol movement for steroidogenesis. Molecular and cellular endocrinology Kraemer, F. B., Shen, W., Azhar, S. 2016


    Steroidogenesis is a complex process through which cholesterol traffics to mitochondria and is converted via a series of enzymatic steps to steroid hormones. Although the rate-limiting step in this process is the movement of cholesterol from the outer to the inner mitochondrial membrane via the actions of StAR, a continuous supply of cholesterol must be delivered to the outer mitochondrial membrane during active steroidogenesis and this is derived from multiple sources, including lipoprotein uptake, endogenous cholesterol synthesis and release from stores within cytoplasmic lipid droplets. A number of mechanisms have been suggested to contribute to cholesterol trafficking to mitochondria; however, there is no definitive consensus and this is particularly so in regards to trafficking from cytoplasmic lipid droplets. In this paper we review experiments in which we have surveyed the expression of SNARE proteins in steroidogenic tissue and cells and examined the role of SNAREs in mediating cholesterol movement from lipid droplets to the mitochondria based on multiple studies that identified SNAREs as components of cytoplasmic lipid droplets. We established and characterized an in vitro mitochondria reconstitution assay system that enabled us to examine the impact of adding recombinant SNARE proteins specifically on the movement of cholesterol from model lipid droplets to the outer mitochondrial membrane. Using this reconstitution assay system in combination with siRNA knockdown experiments in rat primary granulosa cells or in steroidogenic cell lines, we showed that several SNARE proteins are important components in the trafficking of cholesterol from lipid droplets to the mitochondria for steroidogenesis.

    View details for DOI 10.1016/j.mce.2016.07.034

    View details for PubMedID 27477781

  • ACTH Regulation of Adrenal SR-B1 FRONTIERS IN ENDOCRINOLOGY Shen, W., Azhar, S., Kraemer, F. B. 2016; 7


    The adrenal gland is one of the prominent sites for steroid hormone synthesis. Lipoprotein-derived cholesterol esters (CEs) delivered via SR-B1 constitute the dominant source of cholesterol for steroidogenesis, particularly in rodents. Adrenocorticotropic hormone (ACTH) stimulates steroidogenesis through downstream actions on multiple components involved in steroidogenesis. Both acute and chronic ACTH treatments can modulate SR-B1 function, including its transcription, posttranscriptional stability, phosphorylation and dimerization status, as well as the interaction with other protein partners, all of which result in changes in the ability of SR-B1 to mediate HDL-CE uptake and the supply of cholesterol for conversion to steroids. Here, we provide a review of the recent findings on the regulation of adrenal SR-B1 function by ACTH.

    View details for DOI 10.3389/fendo.2016.00042

    View details for Web of Science ID 000378509500001

    View details for PubMedID 27242666

    View details for PubMedCentralID PMC4865504

  • SNARE-Mediated Cholesterol Movement to Mitochondria Supports Steroidogenesis in Rodent Cells. Molecular endocrinology Lin, Y., Hou, X., Shen, W., Hanssen, R., Khor, V. K., Cortez, Y., Roseman, A. N., Azhar, S., Kraemer, F. B. 2016; 30 (2): 234-247


    Vesicular transport involving soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins is known to be responsible for many major cellular activities. In steroidogenic tissues, chronic hormone stimulation results in increased expression of proteins involved in the steroidogenic pathway, whereas acute hormone stimulation prompts the rapid transfer of cholesterol to the inner mitochondrial membrane to be utilized as substrate for steroid hormone production. Several different pathways are involved in supplying cholesterol to mitochondria, but mobilization of stored cholesteryl esters appears to initially constitute the preferred source; however, the mechanisms mediating this cholesterol transfer are not fully understood. To study the potential contribution of SNARE proteins in steroidogenesis, we examined the expression levels of various SNARE proteins in response to hormone stimulation in steroidogenic tissues and cells and established an in vitro mitochondria reconstitution assay system to assess the contribution of various SNARE proteins on cholesterol delivery for steroidogenesis. Our results from reconstitution experiments along with knockdown studies in rat primary granulosa cells and in a Leydig cell line show that soluble N-ethylmaleimide sensitive factor attachment protein-α, synaptosomal-associated protein of 25 kDa, syntaxin-5, and syntaxin-17 facilitate the transport of cholesterol to mitochondria. Thus, although StAR is required for efficient cholesterol movement into mitochondria for steroidogenesis, specific SNAREs participate and are necessary to mediate cholesterol movement to mitochondria.

    View details for DOI 10.1210/me.2015-1281

    View details for PubMedID 26771535

    View details for PubMedCentralID PMC4792230

  • Lipid droplets and steroidogenic cells EXPERIMENTAL CELL RESEARCH Shen, W., Azhar, S., Kraemer, F. B. 2016; 340 (2): 209-214


    Lipid droplets (LDs) in steroidogenic tissues have a cholesteryl ester (CE) core surrounded by a phospholipid monolayer that is coated with associated proteins. Compared with other tissues, they tend to be smaller in size and more numerous in numbers. These LDs are enriched with PLIN1c, PLIN2 and PLIN3. Both CIDE A and B are found in mouse ovary. Free cholesterol (FC) released upon hormone stimulation from LDs is the preferred source of cholesterol substrate for steroidogenesis, and HSL is the major neutral cholesterol esterase mediating the conversion of CEs to FC. Through the interaction of HSL with vimentin and StAR, FC is translocated to mitochondria for steroid hormone production. Proteomic analyses of LDs isolated from loaded primary ovarian granulosa cells, mouse MLTC-1 Leydig tumor cells and mouse testes revealed LD associated proteins that are actively involved in modulating lipid homeostasis along with a number of steroidogenic enzymes. Microscopy analysis confirmed the localization of many of these proteins to LDs. These studies broaden the role of LDs to include being a platform for functional steroidogenic enzyme activity or as a port for transferring steroidogenic enzymes and/or steroid intermediates, in addition to being a storage depot for CEs.

    View details for DOI 10.1016/j.yexcr.2015.11.024

    View details for Web of Science ID 000370581800008

    View details for PubMedID 26639173

    View details for PubMedCentralID PMC4744538

  • A Novel Role of Salt-Inducible Kinase 1 (SIK1) in the Post-Translational Regulation of Scavenger Receptor Class B Type 1 Activity BIOCHEMISTRY Hu, Z., Hu, J., Shen, W., Kraemer, F. B., Azhar, S. 2015; 54 (46): 6917-6930

    View details for DOI 10.1021/acs.biochem.5b00147

    View details for Web of Science ID 000365464100007

    View details for PubMedID 26567857

  • Effect of Creosote Bush-Derived NDGA on Expression of Genes Involved in Lipid Metabolism in Liver of High-Fructose Fed Rats: Relevance to NDGA Amelioration of Hypertriglyceridemia and Hepatic Steatosis PLOS ONE Zhang, H., Li, Y., Hu, J., Shen, W., Singh, M., Hou, X., Bittner, A., Bittner, S., Cortez, Y., Tabassum, J., Kraemer, F. B., Azhar, S. 2015; 10 (9)

    View details for DOI 10.1371/journal.pone.0138203

    View details for PubMedID 26394137

  • Using SRM-MS to quantify nuclear protein abundance differences between adipose tissue depots of insulin-resistant mice JOURNAL OF LIPID RESEARCH Ota, A., Kovary, K. M., Wu, O. H., Ahrends, R., Shen, W., Costa, M. J., Feldman, B. J., Kraemer, F. B., Teruel, M. N. 2015; 56 (5): 1068-1078


    Insulin resistance underlies metabolic disease. Visceral, but not subcutaneous, white adipose tissue (WAT) has been linked to the development of insulin resistance, potentially due to differences in regulatory protein abundance. Here we investigate how protein levels are changed in insulin resistance in different WAT depots by developing a targeted proteomics approach to quantitatively compare the abundance of 42 nuclear proteins in subcutaneous and visceral WAT from a commonly-used insulin resistant mouse model, Lepr(db/db), and from C57BL/6J control mice. The most differentially-expressed proteins were important in adipogenesis, as confirmed by siRNA-mediated depletion experiments, suggesting a defect in adipogenesis in visceral, but not subcutaneous, insulin-resistant WAT. Furthermore, differentiation of visceral, but not subcutaneous, insulin-resistant stromal vascular cells (SVC) was impaired. In an in vitro approach to understand the cause of this impaired differentiation, we compared insulin-resistant visceral SVC to preadipocyte cell culture models made insulin-resistant by different stimuli. The insulin-resistant visceral SVC protein abundance profile correlated most with preadipocyte cell culture cells treated with both palmitate and TNFα. Together, our study introduces a method to simultaneously measure and quantitatively compare nuclear protein expression patterns in primary adipose tissue and adipocyte cell cultures, which we show can reveal relationships between differentiation and disease states of different adipocyte tissue types.

    View details for DOI 10.1194/jlr.D056317

    View details for Web of Science ID 000353767200012

    View details for PubMedID 25840986

  • Adipose Triglyceride Lipase, Not Hormone-Sensitive Lipase, Is the Primary Lipolytic Enzyme in Fasting Elephant Seals (Mirounga angustirostris) PHYSIOLOGICAL AND BIOCHEMICAL ZOOLOGY Fowler, M. A., Costa, D. P., Crocker, D. E., Shen, W., Kraemer, F. B. 2015; 88 (3): 284-294


    Little is known about the mechanisms that allow capital breeders to rapidly mobilize large amounts of body reserves. Northern elephant seals (Mirounga angustirostris) utilize fat reserves for maternal metabolism and to create high fat milk for the pup. Hormone-sensitive lipase (HSL) has been hypothesized to be an important lipolytic enzyme in fasting seals, but the activity of HSL and adipose triglyceride lipase (ATGL) has not been quantified in fasting adult seals, nor has their relationship to milk lipid content been assessed. Blubber and milk samples were obtained from 18 early lactation and 19 late lactation females, as well as blubber from five early and five late molting female seals. Blubber lipolytic activity was assessed with radiometric assays. HSL activity was negligible in seal blubber at all fasting stages. Total triglyceride lipase activity was stable among early and late lactation and early molt but increased in late molting seals. Relative abundance of ATGL protein increased across fasting, but neither activity nor relative protein levels were related to circulating nonesterified fatty acids or milk lipid content, suggesting the possibility of other regulatory pathways between lipolytic activity and milk lipid content. These results demonstrate that HSL is not the primary lipolytic enzyme in fasting adult female seals and that ATGL contributes more to lipolysis than HSL.

    View details for DOI 10.1086/680079

    View details for Web of Science ID 000352794300005

    View details for PubMedID 25860827

  • Quantification of stromal vascular cell mechanics with a linear cell monolayer rheometer JOURNAL OF RHEOLOGY Elkins, C. M., Shen, W., Khor, V. K., Kraemer, F. B., Fuller, G. G. 2015; 59 (1): 33-50

    View details for DOI 10.1122/1.4902437

    View details for Web of Science ID 000347976300003

  • p38 MAPK regulates steroidogenesis through transcriptional repression of STAR gene. Journal of molecular endocrinology Zaidi, S. K., Shen, W., Bittner, S., Bittner, A., McLean, M. P., Han, J., Davis, R. J., Kraemer, F. B., Azhar, S. 2014; 53 (1): 1-16


    STAR/StarD1, part of a protein complex, mediates the transport of cholesterol from the outer to inner mitochondrial membrane, which is the rate-limiting step for steroidogenesis, and where steroid hormone synthesis begins. Herein, we examined the role of oxidant-sensitive p38 MAPKs in the regulation of STAR gene transcription, using model steroidogenic cell lines. Our data indicate that oxidant activation of p38 MAPK exhibits a negative regulatory role in the induction of functional expression of STAR, as evidenced by enhanced induction of STAR (mRNA/protein) expression and increased steroidogenesis during pharmacological inhibition of p38 MAPK or in cells with increased transient overexpression of a dominant-negative (dn) form of p38 MAPKα or p38 MAPKβ. Studies with rat Star-promoter demonstrated that overexpression of p38 MAPKα-wt, -β, or -γ significantly reduced both basal and cAMP-sensitive promoter activity. In contrast, overexpression of p38 MAPKα-dn, -β, or -γ enhanced the Star promoter activity under basal conditions and in response to cAMP stimulation. Use of various constitutively active and dn constructs and designer knock-out cell lines demonstrated that MKK3 and MKK6, the upstream activators of p38 MAPKs, play a role in p38 MAPKα-mediated inhibition of Star promoter activity. In addition, our studies raised the possibility of CREB being a potential target of the p38 MAPK inhibitory effect on Star promoter activity. Collectively, these data provide novel mechanistic information about how oxidant-sensitive p38 MAPKs, particularly p38 MAPKα, contribute to the negative regulation of Star gene expression and inhibit steroidogenesis.

    View details for DOI 10.1530/JME-13-0287

    View details for PubMedID 24780837

  • Scavenger receptor class B type I (SR-BI): a versatile receptor with multiple functions and actions. Metabolism Shen, W., Hu, J., Hu, Z., Kraemer, F. B., Azhar, S. 2014; 63 (7): 875-886


    Scavenger receptor class B type I (SR-BI), is a physiologically relevant HDL receptor that mediates selective uptake of lipoprotein (HDL)-derived cholesteryl ester (CE) in vitro and in vivo. Mammalian SR-BI is a 509-amino acid, ~82 kDa glycoprotein that contains N- and C-terminal cytoplasmic domains, two-transmembrane domains, as well as a large extracellular domain containing 5-6 cysteine residues and multiple sites for N-linked glycosylation. The size and structural characteristics of SR-BI, however, vary considerably among lower vertebrates and insects. Recently, significant progress has been made in understanding the molecular mechanisms involved in the posttranscriptional/posttranslational regulation of SR-BI in a tissue specific manner. The purpose of this review is to summarize the current body of knowledge about the events and molecules connected with the posttranscriptional/posttranslational regulation of SR-BI and to update the molecular and functional characteristics of the insect SR-BI orthologs.

    View details for DOI 10.1016/j.metabol.2014.03.011

    View details for PubMedID 24854385

  • Gerald M. Reaven, MD: Demonstration of the Central Role of Insulin Resistance in Type 2 Diabetes and Cardiovascular Disease DIABETES CARE Kraemer, F. B., Ginsberg, H. N. 2014; 37 (5): 1178-1181

    View details for DOI 10.2337/dc13-2668

    View details for Web of Science ID 000334840100026

    View details for PubMedID 24757223

  • HPA axis genetic variation, cortisol and psychosis in major depression. Molecular psychiatry Schatzberg, A. F., Keller, J., Tennakoon, L., Lembke, A., Williams, G., Kraemer, F. B., Sarginson, J. E., Lazzeroni, L. C., Murphy, G. M. 2014; 19 (2): 220-227


    Genetic variation underlying hypothalamic pituitary adrenal (HPA) axis overactivity in healthy controls (HCs) and patients with severe forms of major depression has not been well explored, but could explain risk for cortisol dysregulation. In total, 95 participants were studied: 40 patients with psychotic major depression (PMD); 26 patients with non-psychotic major depression (NPMD); and 29 HCs. Collection of genetic material was added one third of the way into a larger study on cortisol, cognition and psychosis in major depression. Subjects were assessed using the Brief Psychiatric Rating Scale, the Hamilton Depression Rating Scale and the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders. Blood was collected hourly for determination of cortisol from 1800 to 0900 h and for the assessment of alleles for six genes involved in HPA axis regulation. Two of the six genes contributed significantly to cortisol levels, psychosis measures or depression severity. After accounting for age, depression and psychosis, and medication status, only allelic variation for the glucocorticoid receptor (GR) gene accounted for a significant variance for mean cortisol levels from 1800 to 0100 h (r(2)=0.288) and from 0100 to 0900 h (r(2)=0.171). In addition, GR and corticotropin-releasing hormone receptor 1 (CRHR1) genotypes contributed significantly to psychosis measures and CRHR1 contributed significantly to depression severity rating.Molecular Psychiatry advance online publication, 29 October 2013; doi:10.1038/mp.2013.129.

    View details for DOI 10.1038/mp.2013.129

    View details for PubMedID 24166410

  • The proteome of cholesteryl-ester-enriched versus triacylglycerol-enriched lipid droplets. PloS one Khor, V. K., Ahrends, R., Lin, Y., Shen, W., Adams, C. M., Roseman, A. N., Cortez, Y., Teruel, M. N., Azhar, S., Kraemer, F. B. 2014; 9 (8)


    Within cells, lipids are stored in the form of lipid droplets (LDs), consisting of a neutral lipid core, surrounded by a phospholipid monolayer and an outer layer of protein. LDs typically accumulate either triacylglycerol (TAG) and diacylglycerol or cholesteryl ester (CE), depending on the type of tissue. Recently, there has been an increased interest in the proteins that surround LDs. LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation. Previous proteomics analyses have focused on TAG-enriched LDs, whereas CE-enriched LDs have been largely ignored. Our study has compared the LD proteins from CE-enriched LDs to TAG-enriched LDs in steroidogenic cells. In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts. Protein expression was further validated by selected reaction monitoring (SRM) mass spectrometry (MS). SRM verified expression of 25 of 27 peptides that were previously detected by tandem mass tagging MS. Several proteins were confirmed to be elevated in CE-enriched LDs by SRM including the intermediate filament vimentin. This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.

    View details for DOI 10.1371/journal.pone.0105047

    View details for PubMedID 25111084

    View details for PubMedCentralID PMC4128735

  • The proteome of cholesteryl-ester-enriched versus triacylglycerol-enriched lipid droplets. PloS one Khor, V. K., Ahrends, R., Lin, Y., Shen, W., Adams, C. M., Roseman, A. N., Cortez, Y., Teruel, M. N., Azhar, S., Kraemer, F. B. 2014; 9 (8)

    View details for DOI 10.1371/journal.pone.0105047

    View details for PubMedID 25111084

  • Lipid droplet metabolism. Current opinion in clinical nutrition and metabolic care Khor, V. K., Shen, W., Kraemer, F. B. 2013; 16 (6): 632-637


    With the realization that lipid droplets are not merely inert fat storage organelles, but highly dynamic and actively involved in cellular lipid homeostasis, there has been an increased interest in lipid droplet biology. Recent studies have begun to unravel the roles that lipid dropletss play in cellular physiology and provide insights into the mechanisms by which lipid droplets contribute to cellular homeostasis. This review provides a summary of these recent publications on lipid droplet metabolism.Perilipins have different preferences for associating with triacylglycerol (TAG) or cholesteryl esters, different tissue distributions, and each contributes to lipid metabolism in its unique way. Cell death-inducing DFF45-like effector proteins are not only involved in lipid droplet expansion, but also in the cellular response to stress and lipid secretion. Lipid droplets undergo an active cycle of lipolysis and re-esterification to form microlipid droplets. TAG synthesis for lipid droplet formation and expansion occurs in the endoplasmic reticulum and on lipid droplets, and TAG transfers between lipid droplets during lipid droplet fusion. Lipid droplets interact with the endoplasmic reticulum and mitochondria to facilitate lipid transfer, lipid droplet expansion, and metabolism.Lipid droplets are dynamically active, responding to changes in cellular physiology, as well as interacting with cytosolic proteins and other organelles to control lipid homeostasis.

    View details for DOI 10.1097/MCO.0b013e3283651106

    View details for PubMedID 24100667

  • Hormonal Regulation of MicroRNA Expression in Steroid Producing Cells of the Ovary, Testis and Adrenal Gland PLOS ONE Hu, Z., Shen, W., Cortez, Y., Tang, X., Liu, L., Kraemer, F. B., Azhar, S. 2013; 8 (10)


    Given the emerging roles of miRNAs as potential posttranscriptional/posttranslational regulators of the steroidogenic process in adrenocortical and gonadal cells, we sought to determine miRNA profiles in rat adrenals from animals treated with vehicle, ACTH, 17α-E2 or dexamethasone. Key observations were also confirmed using hormone (Bt2cAMP)-treated mouse Leydig tumor cells, MLTC-1, and primary rat ovarian granulosa cells.RNA was extracted from rat adrenal glands and miRNA profiles were established using microarray and confirmed with qRT-PCR. The expression of some of the hormone-sensitive miRNAs was quantified in MLTC-1 and granulosa cells after stimulation with Bt2cAMP. Targets of hormonally altered miRNAs were explored by qRT-PCR and Western blotting in adrenals and granulosa cells.Adrenals from ACTH, 17α-E2 and dexamethasone treated rats exhibited miRNA profiles distinct from control animals. ACTH up-regulated the expression of miRNA-212, miRNA-182, miRNA-183, miRNA-132, and miRNA-96 and down-regulated the levels of miRNA-466b, miRNA-214, miRNA-503, and miRNA-27a. The levels of miR-212, miRNA-183, miRNA-182, miRNA-132, miRNA-370, miRNA-377, and miRNA-96 were up-regulated, whereas miR-125b, miRNA-200b, miR-122, miRNA-466b, miR-138, miRNA-214, miRNA-503 and miRNA27a were down-regulated in response to 17α-E2 treatment. Dexamethasone treatment decreased miRNA-200b, miR-122, miR-19a, miRNA-466b and miRNA27a levels, but increased miRNA-183 levels. Several adrenal miRNAs are subject to regulation by more than one hormone. Significant cAMP-induced changes in certain miRNAs were also noted in MLTC-1 and granulosa cells. Some of the hormone-induced miRNAs in steroidogenic cells were predicted to target proteins involved in lipid metabolism/steroidogenesis. We also obtained evidence that miR-132 and miRNA-214 inhibit the expression of SREBP-1c and LDLR, respectively.Our results demonstrate that expression of a number of miRNAs in steroidogenic cells of the testis, ovary and adrenal glands is subject to hormonal regulation and that miRNAs and their regulation by specific hormones are likely to play a key role in posttranscriptional/posttranslational regulation of steroidogenesis.

    View details for DOI 10.1371/journal.pone.0078040

    View details for Web of Science ID 000326241200050

    View details for PubMedID 24205079

    View details for PubMedCentralID PMC3810252

  • Cholesterol ester droplets and steroidogenesis. Molecular and cellular endocrinology Kraemer, F. B., Khor, V. K., Shen, W., Azhar, S. 2013; 371 (1-2): 15-19


    Intracellular lipid droplets (LDs) are dynamic organelles that contain a number of associated proteins including perilipin (Plin) and vimentin. Cholesteryl ester (CE)-rich LDs normally accumulate in steroidogenic cells and their mobilization is the preferred initial source of cholesterol for steroidogenesis. Plin1a, 1b and 5 were found to preferentially associate with triacylglycerol-rich LDs and Plin1c and Plin4 to associate with CE-rich LDs, but the biological significance of this remains unanswered. Vimentin null mice were found to have decreased ACTH-stimulated corticosterone levels, and decreased progesterone levels in females, but normal hCG-stimulated testosterone levels in males. Smaller LDs were seen in null cells. Lipoprotein cholesterol delivery to adrenals and ovary was normal, as was the expression of steroidogenic genes; however, the movement of cholesterol to mitochondria was reduced in vimentin null mice. These results suggest that vimentin is important in the maintenance of CE-rich LDs and in the movement of cholesterol for steroidogenesis.

    View details for DOI 10.1016/j.mce.2012.10.012

    View details for PubMedID 23089211

  • Regulation of Expression and Function of Scavenger Receptor Class B, Type I (SR-BI) by Na+/H+ Exchanger Regulatory Factors (NHERFs). journal of biological chemistry Hu, Z., Hu, J., Zhang, Z., Shen, W., Yun, C. C., Berlot, C. H., Kraemer, F. B., Azhar, S. 2013; 288 (16): 11416-11435


    Scavenger receptor class B, type I (SR-BI) binds HDL and mediates selective delivery of cholesteryl esters (CEs) to the liver, adrenals, and gonads for product formation (bile acids and steroids). Because relatively little is known about SR-BI posttranslational regulation in steroidogenic cells, we examined the roles of Na(+)/H(+) exchanger regulatory factors (NHERFs) in regulating SR-BI expression, SR-BI-mediated selective CE uptake, and steroidogenesis. NHERF1 and NHERF2 mRNA and protein are expressed at varying levels in model steroidogenic cell lines and the adrenal, with only low expression of PDZK1 (NHERF3) and NHERF4. Dibutyryl cyclic AMP decreased NHERF1 and NHERF2 and increased SR-BI mRNA expression in primary rat granulosa cells and MLTC-1 cells, whereas ACTH had no effect on NHERF1 and NHERF2 mRNA levels but decreased their protein levels in rat adrenals. Co-immunoprecipitation, colocalization, bimolecular fluorescence complementation, and mutational analysis indicated that SR-BI associates with NHERF1 and NHERF2. NHERF1 and NHERF2 down-regulated SR-BI protein expression through inhibition of its de novo synthesis. NHERF1 and NHERF2 also inhibited SR-BI-mediated selective CE transport and steroidogenesis, which were markedly attenuated by partial deletions of the PDZ1 or PDZ2 domain of NHERF1, the PDZ2 domain of NHERF2, or the MERM domains of NHERF1/2 or by gene silencing of NHERF1/2. Moreover, an intact COOH-terminal PDZ recognition motif (EAKL) in SR-BI is needed. Transient transfection of hepatic cell lines with NHERF1 or NHERF2 caused a significant reduction in endogenous protein levels of SR-BI. Collectively, these data establish NHERF1 and NHERF2 as SR-BI protein binding partners that play a negative role in the regulation of SR-BI expression, selective CE transport, and steroidogenesis.

    View details for DOI 10.1074/jbc.M112.437368

    View details for PubMedID 23482569

  • Mechanisms of Action of Hormone-sensitive Lipase in Mouse Leydig Cells ITS ROLE IN THE REGULATION OF THE STEROIDOGENIC ACUTE REGULATORY PROTEIN JOURNAL OF BIOLOGICAL CHEMISTRY Manna, P. R., Cohen-Tannoudji, J., Counis, R., Garner, C. W., Huhtaniemi, I., Kraemer, F. B., Stocco, D. M. 2013; 288 (12): 8505-8518


    Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of cholesteryl esters in steroidogenic tissues and, thus, facilitates cholesterol availability for steroidogenesis. The steroidogenic acute regulatory protein (StAR) controls the rate-limiting step in steroid biosynthesis. However, the modes of action of HSL in the regulation of StAR expression remain obscure. We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity. Phosphorylation and activation of HSL coincided with increases in StAR, P-StAR (Ser-194), and progesterone levels. Inhibition of HSL activity by CAY10499 effectively suppressed Bt2cAMP-induced StAR expression and progesterone synthesis. Targeted silencing of endogenous HSL, with siRNAs, resulted in increased cholesteryl ester levels and decreased cholesterol content in MA-10 cells. Depletion of HSL affected lipoprotein-derived cellular cholesterol influx, diminished the supply of cholesterol to the mitochondria, and resulted in the repression of StAR and P-StAR levels. Cells overexpressing HSL increased the efficacy of liver X receptor (LXR) ligands on StAR expression and steroid synthesis, suggesting HSL-mediated steroidogenesis entails enhanced oxysterol production. Conversely, cells deficient in LXRs exhibited decreased HSL responsiveness. Furthermore, an increase in HSL was correlated with the LXR target genes, steroid receptor element-binding protein 1c and ATP binding cassette transporter A1, demonstrating HSL-dependent regulation of steroidogenesis predominantly involves LXR signaling. LXRs interact/cooperate with RXRs and result in the activation of StAR gene transcription. These findings provide novel insight and demonstrate the molecular events by which HSL acts to drive cAMP/PKA-mediated regulation of StAR expression and steroidogenesis in mouse Leydig cells.

    View details for DOI 10.1074/jbc.M112.417873

    View details for Web of Science ID 000316564500047

    View details for PubMedID 23362264

  • Hormone-sensitive lipase deficiency disturbs the fatty acid composition of mouse testis PROSTAGLANDINS LEUKOTRIENES AND ESSENTIAL FATTY ACIDS Casado, M. E., Pastor, O., Mariscal, P., Canfran-Duque, A., Martinez-Botas, J., Kraemer, F. B., Lasuncion, M. A., Martin-Hidalgo, A., Busto, R. 2013; 88 (3): 227-233


    Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from intracellular stores. In mice, HSL deficiency results in male sterility caused by a major defect in spermatogenesis. The testes contain high concentrations of PUFA and specific PUFA are essential for spermatogenesis. We investigated the fatty acid composition and the mRNA levels of key enzymes involved in fatty acid metabolism in testis of HSL-knockout mice. HSL deficiency altered fatty acid composition in the testis but not in plasma. The most important changes were decreases in the essential n-6 PUFA LNA and the n-3 PUFA ALA, and an increase in the corresponding synthesis intermediates C22:4n-6 and C22:5n-3 without changes in DPAn-6 or DHA acids. Mead acid, which has been associated with an essential fatty acid deficit leading to male infertility, was increased in the testis from HSL-knockout mice. Moreover, the expression of SCD-1, FADS1, and FADS2 was increased while expression of ELOVL2, an essential enzyme for the formation of very-long PUFA in testis, was decreased. Given the indispensability of these fatty acids for spermatogenesis, the changes in fatty acid metabolism observed in testes from HSL-knockout male mice may underlie the infertility of these animals.

    View details for DOI 10.1016/j.plefa.2012.12.005

    View details for Web of Science ID 000316425700005

    View details for PubMedID 23369366

  • Fat-specific protein 27 modulates nuclear factor of activated T cells 5 and the cellular response to stress JOURNAL OF LIPID RESEARCH Ueno, M., Shen, W., Patel, S., Greenberg, A. S., Azhar, S., Kraemer, F. B. 2013; 54 (3): 734-743


    Fat-specific protein 27 (FSP27), a member of the cell death-inducing DNA fragmentation factor α-like effector (Cide) family, is highly expressed in adipose tissues and is a lipid droplet (LD)-associated protein that induces the accumulation of LDs. Using a yeast two-hybrid system to examine potential interactions of FSP27 with other proteins, a direct interaction with the N-terminal region of nuclear factor of activated T cells 5 (NFAT5) was identified. NFAT5 is a transcription factor that induces osmoprotective and inflammatory genes after its translocation to the nucleus. The interaction between FSP27 and NFAT5 was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation. Using immunocytochemistry, NFAT5 is detected in the cytoplasm and in the nucleus under isotonic conditions; however, overexpression of FSP27 inhibited the hypertonic-induced nuclear translocation of NFAT5. Consistent with the suppression of NFAT5 nuclear translocation, in cells transfected with a reporter construct containing the NFAT5 response element from the monocyte chemoattractant protein 1 (MCP1) promoter, FSP27 overexpression repressed hypertonic-induced luciferase activity and the expression of NFAT5 target genes. Knockdown of FSP27 in differentiated 3T3-L1 adipocytes increased the NFAT5-mediated rise in MCP1. These results suggest that FSP27 not only modulates LD homeostasis but also modulates the response to osmotic stress via a physical interaction with NFAT5 at the LD surface.

    View details for DOI 10.1194/jlr.M033365

    View details for Web of Science ID 000314877000016

    View details for PubMedID 23233732

  • Nordihydroguaiaretic acid improves metabolic dysregulation and aberrant hepatic lipid metabolism in mice by both PPAR alpha-dependent and -independent pathways AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Zhang, H., Shen, W., Cortez, Y., Kraemer, F. B., Azhar, S. 2013; 304 (1): G72-G86


    Creosote bush-derived nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, possesses antioxidant properties and functions as a potent antihyperlipidemic agent in rodent models. Here, we examined the effect of chronic NDGA treatment of ob/ob mice on plasma dyslipidemia, hepatic steatosis, and changes in hepatic gene expression. Feeding ob/ob mice a chow diet supplemented with either low (0.83 g/kg diet) or high-dose (2.5 g/kg diet) NDGA for 16 wk significantly improved plasma triglyceride (TG), inflammatory chemokine levels, hyperinsulinemia, insulin sensitivity, and glucose intolerance. NDGA treatment caused a marked reduction in liver weight and TG content, while enhancing rates of fatty acid oxidation. Microarray analysis of hepatic gene expression demonstrated that NDGA treatment altered genes for lipid metabolism, with genes involved in fatty acid catabolism most significantly increased. NDGA upregulated the mRNA and nuclear protein levels of peroxisome proliferator-activated receptor α (PPARα), and the activated (phosphorylated) form of AMP-activated kinase. NDGA increased PPARα promoter activity in AML12 hepatocytes and also prevented the fatty acid suppression of PPARα expression. In contrast, PPARα siRNA abrogated the stimulatory effect of NDGA on fatty acid catabolism. Likewise, no stimulatory effect of NDGA on hepatic fatty acid oxidation was observed in the livers of PPARα-deficient mice, but the ability of NDGA to reverse fatty liver conditions was unaffected. In conclusion, the beneficial actions of NDGA on dyslipidemia and hepatic steatosis in ob/ob mice are exerted primarily through enhanced fatty acid oxidation via PPARα-dependent pathways. However, PPARα-independent pathways also contribute to NDGA's action to ameliorate hepatic steatosis.

    View details for DOI 10.1152/ajpgi.00328.2012

    View details for Web of Science ID 000313040500008

    View details for PubMedID 23104557

  • Age-related modulation of the effects of obesity on gene expression profiles of mouse bone marrow and epididymal adipocytes. PloS one Liu, L., Shen, W., Ueno, M., Patel, S., Azhar, S., Kraemer, F. B. 2013; 8 (8)


    This study aimed to characterize and compare the effects of obesity on gene expression profiles in two distinct adipose depots, epididymal and bone marrow, at two different ages in mice. Alterations in gene expression were analyzed in adipocytes isolated from diet-induced obese (DIO) C57BL/6J male mice at 6 and 14 months of age and from leptin deficient mice (ob/ob) at 6 months of age using microarrays. DIO affected gene expression in both depots at 6 and 14 months, but more genes were altered in epididymal than bone marrow adipocytes at each age and younger mice displayed more changes than older animals. In epididymal adipocytes a total of 2789 (9.6%) genes were differentially expressed at 6-months with DIO, whereas 952 (3.3%) were affected at 14-months. In bone marrow adipocytes, 347 (1.2%) genes were differentially expressed at 6-months with DIO, whereas only 189 (0.66%) were changed at 14-months. 133 genes were altered by DIO in both fat depots at 6-months, and 37 genes at 14-months. Only four genes were altered in both depots at both ages with DIO. Bone marrow adipocytes are less responsive to DIO than epididymal adipocytes and the response of both depots to DIO declines with age. This loss of responsiveness with age is likely due to age-associated changes in expression of genes related to adipogenesis, inflammation and mitochondrial function that are similar to and obscure the changes commonly associated with DIO. Patterns of gene expression were generally similar in epididymal adipocytes from ob/ob and DIO mice; however, several genes were differentially expressed in bone marrow adipocytes from ob/ob and DIO mice, perhaps reflecting the importance of leptin signaling for bone metabolism. In conclusion, obesity affects age-associated alterations in gene expression in both epididymal and bone marrow adipocytes regardless of diet or genetic background.

    View details for DOI 10.1371/journal.pone.0072367

    View details for PubMedID 23967297

    View details for PubMedCentralID PMC3743818

  • The mineralocorticoid receptor agonist, fludrocortisone, differentially inhibits pituitary-adrenal activity in humans with psychotic major depression PSYCHONEUROENDOCRINOLOGY Lembke, A., Gomez, R., Tenakoon, L., Keller, J., Cohen, G., Williams, G. H., Kraemer, F. B., Schatzberg, A. F. 2013; 38 (1): 115-121


    Hypothalamic-pituitary-adrenal (HPA) axis dysregulation has been linked with major depression, particularly psychotic major depression (PMD), with mineralocorticoid receptors (MRs) playing a role in HPA-axis regulation and the pathophysiology of depression. Herein we hypothesize that the MR agonist fludrocortisone differentially inhibits the HPA axis of psychotic major depression subjects (PMDs), non-psychotic major depression subjects (NPMDs), and healthy control subjects (HCs).Fourteen PMDs, 16 NPMDs, and 19 HCs were admitted to the Stanford University Hospital General Clinical Research Center. Serum cortisol levels were sampled at baseline and every hour from 18:00 to 23:00h, when greatest MR activity is expected, on two consecutive nights. On the second afternoon at 16:00h all subjects were given 0.5mg fludrocortisone. Mean cortisol levels pre- and post-fludrocortisone and percent change in cortisol levels were computed.There were no significant group differences for cortisol at baseline: F(2,47)=.19, p=.83. There were significant group differences for post-fludrocortisone cortisol: F(2,47)=5.13, p=.01, which were significantly higher in PMDs compared to HCs (p=.007), but not compared to NPMDs (p=.18). There were no differences between NPMD's and HC's (p=.61). Also, PMDs had a lower percent change from baseline in cortisol levels at 2200h than NPMDs (p=.01) or HCs (p=.009).Individuals with psychotic major depression compared to healthy control subjects have diminished feedback inhibition of the hypothalamic-pituitary-adrenal (HPA) axis in response to the mineralocorticoid receptor agonist fludrocortisone. To our knowledge, this is the first study to examine HPA axis response to MR stimulation in major depression (with and without psychosis), and only the third study to demonstrate that exogenously administered fludrocortisone can down-regulate the HPA axis in humans.

    View details for DOI 10.1016/j.psyneuen.2012.05.006

    View details for Web of Science ID 000313761000011

    View details for PubMedID 22727477

    View details for PubMedCentralID PMC3633490

  • MicroRNAs 125a and 455 Repress Lipoprotein-Supported Steroidogenesis by Targeting Scavenger Receptor Class B Type I in Steroidogenic Cells MOLECULAR AND CELLULAR BIOLOGY Hu, Z., Shen, W., Kraemer, F. B., Azhar, S. 2012; 32 (24): 5035-5045


    We sought to identify and characterize microRNA (miRNAs) that posttranscriptionally regulate the expression of scavenger receptor class B type I (SR-BI) and SR-BI-linked selective high-density lipoprotein (HDL) cholesteryl ester (CE) transport and steroidogenesis. Four miRNAs (miRNA-125a, miRNA-125b, miRNA-145, and miRNA-455) with a potential to regulate SR-BI were identified in silico and validated by quantitative real-time PCR (qRT-PCR), Western blot analysis, and SR-BI 3' untranslated region (UTR) reporter assays. In vitro treatment of primary rat granulosa cells and MLTC-1 cells with cyclic AMP (cAMP) or in vivo treatment of rat adrenals with adrenocorticotropic hormone (ACTH) decreased the expression of miRNA-125a, miRNA-125b, and miRNA-455 and reciprocally increased SR-BI expression. Using luciferase constructs containing the 3' untranslated region of SR-BI combined with miRNA overexpression and mutagenesis, we have provided evidence that steroidogenic SR-BI is a direct target of miRNA-125a and miRNA-455. Moreover, the transfection of Leydig tumor cells with precursor miRNA 125a (pre-miRNA-125a) or pre-miRNA-455 resulted in the suppression of SR-BI at both the transcript and protein levels and reduced selective HDL CE uptake and HDL-stimulated progesterone production. Transfection of liver Hepa 1-6 cells with pre-miRNA-125a significantly reduced SR-BI expression and its selective transport function. In contrast, overexpression of miRNA-145 did not affect SR-BI expression or selective HDL CE uptake mediated by SR-BI in steroidogenic cell lines. These data suggest that a trophic hormone and cAMP inversely regulate the expression of SR-BI and miRNA-125a and miRNA-455 in steroidogenic tissues/cells and that both miRNA-125a and miRNA-455, by targeting steroidogenic SR-BI, negatively regulate selective HDL CE uptake and HDL CE-supported steroid hormone production.

    View details for DOI 10.1128/MCB.01002-12

    View details for Web of Science ID 000311492200013

    View details for PubMedID 23045399

  • HSL-knockout mouse testis exhibits class B scavenger receptor upregulation and disrupted lipid raft microdomains JOURNAL OF LIPID RESEARCH Emilia Casado, M., Huerta, L., Isabel Ortiz, A., Perez-Crespo, M., Gutierrez-Adan, A., Kraemer, F. B., Angel Lasuncion, M., Busto, R., Martin-Hidalgo, A. 2012; 53 (12): 2586-2597
  • HSL-knockout mouse testis exhibits class B scavenger receptor upregulation and disrupted lipid raft microdomains. Journal of lipid research Casado, M. E., Huerta, L., Ortiz, A. I., Pérez-Crespo, M., Gutiérrez-Adán, A., Kraemer, F. B., Lasunción, M. Á., Busto, R., Martín-Hidalgo, A. 2012; 53 (12): 2586-2597


    There is a tight relationship between fertility and changes in cholesterol metabolism during spermatogenesis. In the testis, class B scavenger receptors (SR-B) SR-BI, SR-BII, and LIMP II mediate the selective uptake of cholesterol esters from HDL, which are hydrolyzed to unesterified cholesterol by hormone-sensitive lipase (HSL). HSL is critical because HSL knockout (KO) male mice are sterile. The aim of the present work was to determine the effects of the lack of HSL in testis on the expression of SR-B, lipid raft composition, and related cell signaling pathways. HSL-KO mouse testis presented altered spermatogenesis associated with decreased sperm counts, sperm motility, and infertility. In wild-type (WT) testis, HSL is expressed in elongated spermatids; SR-BI, in Leydig cells and spermatids; SR-BII, in spermatocytes and spermatids but not in Leydig cells; and LIMP II, in Sertoli and Leydig cells. HSL knockout male mice have increased expression of class B scavenger receptors, disrupted caveolin-1 localization in lipid raft plasma membrane microdomains, and activated phospho-ERK, phospho-AKT, and phospho-SRC in the testis, suggesting that class B scavenger receptors are involved in cholesterol ester uptake for steroidogenesis and spermatogenesis in the testis.

    View details for DOI 10.1194/jlr.M028076

    View details for PubMedID 22988039

    View details for PubMedCentralID PMC3494238

  • Ablation of Vimentin Results in Defective Steroidogenesis ENDOCRINOLOGY Shen, W., Zaidi, S. K., Patel, S., Cortez, Y., Ueno, M., Azhar, R., Azhar, S., Kraemer, F. B. 2012; 153 (7): 3249-3257


    In steroidogenic tissues, cholesterol must be transported to the inner mitochondrial membrane to be converted to pregnenolone as the first step of steroidogenesis. Whereas steroidogenic acute regulatory protein has been shown to be responsible for the transport of cholesterol from the outer to the inner mitochondrial membrane, the process of how cholesterol moves to mitochondria from the cytoplasm is not clearly defined. The involvement of the cytoskeleton has been suggested; however, no specific mechanism has been confirmed. In this paper, using genetic ablation of an intermediate filament protein in mice, we present data demonstrating a marked defect in adrenal and ovarian steroidogenesis in the absence of vimentin. Cosyntropin-stimulated corticosterone production is decreased 35 and 50% in male and female Vimentin null (Vim(-/-)) mice, respectively, whereas progesterone production is decreased 70% in female Vim(-/-) mice after pregnant mare's serum gonadotropin and human chorionic gonadotropin stimulation, but no abnormalities in human chorionic gonadotropin-stimulated testosterone production is observed in male Vim(-/-) mice. These defects in steroid production are also seen in isolated adrenal and granulosa cells in vitro. Further studies show a defect in the movement of cholesterol from the cytosol to mitochondria in Vim(-/-) cells. Because the mobilization of cholesterol from lipid droplets and its transport to mitochondria is a preferred pathway for the initiation of steroid production in the adrenal and ovary but not the testis and vimentin is a droplet-associated protein, our results suggest that vimentin is involved in the movement of cholesterol from its storage in lipid droplets to mitochondria for steroidogenesis.

    View details for DOI 10.1210/en.2012-1048

    View details for Web of Science ID 000305781800031

    View details for PubMedID 22535769

    View details for PubMedCentralID PMC3380307

  • Evidence for protein-mediated fatty acid efflux by adipocytes ACTA PHYSIOLOGICA Henkin, A. H., Ortegon, A. M., Cho, S., Shen, W., Falcon, A., Kraemer, F. B., Lee, S., Stahl, A. 2012; 204 (4): 562-570


    The hormonally controlled mobilization and release of fatty acids from adipocytes into the circulation is an important physiological process required for energy homeostasis. While uptake of fatty acids by adipocytes has been suggested to be predominantly protein-mediated, it is unclear whether the efflux of fatty acids also requires membrane proteins.We used fluorescent fatty acid efflux assays and colorimetric assays for free fatty acids and glycerol to identify inhibitors with effects on fatty acid efflux, but not lipolysis, in 3T3-L1 adipocytes. We assessed the effect of these inhibitors on a fibroblast-based cell line expressing fatty acid transport protein 1, hormone-sensitive lipase and perilipin, which presumably lacks adipocyte-specific proteins for fatty acid efflux.We identified 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) as an inhibitor of fatty acid efflux that did not impair lipolysis or the cellular exit of glycerol but lead to an accumulation of intracellular fatty acids. In contrast, fatty acid efflux by the reconstituted cellular model for fatty acid efflux was responsive to lipolytic stimuli, but insensitive to DIDS inhibition.We propose that adipocytes specifically express an as yet unidentified DIDS-sensitive protein that enhances the efflux of fatty acids and therefore may lead to novel treatment approaches for obesity-related disorders characterized by abnormal lipid fluxes and ectopic triglyceride accumulation.

    View details for DOI 10.1111/j.1748-1716.2011.02367.x

    View details for Web of Science ID 000300716400014

    View details for PubMedID 21951599

    View details for PubMedCentralID PMC3271185

  • Lipid droplet meets a mitochondrial protein to regulate adipocyte lipolysis EMBO JOURNAL Greenberg, A. S., Kraemer, F. B., Soni, K. G., Jedrychowski, M. P., Yan, Q., Graham, C. E., Bowman, T. A., Mansur, A. 2011; 30 (21): 4337-4339

    View details for DOI 10.1038/emboj.2011.371

    View details for Web of Science ID 000296716600001

    View details for PubMedID 22048333

  • Hormone-sensitive lipase-knockout mice maintain high bone density during aging FASEB JOURNAL Shen, W., Liu, L., Patel, S., Kraemer, F. B. 2011; 25 (8): 2722-2730


    We tested the hypothesis that the actions of hormone-sensitive lipase (HSL) affect the microenvironment of the bone marrow and that removal of HSL function by gene deletion maintains high bone mass in aging mice. We compared littermate control wild-type (WT) and HSL(-/-) mice during aging for changes in serum biochemical values, trabecular bone density using micro-computed tomography, bone histomorphometry, and characteristics of primary bone marrow cells and preosteoblasts. There is a regulated expression of HSL and genes involved in lipid metabolism in the bone marrow during aging. HSL(-/-) mice have increased serum levels of insulin and osteocalcin with decreased leptin levels. Compared with the marked adipocyte infiltration in WT bone marrow (65% by area) at 14 mo, HSL(-/-) mice have fewer (16%, P<0.05) and smaller adipocytes in bone marrow. While peak bone density is similar, HSL(-/-) mice maintain a higher bone density (bone volume/total volume 6.1%) with age than WT mice (2.6%, P<0.05). Primary osteoblasts from HSL(-/-) mice show increased growth rates and higher osteogenic potential, manifested by increased expression of Runx2 (3.5-fold, P<0.05) and osteocalcin (4-fold, P<0.05). The absence of HSL directs cells within the bone marrow toward osteoblast differentiation and favors the maintenance of bone density with aging.

    View details for DOI 10.1096/fj.11-181016

    View details for Web of Science ID 000293337800021

    View details for PubMedID 21566206

    View details for PubMedCentralID PMC3136346

  • The role of lipid droplets in metabolic disease in rodents and humans JOURNAL OF CLINICAL INVESTIGATION Greenberg, A. S., Coleman, R. A., Kraemer, F. B., McManaman, J. L., Obin, M. S., Puri, V., Yan, Q., Miyoshi, H., Mashek, D. G. 2011; 121 (6): 2102-2110


    Lipid droplets (LDs) are intracellular organelles that store neutral lipids within cells. Over the last two decades there has been a dramatic growth in our understanding of LD biology and, in parallel, our understanding of the role of LDs in health and disease. In its simplest form, the LD regulates the storage and hydrolysis of neutral lipids, including triacylglycerol and/or cholesterol esters. It is becoming increasingly evident that alterations in the regulation of LD physiology and metabolism influence the risk of developing metabolic diseases such as diabetes. In this review we provide an update on the role of LD-associated proteins and LDs in metabolic disease.

    View details for DOI 10.1172/JCI46069

    View details for Web of Science ID 000291234300007

    View details for PubMedID 21633178

  • Characterization of age-related gene expression profiling in bone marrow and epididymal adipocytes BMC GENOMICS Liu, L., Shen, W., Ueno, M., Patel, S., Kraemer, F. B. 2011; 12


    While an increase in bone marrow adiposity is associated with age-related bone disease, the function of bone marrow adipocytes has not been studied. The aim of this study was to characterize and compare the age-related gene expression profiles in bone marrow adipocytes and epididymal adipocytes.A total of 3918 (13.7%) genes were differentially expressed in bone marrow adipocytes compared to epididymal adipocytes. Bone marrow adipocytes revealed a distinct gene profile with low expression of adipocyte-specific genes peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid binding protein 4 (FABP4), perilipin (Plin1), adipsin (CFD) and high expression of genes associated with early adipocyte differentiation (CCAAT/enhancer binding protein beta (C/EBPβ), regulator of G-protein signaling 2 (RGS2). In addition, a number of genes including secreted frizzled related protein 4 (SFRP4), tumor necrosis factor α (TNFα), transforming growth factor beta 1(TGFβ1), G-protein coupled receptor 109A (GPR109A) and interleukin 6 (IL-6), that could affect adipose-derived signaling to bone are markedly increased in bone marrow adipocytes. Age had a substantial effect on genes associated with mitochondria function and inflammation in bone marrow adipocytes. Twenty seven genes were significantly changed with age in both adipocyte depots. Among these genes, IL6 and GPR109A were significantly reduced with age in both adipocyte depots.Overall, gene profiling reveals a unique phenotype for primary bone marrow adipocytes characterized by low adipose-specific gene expression and high expression of inflammatory response genes. Bone marrow and epididymal adipocytes share a common pathway in response to aging in mice, but age has a greater impact on global gene expression in epididymal than in bone marrow adipocytes. Genes that are differentially expressed at greater levels in the bone marrow are highly regulated with age.

    View details for DOI 10.1186/1471-2164-12-212

    View details for Web of Science ID 000291688000001

    View details for PubMedID 21545734

    View details for PubMedCentralID PMC3113784

  • Hormone-sensitive lipase modulates adipose metabolism through PPAR gamma BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS Shen, W., Yu, Z., Patel, S., Jue, D., Liu, L., Kraemer, F. B. 2011; 1811 (1): 9-16


    Hormone-sensitive lipase (HSL) is rate limiting for diacylglycerol and cholesteryl ester hydrolysis in adipose tissue and essential for complete hormone-stimulated lipolysis. Gene expression profiling in HSL-/- mice suggests that HSL is important for modulating adipogenesis and adipose metabolism. To test whether HSL is required for the supply of intrinsic ligands for PPARγ for normal adipose differentiation, HSL-/- and wild-type (WT) littermates were fed normal chow (NC) and high-fat (HF) diets supplemented with or without rosiglitazone (200 mg/kg) for 16 weeks. Results show that supplementing rosiglitazone to an NC diet completely normalized the decreased body weight and adipose depots in HSL-/- mice. Additionally, rosiglitazone resulted in similar serum glucose, total cholesterol, FFA, and adiponectin values in WT and HSL-/- mice. Furthermore, rosiglitazone normalized the expression of genes involved in adipocyte differentiation, markers of adipocyte differentiation, and enzymes involved in triacylglycerol synthesis and metabolism, and cholesteryl ester homeostasis, in HSL-/- mice. Supplementing rosiglitazone to an HF diet resulted in improved glucose tolerance in both WT and HSL-/- animals and also partial normalization in HSL-/- mice of abnormal WAT gene expression, serum chemistries, organ and body weight changes. In vitro studies showed that adipocytes from WT animals can provide ligands for activation of PPARγ and that activation is further boosted following lipolytic stimulation, whereas adipocytes from HSL-/- mice displayed attenuated activation of PPARγ, with no change following lipolytic stimulation. These results suggest that one of the mechanisms by which HSL modulates adipose metabolism is by providing intrinsic ligands or pro-ligands for PPARγ.

    View details for DOI 10.1016/j.bbalip.2010.10.001

    View details for Web of Science ID 000286027400002

    View details for PubMedID 20950707

    View details for PubMedCentralID PMC2998198

  • IL-17 Regulates Adipogenesis, Glucose Homeostasis, and Obesity JOURNAL OF IMMUNOLOGY Zuniga, L. A., Shen, W., Joyce-Shaikh, B., Pyatnova, E. A., Richards, A. G., Thom, C., Andrade, S. M., Cua, D. J., Kraemer, F. B., Butcher, E. C. 2010; 185 (11): 6947-6959


    Inflammatory mediators have the potential to impact a surprising range of diseases, including obesity and its associated metabolic syndrome. In this paper, we show that the proinflammatory cytokine IL-17 inhibits adipogenesis, moderates adipose tissue (AT) accumulation, and regulates glucose metabolism in mice. IL-17 deficiency enhances diet-induced obesity in mice and accelerates AT accumulation even in mice fed a low-fat diet. In addition to potential systemic effects, IL-17 is expressed locally in AT by leukocytes, predominantly by γδ T cells. IL-17 suppresses adipocyte differentiation from mouse-derived 3T3-L1 preadipocytes in vitro, and inhibits expression of genes encoding proadipogenic transcription factors, adipokines, and molecules involved in lipid and glucose metabolism. IL-17 also acts on differentiated adipocytes, impairing glucose uptake, and young IL-17-deficient mice show enhanced glucose tolerance and insulin sensitivity. Our findings implicate IL-17 as a negative regulator of adipogenesis and glucose metabolism in mice, and show that it delays the development of obesity.

    View details for DOI 10.4049/jimmunol.1001269

    View details for Web of Science ID 000284311500062

    View details for PubMedID 21037091

    View details for PubMedCentralID PMC3001125

  • Adipocytes Decrease Runx2 Expression in Osteoblastic Cells: Roles of PPAR gamma and Adiponectin JOURNAL OF CELLULAR PHYSIOLOGY Liu, L., Shen, W., Zhang, Z. H., Wang, L. J., Kraemer, F. B. 2010; 225 (3): 837-845


    The mechanisms through which bone marrow adipocytes might influence differentiation and function of osteoblasts are not completely understood. To investigate the direct effects of bone marrow fat cells on osteoblast function, an ex vivo co-culture system was utilized comprising either primary fat cells or differentiated 3T3-L1 adipocytes and osteoblastic cells on transwells. In co-culture, both adipocytes and osteoblastic cells were differentiated into adipocytes or osteoblasts, respectively, before culturing on transwells. Co-culture with either primary fat cells or fully differentiated 3T3-L1 adipocytes significantly decreased mRNA and protein expression of runt-related transcription factor 2 (Runx2) in osteoblastic cells. An increase in mRNA and protein expression of peroxisome proliferator-activated receptor γ (PPARγ) occurred concomitantly with the reduction of Runx2 expression. Adiponectin concentration was increased in the media by co-culture. In addition, co-culture with conditioned media from fat cells increased PPARγ promoter activity and decreased Runx2 promoter activity. Knockdown of PPARγ or adiponectin receptor 1 in osteoblastic cells by siRNA prevented the down-regulation of mRNA expression of Runx2 in osteoblastic cells cultured with fully differentiated 3T3-L1 cells. Furthermore, co-transfection with PPARγ decreased Runx2 promoter activity. A marker of osteogenesis, alkaline phosphatase activity in osteoblastic cells was significantly decreased by co-culture. Annexin V/propidium iodide staining showed that co-culture did not induce apoptosis in osteoblastic cells. Thus, we conclude that adipocytes modulate key metabolic functions of osteoblasts through the release of secretory products. PPARγ plays a key role in mediating the effects of adipocytes on osteoblasts.

    View details for DOI 10.1002/jcp.22291

    View details for Web of Science ID 000284191100024

    View details for PubMedID 20589837

  • Strong induction of PCSK9 gene expression through HNF1 alpha and SREBP2: mechanism for the resistance to LDL-cholesterol lowering effect of statins in dyslipidemic hamsters JOURNAL OF LIPID RESEARCH Dong, B., Wu, M., Li, H., Kraemer, F. B., Adeli, K., Seidah, N. G., Park, S. W., Liu, J. 2010; 51 (6): 1486-1495


    We investigated the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the resistance of dyslipidemic hamsters to statin-induced LDL-cholesterol (LDL-C) reduction and the molecular mechanism by which statins modulated PCSK9 gene expression in vivo. We utilized the fructose diet-induced dyslipidemic hamsters as an in vivo model and rosuvastatin to examine its effects on liver PCSK9 and LDL receptor (LDLR) expression and serum lipid levels. We showed that rosuvastatin induced PCSK9 mRNA to a greater extent than LDLR mRNA in the hamster liver. The net result was that hepatic LDLR protein level was reduced. This correlated closely with an increase in serum LDL-C with statin treatment. More importantly, we demonstrated that in addition to an increase in sterol response element binding protein 2 (SREBP2) expression, rosuvastatin treatment increased the liver expression of hepatocyte nuclear factor 1 alpha (HNF1alpha), the newly identified key transactivator for PCSK9 gene expression. Our study suggests that the inducing effect of rosuvastatin on HNF1alpha is likely a underlying mechanism accounting for the higher induction of PCSK9 than LDLR because of the utilization of two transactivators (HNF1alpha and SREBP2) in PCSK9 transcription versus one (SREBP2) in LDLR transcription. Thus, the net balance is in favor of PCSK9-induced degradation of LDLR in the hamster liver, abrogating the effect of rosuvastatin on LDL-C lowering.

    View details for DOI 10.1194/jlr.M003566

    View details for Web of Science ID 000277564700024

    View details for PubMedID 20048381

  • Vimentin Is a Functional Partner of Hormone Sensitive Lipase And Facilitates Lipolysis JOURNAL OF PROTEOME RESEARCH Shen, W., Patel, S., Eriksson, J. E., Kraemer, F. B. 2010; 9 (4): 1786-1794


    Lipolysis involves a number of components including signaling pathways, droplet-associated proteins, and lipases such as hormone-sensitive lipase (HSL). We used surface enhanced laser desorption/ionization time-of-flight mass spectroscopy to identify cellular proteins that might interact with HSL and potentially influence lipolysis. Using recombinant HSL as bait on protein chips, clusters of proteins of 14.7-18.9, 25.8-26.8, 36.1, 44.3-49.1, and 53.7 kDa were identified that interact with HSL, particularly when lysates were examined from beta-agonist treated mouse adipocytes. The ability to detect these interacting proteins was markedly diminished when the adipocytes were treated with insulin. A very similar pattern of proteins was identified when anti-HSL IgG was used as the bait. Following immunocapture, the identification of the prominent 53.7 kDa protein was carried out by tryptic digestion and MS analysis and determined to be vimentin. The interaction of HSL with vimentin, and its hormonal dependence, was confirmed by coimmunoprecipitation. beta-Agonist stimulated lipolysis and the rate of HSL translocation were impaired in vimentin null adipocytes, even though normal amounts of lipases and droplet-associated proteins are expressed. The current studies provide evidence that vimentin participates in lipolysis through direct, hormonally regulated interactions with HSL.

    View details for DOI 10.1021/pr900909t

    View details for Web of Science ID 000276215700015

    View details for PubMedID 20143880

    View details for PubMedCentralID PMC2849902

  • Functional interaction of hormone-sensitive lipase and perilipin in lipolysis JOURNAL OF LIPID RESEARCH Shen, W., Patel, S., Miyoshi, H., Greenberg, A. S., Kraemer, F. B. 2009; 50 (11): 2306-2313


    Adipocyte lipolysis is controlled by complex interactions of lipases, cofactors, and structural proteins associated with lipid droplets. Perilipin (Plin) A is a major droplet-associated protein that functions as a scaffold, both suppressing basal and facilitating cAMP-dependent protein kinase (PKA)-stimulated lipolysis. Plin is required for the translocation of hormone-sensitive lipase (HSL) from the cytosol to lipid droplets upon stimulation. In these studies, we provide direct evidence for a physical interaction of HSL with Plin. By coexpressing HSL with truncation mutations of Plin, we demonstrate using coimmunoprecipitation that HSL can interact with an N-terminal region located between amino acids 141 and 200 of Plin A as well as with a C-terminal region located between amino acids 406 and 480. The N-terminal construct, Plin 1-200, which does not associate with lipid droplets but interacts with HSL, can function as a dominant negative for PKA-stimulated lipolysis. Using confocal microscopy of Plin truncations, we demonstrate that sequences between amino acids 463 and 517 may be important for or participate in lipid targeting. The results suggest the translocation of HSL to the lipid droplet occurs by virtue of Plin localization to the surface of lipid droplets and a physical interaction of HSL occurring with sequences within the N-terminal region of Plin.

    View details for DOI 10.1194/jlr.M900176-JLR200

    View details for Web of Science ID 000270860900020

    View details for PubMedID 19515989

    View details for PubMedCentralID PMC2759837

  • Effects of hormone-sensitive lipase disruption on cardiac energy metabolism in response to fasting and refeeding AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Suzuki, J., Ueno, M., Uno, M., Hirose, Y., Zenimaru, Y., Takahashi, S., Osuga, J., Ishibashi, S., Takahashi, M., Hirose, M., Yamada, M., Kraemer, F. B., Miyamori, I. 2009; 297 (5): E1115-E1124


    Increased fatty acid (FA) flux and intracellular lipid accumulation (steatosis) give rise to cardiac lipotoxicity in both pathological and physiological conditions. Since hormone-sensitive lipase (HSL) contributes to intracellular lipolysis in adipose tissue and heart, we investigated the impact of HSL disruption on cardiac energy metabolism in response to fasting and refeeding. HSL-knockout (KO) mice and wild-type (WT) littermates were fasted for 24 h, followed by ∼6 h of refeeding. Plasma FA concentration in WT mice was elevated twofold with fasting, whereas KO mice lacked this elevation, resulting in twofold lower cardiac FA uptake compared with WT mice. Echocardiography showed that fractional shortening was 15% decreased during fasting in WT mice and was associated with steatosis, whereas both of these changes were absent in KO mice. Compared with Langendorff-perfused hearts isolated from fasted WT mice, the isolated KO hearts also displayed higher contractile function and a blunted response to FA. Although cardiac glucose uptake in KO mice was comparable with WT mice under all conditions tested, cardiac VLDL uptake and lipoprotein lipase (LPL) activity were twofold higher in KO mice during fasting. The KO hearts showed undetectable activity of neutral cholesteryl esterase and 40% lower non-LPL triglyceride lipase activity compared with WT hearts in refed conditions accompanied by overt steatosis, normal cardiac function, and increased mRNA expression of adipose differentiation-related protein. Thus, the dissociation between cardiac steatosis and functional sequelae observed in HSL-KO mice suggests that excess FA influx, rather than steatosis per se, appears to play an important role in the pathogenesis of cardiac lipotoxicity.

    View details for DOI 10.1152/ajpendo.91031.2008

    View details for Web of Science ID 000270990500017

    View details for PubMedID 19706782

  • Identification of mRNA binding proteins that regulate the stability of LDL receptor mRNA through AU-rich elements JOURNAL OF LIPID RESEARCH Li, H., Chen, W., Zhou, Y., Abidi, P., Sharpe, O., Robinson, W. H., Kraemer, F. B., Liu, J. 2009; 50 (5): 820-831


    The 3'untranslated region (UTR) of human LDL receptor (LDLR) mRNA contains three AU-rich elements (AREs) responsible for rapid mRNA turnover and mediates the stabilization induced by berberine (BBR). However, the identities of the specific RNA binding proteins involved in the regulation of LDLR mRNA stability at the steady state level or upon BBR treatment are unknown. By conducting small interfering RNA library screenings, biotinylated RNA pull-down, mass spectrometry analysis, and functional assays, we now identify heterogeneous nuclear ribonucleoprotein D (hnRNP D), hnRNP I, and KH-type splicing regulatory protein (KSRP) as key modulators of LDLR mRNA stability in liver cells. We show that hnRNP D, I, and KSRP interact with AREs of the LDLR 3'UTR with sequence specificity. Silencing the expression of these proteins increased LDLR mRNA and protein levels. We further demonstrate that BBR-induced mRNA stabilization involves hnRNP I and KSRP, as their cellular depletions abolished the BBR effect and BBR treatment reduced the binding of hnRNP I and KSRP to the LDLR mRNA 3'UTR. These new findings demonstrate that LDLR mRNA stability is controlled by a group of ARE binding proteins, including hnRNP D, hnRNP I, and KSRP. Our results suggest that interference with the ability of destabilizing ARE binding proteins to interact with LDLR-ARE motifs is likely a mechanism for regulating LDLR expression by compounds such as BBR and perhaps others.

    View details for DOI 10.1194/jlr.M800375-JLR200

    View details for Web of Science ID 000264969300006

    View details for PubMedID 19141871

    View details for PubMedCentralID PMC2666168

  • Cardiac overexpression of hormone-sensitive lipase inhibits myocardial steatosis and fibrosis in streptozotocin diabetic mice AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Ueno, M., Suzuki, J., Zenimaru, Y., Takahashi, S., Koizumi, T., Noriki, S., Yamaguchi, O., Otsu, K., Shen, W., Kraemer, F. B., Miyamori, I. 2008; 294 (6): E1109-E1118


    Intracellular lipid accumulation (steatosis) and resultant lipotoxicity are key features of diabetic cardiomyopathy. Since cardiac hormone-sensitive lipase (HSL) is activated in diabetic mice, we sought to explore a pathophysiological function of cardiac HSL in the development of diabetic cardiomyopathy. Transgenic (Tg) mice with heart-specific HSL overexpression were generated, and cardiac histology, function, lipid profile, and gene expressions were analyzed after induction of diabetes by streptozotocin. Electron microscopy showed numerous lipid droplets in wild-type (Wt) hearts after 3 wk of diabetes, whereas Tg mice showed no lipid droplet accumulation. Cardiac content of acylglycerides was increased approximately 50% with diabetes in Wt mice, whereas this was blunted in Tg hearts. Cardiac lipid peroxide content was twofold lower in Tg hearts than in Wt hearts. The mRNA expressions for peroxisome proliferator-activated receptor-alpha, genes for triacylglycerol synthesis, and lipoprotein lipase were increased with diabetes in Wt hearts, whereas this induction was absent in Tg hearts. Expression of genes associated with lipoapoptosis was decreased, whereas antioxidant protein metallothioneins were increased in diabetic Tg hearts. Diabetic Wt hearts showed interstitial fibrosis and increased collagen content. However, Tg hearts displayed no overt fibrosis, concomitant with decreased expression of collagens, transforming growth factor-beta, and matrix metalloproteinase 2. Notably, mortality during the experimental period was approximately twofold lower in diabetic Tg mice compared with Wt mice. In conclusion, since HSL overexpression inhibits cardiac steatosis and fibrosis by apparently hydrolyzing toxic lipid metabolites, cardiac HSL could be a therapeutic target for regulating diabetic cardiomyopathy.

    View details for DOI 10.1152/ajpendo.00016.2008

    View details for Web of Science ID 000256469200012

    View details for PubMedID 18413675

  • Analysis of polymorphisms in the 3 ' untranslated region of the LDL receptor gene and their effect on plasma cholesterol levels and drug response INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE Chen, W., Wang, S., Ma, Y., Zhou, Y., Liu, H., Strnad, P., Kraemer, F. B., Krauss, R. M., Liu, J. 2008; 21 (3): 345-353


    The proximal section of the 3' untranslated region (3'UTR) of LDL receptor (LDLR) mRNA contains important regulatory sequences that control the messenger stability and mediate the cholesterol-lowering drug berberine (BBR)-induced increase in LDLR mRNA half-life. In the present study, we examined whether single nucleotide polymorphisms (SNPs) within this region cause a predisposition to the development of coronary heart disease (CHD) and whether they affect the response to BBR treatment. Genomic DNAs were isolated from peripheral blood of a Chinese cohort of 103 normolipidemic subjects and 94 hyperlipidemic CHD patients. The 1.1-kb proximal fragment of LDLR mRNA 3'UTR was PCR-amplified and sequenced. Six SNPs were detected within this region. Among them, the presence of SNP1 and SNP6 in both study groups showed complete association (r2=1). The frequency of individual SNPs and genotypes did not differ between CHD patients and normolipidemic individuals. Allelic variations did not correlate with total and LDL-cholesterol levels. To examine the effects of genetic variations in 3'UTR on BBR treatment, entire 2.5-kb regions of 3'UTR from three common SNP haplotypes were cloned into a luciferase reporter and the reporter constructs were transfected into HepG2 cells. The expression of reporter genes carrying different haplotypes of LDLR 3'UTR was increased to a similar extent upon BBR treatment. Taken together, these findings suggest that the 3'UTR LDLR polymorphisms commonly found in the Chinese population do not cause a predisposition to the development of CHD, nor do they affect the plasma lipid levels or the cholesterol-lowering effect of BBR.

    View details for Web of Science ID 000253507700010

    View details for PubMedID 18288382

  • Transcriptional activation of hepatic ACSL3 and ACSL5 by oncostatin M reduces hypertriglyceridemia through enhanced beta-oxidation ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Zhou, Y., Abidi, P., Kim, A., Chen, W., Huang, T., Kraemer, F. B., Liu, J. 2007; 27 (10): 2198-2205


    In our previous studies that examined in vivo activities of oncostatin M (OM) in upregulation of hepatic LDL receptor (LDLR) expression, we observed reductions of LDL-cholesterol and triglyceride (TG) levels in OM-treated hyperlipidemic hamsters. Interestingly, the OM effect of lowering plasma TG was more pronounced than LDL-cholesterol reduction, suggesting additional LDLR-independent actions. Here, we investigated mechanisms underlying the direct TG-lowering effect of OM.We demonstrate that OM activates transcription of long-chain acyl-coenzymeA (CoA) synthetase isoforms 3 and 5 (ACSL3, ACSL5) in HepG2 cells through the extracellular signal-regulated kinase (ERK) signaling pathway. Increased acyl-CoA synthetase activities in OM-stimulated HepG2 cells and in livers of OM-treated hamsters are associated with decreased TG accumulation and increased fatty acid beta-oxidation. We further show that overexpression of ACSL3 or ACSL5 alone in the absence of OM led to fatty acid partitioning into beta-oxidation. Importantly, we demonstrate that transfection of siRNAs targeted to ACSL3 and ACSL5 abrogated the enhancing effect of OM on fatty acid oxidation in HepG2 cells.These new findings identify ACSL3 and ACSL5 as OM-regulated genes that function in fatty acid metabolism and suggest a novel cellular mechanism by which OM directly lowers the plasma TG in hyperlipidemic animals through stimulating the transcription of ACSL specific isoforms in the liver.

    View details for DOI 10.1161/ATVBAHA.107.148429

    View details for Web of Science ID 000249587000019

    View details for PubMedID 17761945

  • Generation of novel adipocyte monolayer cultures from embryonic stem cells 44th Annual Meeting of the American-Society-for-Cell-Biology Chen, T. L., Shen, W., Qiu, X., Li, T., Hoffman, A. R., Kraemer, F. B. MARY ANN LIEBERT INC. 2007: 371–80


    Here we show a simplified and improved method to produce large quantities of evenly distributed monolayer cultures that display major characteristics of adipocytes. These cultures are applicable for quantitative analysis for biochemical and molecular events in adipogenesis during development and may provide a useful system for high-throughput drug screening assays of antiobesity drugs. In our method, we treated embryoid bodies (EBs) with all-trans retinoic acid (ATRA) for 3 days, 1 day after they attached to the gelatin-coated culture plates without further transfer. The cells were maintained in insulin and trioiodothyronine (T(3))-containing medium until day 12, when they were dispersed by enzymatic digestion and replated onto multiple culture plates. Two days later, adipocyte induction factors were added for 6 days and examined 6 days later. The amount of lipid droplet-laden adipocytes in the culture reached approximately 80%, with a nearly five-fold increase in GPDH activity. The cells expressed high levels of adipose-specific proteins (adipocyte markers), including PPARgamma2, ALBP, LPL, HSL, perilipin, and DGAT1. The adipocytes are functionally active, as evidenced by their response to lipolytic agents, such as forskolin, Bt2-cAMP, and isoproterenol, with more than 20-fold increases in glycerol release.

    View details for DOI 10.1089/scd.2006.0037

    View details for Web of Science ID 000247721800005

    View details for PubMedID 17610367

  • Adrenal cholesterol utilization Joint Meeting of the 6th Conference on the Adrenal Cortex/Molecular Steroidogenesis Workshop Kraemer, F. B. ELSEVIER IRELAND LTD. 2007: 42–45


    There are multiple systems for cellular cholesterol delivery for steroidogenesis, including uptake of lipoprotein-derived cholesterol via LDL receptor mediated endocytic pathways and SR-BI mediated "selective" pathways, as well as from endogenous cholesterol synthesis and the mobilization of stored cholesteryl esters. The vast majority of lipoprotein-derived cholesterol utilized for murine adrenal steroidogenesis is obtained via SR-BI mediated "selective" uptake of cholesteryl esters. Hormone-sensitive lipase (HSL) is responsible for neutral cholesteryl ester hydrolase activity in the adrenal and is critical for hydrolyzing stored cholesteryl esters, as well as cholesteryl esters that are selectively delivered from lipoproteins via SR-BI. Marked defects in steroid production are observed in adrenal cells from HSL knockout mice, due to an inability to process and utilize cholesteryl esters selectively derived from lipoproteins. Although the LDL receptor is responsible for receptor-mediated endocytic delivery of cholesteryl esters, adrenal steroid hormone production is normal in mice lacking LDL receptors.

    View details for DOI 10.1016/j.mce.2006.12.001

    View details for Web of Science ID 000245046400008

  • Effects of rosightazone and high fat diet on lipase/esterase expression in adipose tissue BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS Shen, W., Patel, S., Yu, Z., Jue, D., Kraemer, F. B. 2007; 1771 (2): 177-184


    A number of intracellular lipase/esterase have been reported in adipose tissue either by functional assays of activity or through proteomic analysis. In the current work, we have studied the relative expression level of 12 members of the lipase/esterase family that are found in white adipose tissue. We found that the relative mRNA levels of ATGL and HSL are the most abundant, being 2-3 fold greater than TGH or ADPN; whereas other intracellular neutral lipase/esterases were expressed at substantially lower levels. High fat feeding did not alter the mRNA expression levels of most lipase/esterases, but did reduce CGI-58 and WBSCR21. Likewise, rosiglitazone treatment did not alter the mRNA expression levels of most lipase/esterases, but did increase ATGL, TGH, CGI-58 and WBSCR21, while reducing ADPN. WAT from HSL-/- mice showed no compensatory increase in any lipase/esterases, rather mRNA levels of most lipase/esterases were reduced. In contrast, BAT from HSL-/- mice showed an increase in ATGL expression, as well as a decrease in ES-1, APEH and WBSCR21. Analysis of the immunoreactive protein levels of some of the lipases confirmed the results seen with mRNA. In conclusion, these data highlight the complexity of the regulation of the expression of intracellular neutral lipase/esterases involved in lipolysis.

    View details for DOI 10.1016/j.bbalip.2006.11.009

    View details for Web of Science ID 000244639100006

    View details for PubMedID 17215164

    View details for PubMedCentralID PMC1933526

  • The LDL receptor is not necessary for acute adrenal steroidogenesis in mouse adrenocortical cells AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Kraemer, F. B., Shen, W., Patel, S., Osuga, J., Ishibashi, S., Azhar, S. 2007; 292 (2): E408-E412


    Steroid hormones are synthesized using cholesterol as precursor. To determine the functional importance of the low density lipoprotein (LDL) receptor and hormone-sensitive lipase (HSL) in adrenal steroidogenesis, adrenal cells were isolated from control, HSL(-/-), LDLR(-/-), and double LDLR/HSL(-/-) mice. The endocytic and selective uptake of apolipoprotein E-free human high density lipoprotein (HDL)-derived cholesteryl esters did not differ among the mice, with selective uptake accounting for >97% of uptake. In contrast, endocytic uptake of either human LDL- or rat HDL-derived cholesteryl esters was reduced 80-85% in LDLR(-/-) and double-LDLR/HSL(-/-) mice. There were no differences in the selective uptake of either human LDL- or rat HDL-derived cholesteryl esters among the mice. Maximum corticosterone production induced by ACTH or dibutyryl cyclic AMP and lipoproteins was not altered in LDLR(-/-) mice but was reduced 80-90% in HSL(-/-) mice. Maximum corticosterone production was identical in HSL(-/-) and double-LDLR/HSL(-/-) mice. These findings suggest that, although the LDL receptor is responsible for endocytic delivery of cholesteryl esters from LDL and rat HDL to mouse adrenal cells, it appears to play a negligible role in the delivery of cholesterol for acute adrenal steroidogenesis in the mouse. In contrast, HSL occupies a vital role in adrenal steroidogenesis because of its link to utilization of selectively delivered cholesteryl esters from lipoproteins.

    View details for DOI 10.1152/ajpendo.00428.2006

    View details for Web of Science ID 000243997100005

    View details for PubMedID 16985254

  • Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes JOURNAL OF BIOLOGICAL CHEMISTRY Miyoshi, H., Perfield, J. W., Souza, S. C., Shen, W., Zhang, H., Stancheva, Z. S., Kraemer, F. B., Obin, M. S., Greenberg, A. S. 2007; 282 (2): 996-1002


    Phosphorylation of the lipid droplet-associated protein perilipin A (Peri A) mediates the actions of cyclic AMP-dependent protein kinase A (PKA) to stimulate triglyceride hydrolysis (lipolysis) in adipocytes. Studies addressing how Peri A PKA sites regulate adipocyte lipolysis have relied on non-adipocyte cell models, which express neither adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triglyceride catabolism in mice, nor the "downstream" lipase, hormone-sensitive lipase (HSL). ATGL and HSL are robustly expressed by adipocytes that we generated from murine embryonic fibroblasts of perilipin knock-out mice. Adenoviral expression of Peri A PKA site mutants in these cells reveals that mutation of serine 517 alone is sufficient to abrogate 95% of PKA (forskolin)-stimulated fatty acid (FA) and glycerol release. Moreover, a "phosphomimetic" (aspartic acid) substitution at serine 517 enhances PKA-stimulated FA release over levels obtained with wild type Peri A. Studies with ATGL-and HSL-directed small hairpin RNAs demonstrate that 1) ATGL activity is required for all PKA-stimulated FA and glycerol release in murine embryonic fibroblast adipocytes and 2) all PKA-stimulated FA release in the absence of HSL activity requires serine 517 phosphorylation. These results provide the first demonstration that Peri A regulates ATGL-dependent lipolysis and identify serine 517 as the Peri A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphorylated or phosphomimetic serine 517. Thus, serine 517 is a novel "master regulator" of PKA-stimulated adipocyte lipolysis.

    View details for DOI 10.1074/jbc.M605770200

    View details for Web of Science ID 000243295200022

    View details for PubMedID 17114792

  • Regulation of hormone-sensitive lipase in islets DIABETES RESEARCH AND CLINICAL PRACTICE Shen, W., Liang, Y., Wang, J., Harada, K., Patel, S., Michie, S. A., Osuga, J., Ishibashi, S., Kraemer, F. B. 2007; 75 (1): 14-26


    An unique isoform of hormone-sensitive lipase (HSL) is expressed in beta-cells. Recent findings suggest that HSL could be involved in the regulation of glucose stimulated insulin secretion (GSIS), however, these findings are controversial. To test the hypothesis that HSL is involved in control of normal GSIS via changes in its expression and/or activity in response to stimuli, we examined the effects of free fatty acid (FFA) loading and glucagon like peptide-1 (GLP-1) stimulation on the regulation of HSL expression and activity. With prolonged FFA loading, there was increased expression of beta-cell HSL and increased HSL hydrolytic activity in clonal beta-cells. Short-term treatment with GLP-1 increased HSL activity without changing the expression of the beta-cell isoform of HSL. Basal insulin secretion was increased, whereas GLP-1 potentiation of GSIS was decreased in islets isolated from HSL-/- mice, as compared to islets from wild type mice. Furthermore, using PancChip 2.2 cDNA microarrays (NIDDK consortium), the gene expression profile in the islets of HSL-/- mice was compared with wild type mice. Results showed changes in several metabolic pathways due to changes in lipid homeostasis caused by inactivation of HSL. Quantitative PCR for selected genes also revealed changes in genes that are related to insulin secretion, such as UCP-2. Therefore, these results suggest that the beta-cell isoform of HSL is involved in maintaining lipid homeostasis in islets and contributes to the proper control of GSIS.

    View details for DOI 10.1016/j.diabres.2006.05.001

    View details for Web of Science ID 000243470700003

    View details for PubMedID 16765472

  • The medicinal plant goldenseal is a natural LDL-lowering agent with multiple bioactive components and new action mechanisms JOURNAL OF LIPID RESEARCH Abidi, P., Chen, W., Kraemer, F. B., Li, H., Liu, J. 2006; 47 (10): 2134-2147


    Our previous studies have identified berberine (BBR), an alkaloid isolated from the Chinese herb huanglian, as a unique cholesterol-lowering drug that upregulates hepatic low density lipoprotein receptor (LDLR) expression through a mechanism of mRNA stabilization. Here, we demonstrate that the root extract of goldenseal, a BBR-containing medicinal plant, is highly effective in upregulation of liver LDLR expression in HepG2 cells and in reducing plasma cholesterol and low density lipoprotein cholesterol (LDL-c) in hyperlipidemic hamsters, with greater activities than the pure compound BBR. By conducting bioassay-driven semipurifications, we demonstrate that the higher potency of goldenseal is achieved through concerted actions of multiple bioactive compounds in addition to BBR. We identify canadine (CND) and two other constituents of goldenseal as new upregulators of LDLR expression. We further show that the activity of BBR on LDLR expression is attenuated by multiple drug resistance-1 (MDR1)-mediated efflux from liver cells, whereas CND is resistant to MDR1. This finding defines a molecular mechanism for the higher activity of CND than BBR. We also provide substantial evidence to show that goldenseal contains natural MDR1 antagonist(s) that accentuate the upregulatory effect of BBR on LDLR mRNA expression. These new findings identify goldenseal as a natural LDL-c-lowering agent, and our studies provide a molecular basis for the mechanisms of action.

    View details for DOI 10.1194/jlr.M600195-JLR200

    View details for Web of Science ID 000240847600004

    View details for PubMedID 16885565

  • Elucidation of an SRE-1/SREBP-independent cellular pathway for LDL-receptor regulation: from the cell surface to the nucleus. Future cardiology Liu, J., Briggs, M. R., Kraemer, F. B. 2006; 2 (5): 605-612


    Reduction in blood levels of low-density lipoprotein (LDL) cholesterol lowers the risk of coronary heart disease. The elucidation of cellular pathways that control LDL-receptor expression through a cholesterol-mediated negative feedback mechanism has provided a crucial molecular basis for the development and clinical applications of statins in the treatment of hypercholesterolemia. The characterization of signaling transduction pathways elicited by cytokine oncostatin M (OM) in liver cells has revealed a novel cellular pathway that activates LDL-receptor transcription independent of intracellular levels of cholesterol and sterol-regulatory element binding proteins. This transcriptional activation is achieved through interactions of the sterol-independent regulatory element of LDL-receptor promoter and transcription factors Egr1 and c/EBPbeta, and is dependent upon the activation of the extracellular signal-regulated kinase signaling cascade by OM. In vivo OM administration in hyperlipidemic animals reduces circulating cholesterol and prevents lipid accumulation in the liver. Exploring this sterol-independent cellular pathway may lead to new therapeutic advances.

    View details for DOI 10.2217/14796678.2.5.605

    View details for PubMedID 19804198

  • Perilipin promotes hormone-sensitive lipase-mediated adipocyte lipolysis via phosphorylation-dependent and -independent mechanisms JOURNAL OF BIOLOGICAL CHEMISTRY Miyoshi, H., Souza, S. C., Zhang, H., Strissel, K. J., Christoffolete, M. A., Kovsan, J., Rudich, A., Kraemer, F. B., Bianco, A. C., Obin, M. S., Greenberg, A. S. 2006; 281 (23): 15837-15844


    Hormone-sensitive lipase (HSL) is the predominant lipase effector of catecholamine-stimulated lipolysis in adipocytes. HSL-dependent lipolysis in response to catecholamines is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin A (Peri A), an essential lipid droplet (LD)-associated protein. It is believed that perilipin phosphorylation is essential for the translocation of HSL from the cytosol to the LD, a key event in stimulated lipolysis. Using adipocytes retrovirally engineered from murine embryonic fibroblasts of perilipin null mice (Peri-/- MEF), we demonstrate by cell fractionation and confocal microscopy that up to 50% of cellular HSL is LD-associated in the basal state and that PKA-stimulated HSL translocation is fully supported by adenoviral expression of a mutant perilipin lacking all six PKA sites (Peri Adelta1-6). PKA-stimulated HSL translocation was confirmed in differentiated brown adipocytes from perilipin null mice expressing an adipose-specific Peri Adelta1-6 transgene. Thus, PKA-induced HSL translocation was independent of perilipin phosphorylation. However, Peri Adelta1-6 failed to enhance PKA-stimulated lipolysis in either MEF adipocytes or differentiated brown adipocytes. Thus, the lipolytic action(s) of HSL at the LD surface requires PKA-dependent perilipin phosphorylation. In Peri-/- MEF adipocytes, PKA activation significantly enhanced the amount of HSL that could be cross-linked to and co-immunoprecipitated with ectopic Peri A. Notably, this enhanced cross-linking was blunted in Peri-/- MEF adipocytes expressing Peri Adelta1-6. This suggests that PKA-dependent perilipin phosphorylation facilitates (either direct or indirect) perilipin interaction with LD-associated HSL. These results redefine and expand our understanding of how perilipin regulates HSL-mediated lipolysis in adipocytes.

    View details for DOI 10.1074/jbc.M601097200

    View details for Web of Science ID 000237996000032

    View details for PubMedID 16595669

  • Hormone-sensitive lipase knockouts NUTRITION & METABOLISM Kraemer, F. B., Shen, W. 2006; 3


    All treatments for obesity, including dietary restriction of carbohydrates, have a goal of reducing the storage of fat in adipocytes. The chief enzyme responsible for the mobilization of FFA from adipose tissue, i.e., lipolysis, is thought to be hormone-sensitive lipase (HSL). Studies of HSL knockouts have provided important insights into the functional significance of HSL and into adipose metabolism in general. Studies have provided evidence that HSL, though possessing triacylglycerol lipase activity, appears to be the rate-limiting enzyme for cholesteryl ester and diacylglycerol hydrolysis in adipose tissue and is essential for complete hormone stimulated lipolysis, but other triacylglycerol lipases are important in mediating triacylglycerol hydrolysis in lipolysis. HSL knockouts are resistant to both high fat diet-induced and genetic obesity, displaying reduced quantities of white with increased amounts of brown adipose tissue, increased numbers of adipose macrophages, and have multiple alterations in the expression of genes involved in adipose differentiation, including transcription factors, markers of adipocyte differentiation, and enzymes of fatty acid and triglyceride synthesis. With disruption of lipolysis by removal of HSL, there is a drastic reduction in lipogenesis and alteration in adipose metabolism.

    View details for DOI 10.1186/1743-7075-3-12

    View details for Web of Science ID 000239134300001

    View details for PubMedID 16472389

    View details for PubMedCentralID PMC1391915

  • In vivo activities of cytokine oncostatin M in the regulation of plasma lipid levels JOURNAL OF LIPID RESEARCH Kong, W., Abidi, P., Kraemer, F. B., Jiang, J. D., Liu, J. W. 2005; 46 (6): 1163-1171


    Our previous studies have demonstrated the activity of oncostatin M (OM) in stimulating the transcription of the human LDL receptor (LDLR) gene in HepG2 cells through a sterol-independent regulatory mechanism. The current studies were designed to determine whether this in vitro property of OM could be recapitulated in vivo to increase LDLR expression in cholesterol-loaded livers and consequently decrease plasma levels of LDL-cholesterol (LDL-C) and total cholesterol (TC) using hypercholesterolemic hamsters as an experimental model. We show that administration of human recombinant OM for 7 days in hamsters fed a high-fat diet significantly reduced plasma levels of TC, LDL-C, and triglyceride in dose- and time-dependent manners. This lipid-lowering effect was associated with increased hepatic LDLR mRNA expression, as determined by quantitative real-time RT-PCR. Additionally, hepatic fat storage and cholesterol content in the hypercholesterolemic animals were substantially reduced by OM treatment. As a consequence, the increased aminotransferase levels in the high-fat diet-fed hamsters were normalized nearly to baseline values. These results not only corroborate the in vitro finding of OM in the regulation of LDLR but also, for the first time, demonstrate that OM has a strong lipid-lowering effect under in vivo conditions in which the levels of circulating LDL-C are high and liver LDLR transcription is repressed.

    View details for DOI 10.1194/jlr.M400425-JLR200

    View details for Web of Science ID 000229061100009

    View details for PubMedID 15772430

  • Mutational analysis of the "regulatory module" of hormone-sensitive lipase BIOCHEMISTRY Wang, J., Shen, W. J., Patel, S., Harada, K., Kraemer, F. B. 2005; 44 (6): 1953-1959


    Hormone-sensitive lipase (HSL) is a rate-limiting enzyme in lipolysis that displays broad substrate specificity. HSL function is regulated by reversible phosphorylation that occurs within a 150 aa "regulatory module" of the protein. The current studies used mutational analysis to dissect the contribution of the "regulatory module" in HSL activity and substrate specificity. Deletion of the entire "regulatory module" or replacement of the "regulatory module" with the "lid" of lipoprotein lipase resulted in enzymatically inactive proteins. Deletion of sequentially longer stretches of the "regulatory module" resulted in a stepwise reduction in hydrolytic activity. Analysis of 7-19 amino acid deletional mutants that spanned the "regulatory module" showed that the N-terminal partial deletion mutants retained normal hydrolytic activity and activation by PKA. In contrast, the C-terminal partial deletion mutants displayed reduced hydrolytic activities, with preferential loss of activity against lipid-, as opposed to water-soluble, substrates. Single amino acid mutations of F650C, P651A, and F654D reduced activity against lipid-, but not water-soluble, substrates. The current results suggest that the length of the "regulatory module" and specific sequences within the C-terminal portion of the "regulatory module" of HSL (amino acids 644-683) are crucial for activity and appear to be responsible for determining lipase activity.

    View details for DOI 10.1021/bi049206t

    View details for Web of Science ID 000226969400020

    View details for PubMedID 15697220

  • Hormone-sensitive lipase is reduced in the adipose tissue of patients with type 2 diabetes mellitus: influence of IL-6 infusion DIABETOLOGIA Watt, M. J., Carey, A. L., Wolsk-Petersen, E., Kraemer, F. B., Pedersen, B. K., Febbraio, M. A. 2005; 48 (1): 105-112


    Type 2 diabetes mellitus is characterised by increased plasma NEFA and IL-6 concentrations, and IL-6 increases lipolysis in healthy men. We assessed the adipose tissue hormone-sensitive lipase (HSL) mRNA expression, protein expression and HSL activity in patients with type 2 diabetes mellitus, and determined the effect of IL-6 administration on these measures.Seven patients with type 2 diabetes mellitus (age 67+/-4 years, weight 87+/-7 kg) and six age- and weight-matched individuals visited the laboratory on two occasions. Subcutaneous adipose tissue biopsies and blood samples were obtained prior to and during 3 h of either saline or recombinant human IL-6 infusion.HSL mRNA was reduced (p<0.05) by approximately 40% in type 2 diabetes mellitus relative to control subjects, while HSL protein expression showed a tendency to be decreased (35%, p=0.09). HSL activity averaged 8.87+/-1.25 and 6.91+/-1.20 nmol min(-1) mg(-1) protein for control and type 2 diabetic subjects respectively (p<0.05). IL-6 administration increased (p<0.05) HSL mRNA 2-fold at 60 min in both groups, whereas HSL protein and activity were unaffected by IL-6. Plasma insulin was elevated (p<0.05) in patients with type 2 diabetes mellitus at rest and was blunted (p<0.05) during IL-6 infusion in both groups. Plasma glucagon and cortisol were elevated (p<0.05) by IL-6 in both groups.Our data demonstrate that basal HSL is decreased in patients with type 2 diabetes mellitus, and this may be a consequence of elevated plasma insulin levels. We have also shown that IL-6 administration increases HSL gene expression, although it exerted no effect on HSL protein and activity. This disparity between mRNA, protein and enzyme activity may be a function either of the marked alterations in the hormonal milieu induced by IL-6 administration and/or of post-transcriptional events.

    View details for DOI 10.1007/s00125-004-1598-x

    View details for Web of Science ID 000226459700015

    View details for PubMedID 15609025

  • Physical association between the adipocyte fatty acid-binding protein and hormone-sensitive lipase - A fluorescence resonance energy transfer analysis JOURNAL OF BIOLOGICAL CHEMISTRY SMITH, A. J., Sanders, M. A., Thompson, B. R., Londos, C., Kraemer, F. B., Bernlohr, D. A. 2004; 279 (50): 52399-52405


    Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 microm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet.

    View details for DOI 10.1074/jbc.M410301200

    View details for Web of Science ID 000225493400084

    View details for PubMedID 15456755

  • Function of hormone-sensitive lipase in diacylglycerol-protein kinase C pathway 63rd Annual Meeting of the American-Diabetes-Association Kanehara, H., Suzuki, J., Zenimaru, Y., Takahashi, S., Oida, K., Shen, W. J., Kraemer, F. B., Miyamori, I. ELSEVIER IRELAND LTD. 2004: 209–15


    To explore the functional effects of hormone-sensitive lipase (HSL) in diacylglycerol (DAG) metabolism, Chinese hamster ovary cells were stably transfected with rat HSL cDNA (wt-HSL), inactive mutant S423A-HSL cDNA (S423A) and pcDNA3 vector alone (Ct). [(14)C]Glucose-incorporation into triglyceride (TG) was 75% lower in the presence or absence of insulin in cells expressing wt-HSL compared to Ct or S423A. [(14)C]Glucose-incorporation into DAG was 33% lower without insulin and 51% lower with insulin in cells expressing wt-HSL compared to Ct or S423A. Insulin stimulated glucose-incorporation into DAG 2.2-fold in S423A and Ct cells, whereas only a 50% increase was observed in cells expressing wt-HSL. Phospholipase C-mediated release of DAG from membrane phospholipids was reduced 70% in cells expressing wt-HSL compared to Ct or S423A. Western blot analysis showed that membrane-bound protein kinase C (PKC)-alpha and -epsilon were decreased 40-50% in cells expressing wt-HSL grown in high glucose with insulin. These data show that HSL potentially hydrolyzes cellular DAG generated either by de novo synthesis from glucose or release from membrane phospholipids by phospholipase C, resulting in a reduction in the translocation of DAG-sensitive PKCs.

    View details for DOI 10.1016/j.diabres.2004.02.006

    View details for Web of Science ID 000224016000003

    View details for PubMedID 15331200

  • Absence of hormone-sensitive lipase inhibits obesity and adipogenesis in Lep(ob/ob) mice JOURNAL OF BIOLOGICAL CHEMISTRY Sekiya, M., Osuga, J. I., Okazaki, H., Yahagi, N., Harada, K., Shen, W. J., Tamura, Y., Tomita, S., Iizuka, Y., Ohashi, K., Okazaki, M., Sata, M., Nagai, R., Fujita, T., Shimano, H., Kraemer, F. B., Yamada, N., Ishibashi, S. 2004; 279 (15): 15084-15090


    Hormone-sensitive lipase (HSL) plays a crucial role in the hydrolysis of triacylglycerol and cholesteryl ester in various tissues including adipose tissues. To explore the role of HSL in the metabolism of fat and carbohydrate, we have generated mice lacking both leptin and HSL (Lep(ob/ob)/HSL(-/-)) by cross-breeding HSL(-/-) mice with genetically obese Lep(ob/ob) mice. Unexpectedly, Lep(ob/ob)/HSL(-/-) mice ate less food, gained less weight, and had lower adiposity than Lep(ob/ob)/HSL(+/+) mice. Lep(ob/ob)/HSL(-/-) mice had massive accumulation of preadipocytes in white adipose tissues with increased expression of preadipocyte-specific genes (CAAT/enhancer-binding protein beta and adipose differentiation-related protein) and decreased expression of genes characteristic of mature adipocytes (CCAAT/enhancer-binding protein alpha, peroxisome proliferator activator receptor gamma, and adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1). Consistent with the reduced food intake, hypothalamic expression of neuropeptide Y and agouti-related peptide was decreased. Since HSL is expressed in hypothalamus, we speculate that defective generation of free fatty acids in the hypothalamus due to the absence of HSL mediates the altered expression of these orexigenic neuropeptides. Thus, deficiency of both leptin and HSL has unmasked novel roles of HSL in adipogenesis as well as in feeding behavior.

    View details for DOI 10.1074/jbc.M310985200

    View details for Web of Science ID 000220594700073

    View details for PubMedID 14752112

  • Hormone-sensitive lipase is required for high-density lipoprotein cholesteryl ester-supported adrenal steroidogenesis MOLECULAR ENDOCRINOLOGY Kraemer, F. B., Shen, W. J., Harada, K., Patel, S., Osuga, J. I., Ishibashi, S., Azhar, S. 2004; 18 (3): 549-557


    Steroid hormones are synthesized using cholesterol as precursor, with a substantial portion supplied by the selective uptake of lipoprotein-derived cholesteryl esters. Adrenals express a high level of neutral cholesteryl ester hydrolase activity, and recently hormone-sensitive lipase (HSL) was shown to be responsible for most adrenal neutral cholesteryl ester hydrolase activity. To determine the functional importance of HSL in adrenal steroidogenesis, adrenal cells were isolated from control and HSL-/- mice, and the in vitro production of corticosterone was quantified. Results show that, even though adrenal cholesteryl ester content was substantially elevated in both male and female HSL-/- mice, basal corticosterone production was reduced approximately 50%. The maximum corticosterone production induced by dibutyryl cAMP, and lipoproteins was approximately 75-85% lower in adrenal cells from HSL-/- mice compared with control. There is no intrinsic defect in the conversion of cholesterol into steroids in HSL-/- mice. Dibutyryl cAMP-stimulated conversion of high-density lipoprotein cholesteryl esters into corticosterone was reduced 97% in HSL-/- mice. An increase in low-density lipoprotein receptor expression appears to be one of the compensatory mechanisms for cholesterol delivery in HSL-/- mice. These findings suggest that HSL is functionally linked to the selective pathway and is critically involved in the intracellular processing and availability of cholesterol for adrenal steroidogenesis.

    View details for DOI 10.1210/me.2003-0179

    View details for Web of Science ID 000189299100006

    View details for PubMedID 14657254

  • Overexpression of leptin in transgenic mice leads to decreased basal lipolysis, PKA activity, and perilipin levels BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Ke, Y. H., Qiu, J., Ogus, S., Shen, W. J., Kraemer, F. B., Chehab, F. F. 2003; 312 (4): 1165-1170


    Transgenic mice overexpressing leptin (LepTg) exhibit substantial reductions in adipose mass. Since the binding of leptin to its receptor activates the sympathetic nervous system, we reasoned that the lean state of the LepTg mice could be caused by chronic lipolysis. Instead, the LepTg mice exhibited a low basal lipolysis state and their lean phenotype was not dependent on the presence of beta3-adrenergic receptors. In their white adipose tissue, protein levels of protein kinase A, hormone-sensitive lipase, and ADRP were not impaired. However, compared to normal mice, perilipin, perilipin mRNA, and cAMP-stimulated PKA activity were significantly attenuated. Overall, we demonstrate that the lean phenotype of the LepTg mice does not result in a chronically elevated lipolytic state, but instead in a low basal lipolysis state characterized by a decrease in perilipin and PKA activity in white fat.

    View details for DOI 10.1016/j.bbrc.2003.11.053

    View details for Web of Science ID 000187252300045

    View details for PubMedID 14651995

  • Lipase-selective functional domains of perilipin A differentially regulate constitutive and protein kinase A-stimulated lipolysis 63rd Annual Meeting of the American-Diabetes-Association Zhang, H. H., Souza, S. C., Muliro, K. V., Kraemer, F. B., Obin, M. S., Greenberg, A. S. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. 2003: 51535–42


    Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMP-dependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1-300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301-517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1-300 (Ser81, Ser222, and Ser276). PKA-stimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301-517 (Ser433, Ser492, and Ser517). Peri 301-517 promoted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301-517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific "barrier" domains block basal lipolysis by HSL and non-HSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity.

    View details for DOI 10.1074/jbc.M309591200

    View details for Web of Science ID 000187206300085

    View details for PubMedID 14527948

  • Resistance to high-fat diet-induced obesity and altered expression of adipose-specific genes in HSL-deficient mice AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Harada, K., Shen, W. J., Patel, S., Natu, V., Wang, J. N., Osuga, J., Ishibashi, S., Kraemer, F. B. 2003; 285 (6): E1182-E1195


    To elucidate the role of hormone-sensitive lipase (HSL) in diet-induced obesity, HSL-deficient (HSL-/-) and wild-type mice were fed normal chow or high-fat diets. HSL-/- mice were resistant to diet-induced obesity showing higher core body temperatures. Weight and triacylglycerol contents were decreased in white adipose tissue (WAT) but increased in both brown adipose tissue (BAT) and liver of HSL-/- mice. Serum insulin levels in the fed state and tumor necrosis factor-alpha mRNA levels in adipose tissues were higher, whereas serum levels of adipocyte complement-related protein of 30 kDa (ACRP30)/adiponectin and leptin, as well as mRNA levels of ACRP30/adiponectin, leptin, resistin, and adipsin in WAT, were lower in HSL-/- mice than in controls. Expression of transcription factors associated with adipogenesis (peroxisome proliferator-activated receptor-gamma, CAAT/enhancer-binding protein-alpha) and lipogenesis (carbohydrate response element-binding protein, adipocyte determination- and differentiation-dependent factor-1/sterol regulatory element-binding protein-1c), as well as of adipose differentiation markers (adipocyte lipid-binding protein, perilipin, lipoprotein lipase), lipogenic enzymes (glycerol-3-phosphate acyltransferase, acyl-CoA:diacylglycerol acyltransferase-1 and -2, fatty acid synthase, ATP citrate lyase) and insulin signaling proteins (insulin receptor, insulin receptor substrate-1, GLUT4), was suppressed in WAT but not in BAT of HSL-/- mice. In contrast, expression of genes associated with cholesterol metabolism (sterol-regulatory element-binding protein-2, 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA:cholesterol acyltransferase-1) and thermogenesis (uncoupling protein-2) was upregulated in both WAT and BAT of HSL-/- mice. Our results suggest that impaired lipolysis in HSL deficiency affects lipid metabolism through alterations of adipose differentiation and adipose-derived hormone levels.

    View details for DOI 10.1152/ajpendo.00259.2003

    View details for Web of Science ID 000186474500005

    View details for PubMedID 12954598

  • Fatty acid-binding protein-hormone-sensitive lipase interaction - Fatty acid dependence on binding JOURNAL OF BIOLOGICAL CHEMISTRY Jenkins-Kruchten, A. E., Bennaars-Eiden, A., Ross, J. R., Shen, W. J., Kraemer, F. B., Bernlohr, D. A. 2003; 278 (48): 47636-47643


    Adipose lipolysis is mediated, in part, via interaction of fatty acid-binding protein (FABP) with hormone-sensitive lipase (HSL). Mice with reduced FABP content in fat (adipocyte FABP null) exhibit diminished fat cell lipolysis, whereas transgenic mice with increased FABP content in fat (epithelial FABP transgenic) exhibit enhanced lipolysis. To examine the relationship between the binding of FABP to HSL and activation of catalytic activity, isothermal titration microcalorimetry as well as kinetic analysis using a variety of FABP isoforms have been employed. In the absence of fatty acids, no FABP-HSL association could be demonstrated for any FABP form. However, in the presence of 10 microm oleate, A-FABP and E-FABP each bound to HSL with high affinity (Kd of 0.5 and 3 nM, respectively) in a approximately 1:1 molar stoichiometry, whereas liver FABP and intestinal FABP did not exhibit any association. To compare binding to catalysis, each FABP isoform was incubated with HSL in vitro, and enzymatic activity was assessed. Importantly, each FABP form stimulated HSL activity approximately 2-fold using cholesteryl oleate as substrate but exhibited no activation using p-nitrophenyl butyrate. The activation by A-FABP was dependent upon its fatty acid binding properties because a non-fatty acid binding mutant, R126Q, failed to activate HSL. These results suggest that binding and activation of HSL by FABPs are separate and distinct functions and that HSL contains a site for fatty acid binding that allows for FABP association.

    View details for DOI 10.1074/jbc.M307680200

    View details for Web of Science ID 000186731400037

    View details for PubMedID 13129924

  • Angiotensin II activates cholesterol ester hydrolase in bovine adrenal glomerulosa cells through phosphorylation mediated by p42/p44 mitogen-activated protein kinase ENDOCRINOLOGY Cherradi, N., Pardo, B., Greenberg, A. S., Kraemer, F. B., Capponi, A. M. 2003; 144 (11): 4905-4915


    In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) occurs via activation of the Ca2+ messenger system, increased expression of the steroidogenic acute regulatory protein, and enhanced transfer of cholesterol to the inner mitochondrial membrane. We examined here whether Ang II affects the activity of cholesterol ester hydrolase (CEH), also named hormone-sensitive lipase, the enzyme recruiting cholesterol from intracellular pools, in bovine adrenal glomerulosa cells. In bovine adrenal tissue, CEH activity was detected with characteristics similar to those reported in other tissues (Michaelis constant = 46.3 +/- 6.7 microM, n = 3; maximal velocity = 1 nmol/mg.min). This activity was significantly enhanced in isolated bovine glomerulosa cells challenged for 2 h with 10 nM Ang II (to 149 +/- 11% of controls, n = 3). Similarly, 25 microM forskolin raised CEH activity to 151 +/- 5% of controls (n = 3). This increase in activity of CEH was not due to an increase in the amount of enzyme protein but was associated with an increased phosphorylation of the enzyme to 337 +/- 33% of controls (n = 9, P < 0.0001). Potassium ion (K+) and forskolin also stimulated [32P]orthophosphate incorporation, although to a lesser extent (to 157 +/- 18% and 186 +/- 25% of controls, respectively). On SDS-PAGE, the majority of this radioactivity was associated with a species of 172 kDa, corresponding to a CEH dimer. Both Ang II-induced CEH phosphorylation and pregnenolone production were significantly reduced (to 47 +/- 6% and 50 +/- 8% of controls with Ang II alone, respectively) in the presence of PD098059, an inhibitor of p42/p44 MAPK. Indeed, Ang II challenge led to a rapid 32P incorporation into p42/p44 MAPK. These results demonstrate that, in addition to its known effects on intramitochondrial cholesterol transfer, Ang II also promotes aldosterone biosynthesis by rapidly increasing cholesterol supply to the outer mitochondrial membrane.

    View details for DOI 10.1210/en.2003-0325

    View details for Web of Science ID 000186178300033

    View details for PubMedID 12960096

  • Interaction of hormone-sensitive lipase with steroidogeneic acute regulatory protein - Facilitation of cholesterol transfer in adrenal JOURNAL OF BIOLOGICAL CHEMISTRY Shen, W. J., Patel, S., Natu, V., Hong, R., Wang, J., Azhar, S., Kraemer, F. B. 2003; 278 (44): 43870-43876


    Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis.

    View details for DOI 10.1074/jbc.M303934200

    View details for Web of Science ID 000186157000137

    View details for PubMedID 12925534

  • Gender- and site-related effects on lipolytic capacity of rat white adipose tissue CELLULAR AND MOLECULAR LIFE SCIENCES Pujol, E., Rodriguez-Cuenca, S., Frontera, M., Justo, R., Llado, I., Kraemer, F. B., Gianotti, M., Roca, P. 2003; 60 (9): 1982-1989


    Gender- and site-related differences in the lipolytic capacity, at the different steps of the adrenergic pathway, in gonadal and inguinal white adipose tissue (WAT), were assessed by studying alpha2A-adrenergic receptor (AR), beta3-AR and hormone-sensitive lipase (HSL) protein levels, and by determining the lipolytic response to different agents. Gonadal WAT showed a lower alpha2A/beta3-AR ratio, a greater lipolytic capacity in response to AR agonists, and higher HSL activity and protein levels than inguinal WAT. In female rats, we found greater alpha2A-AR protein levels and alpha2A/beta3-AR ratio compared to their male counterparts, but, on the other hand, a higher lipolytic response to beta-AR agonists and a greater lipolytic capacity at the postreceptor level, including a more activated HSL protein. Thus, the lipolytic capacity was clearly higher in gonadal than in inguinal WAT, at the different steps of the adrenergic pathway studied. Moreover, in both tissues, females showed a greater inhibition of lipolysis via alpha2-AR, which was counteracted by the higher lipolytic capacity at the postreceptor level.

    View details for DOI 10.1007/s00018-003-3125-5

    View details for Web of Science ID 000185743800017

    View details for PubMedID 14523558

  • Sterol-mediated regulation of hormone-sensitive lipase in 3T3-L1 adipocytes LIPIDS Miura, S., Nagura, H., Sawamura, F., Tomita, I., Kawai, E., Mochizuki, N., Ikeda, M., Kraemer, F. B., Tomita, T. 2003; 38 (7): 743-750


    We previously reported that intracellular free cholesterol at physiological concentrations regulates the activity of neutral cholesterol esterase (N-CEase) in macrophages. The objective of the present study is to investigate whether the regulation of N-CEase by cholesterol is generally observed in other types of cells such as adipocytes with high activity of hormone-sensitive lipase (HSL), the same gene product as N-CEase. 3T3-L1 adipocytes were cultured with and without cholesterol (1-30 microg/mL) or 25-hydroxycholesterol (0.1-10 microg/mL), and changes in the N-CEase activity, expression of HSL mRNA, and protein were examined. Incubation (24 h) of cells with cholesterol did not change N-CEase activity, but incubation with 25-hydroxycholesterol decreased the activity in a concentration-dependent manner by 24 (24 h) and 54% (36 h). Quantitative reverse transcription-PCR indicated that 25-hydroxycholesterol (10 microg/mL) did not influence expression of HSL mRNA. However, Western blot analysis showed that this sterol reduced HSL protein by 72 (24 h) and by 93% (36 h), respectively. It was concluded that sterol-mediated regulation of HSL/N-CEase occurs not only in macrophages but also in adipocytes, and regulation appears to occur not at a transcriptional level but by a post-transcriptional process. Sterol-mediated proteolysis may be involved in the loss of HSL protein.

    View details for Web of Science ID 000185058800007

    View details for PubMedID 14506837

  • Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis JOURNAL OF LIPID RESEARCH Kraemer, F. B., Shen, W. J. 2002; 43 (10): 1585-1594


    Hormone-sensitive lipase (HSL) is an intracellular neutral lipase that is capable of hydrolyzing triacylglycerols, diacylglycerols, monoacylglycerols, and cholesteryl esters, as well as other lipid and water soluble substrates. HSL activity is regulated post-translationally by phosphorylation and also by pretranslational mechanisms. The enzyme is highly expressed in adipose tissue and steroidogenic tissues, with lower amounts expressed in cardiac and skeletal muscle, macrophages, and islets. Studies of the structure of HSL have identified several amino acids and regions of the molecule that are critical for enzymatic activity and regulation of HSL. This has led to important insights into its function, including the interaction of HSL with other intracellular proteins, such as adipocyte lipid binding protein. Accumulating evidence has defined important functions for HSL in normal physiology, affecting adipocyte lipolysis, steroidogenesis, spermatogenesis, and perhaps insulin secretion and insulin action; however, direct links between abnormal expression or genetic variations of HSL and human disorders, such as obesity, insulin resistance, type 2 diabetes, and hyperlipidemia, await further clarification. The published reports examining the regulation, and function of HSL in normal physiology and disease are reviewed in this paper.

    View details for DOI 10.1194/jlr.R200009-JLR200

    View details for Web of Science ID 000178598400001

    View details for PubMedID 12364542

  • Identification of Egr1 as the oncostatin M-induced transcription activator that binds to sterol-independent regulatory element of human LDL receptor promoter JOURNAL OF LIPID RESEARCH Zhang, F., Ahlborn, T. E., Li, C., Kraemer, F. B., Liu, J. W. 2002; 43 (9): 1477-1485


    Previously, we identified the low density lipoprotein receptor (LDLR) promoter region -17 to -1 as a novel sterol-independent regulatory element (SIRE) that mediates the stimulating effect of oncostatin M (OM). The goal of this study was to identify the OM-induced transcription activator that binds to the SIRE sequence. By conducting a electrophoretic mobility shift assay (EMSA) followed by UV crosslinking and SDS-PAGE, we show that a protein with a molecular mass of 85 kDa was present in the OM-induced SIRE DNA-protein complex. Western blotting and supershift assays reveal that the 85 kDa factor is early growth response gene 1 (Egr1). The interaction of Egr1 with the SIRE sequence was further confirmed in vivo by chromatin immunoprecipitation assays. The functional role of Egr1 in LDLR transcription was assessed by cotransfection of an Egr1 expression vector with an LDLR promoter reporter construct. We show that overexpression of Egr1 significantly increases LDLR promoter activity when cotransfected with CCAAT/enhancer binding protein beta (c/EBPbeta) or with cAMP-responsive element binding protein (CREB) expression vectors. Our studies clearly demonstrate that Egr1 is the OM-induced transcription factor that binds to the SIRE sequence of the LDLR promoter and also suggest that Egr1 may have a functional role in OM-induced upregulation of LDLR transcription through interaction with other SIRE binding proteins such as c/EBPbeta or CREB.

    View details for DOI 10.1194/jlr.M200126-JLR200

    View details for Web of Science ID 000178252900015

    View details for PubMedID 12235180

  • Cardiac gene expression profile and lipid accumulation in response to starvation AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Suzuki, J., Shen, W. J., Nelson, B. D., Selwood, S. P., Murphy, G. M., Kanefara, H., Takahashi, S., Oida, K., Miyamori, I., Kraemer, F. B. 2002; 283 (1): E94-E102


    Starvation induces many biochemical and histological changes in the heart; however, the molecular events underlying these changes have not been fully elucidated. To explore the molecular response of the heart to starvation, microarray analysis was performed together with biochemical and histological investigations. Serum free fatty acids increased twofold in both 16- and 48-h-fasted mice, and cardiac triglyceride content increased threefold and sixfold in 16- and 48-h-fasted mice, respectively. Electron microscopy showed numerous lipid droplets in hearts of 48-h-fasted mice, whereas fewer numbers of droplets were seen in hearts from 16-h-fasted mice. Expression of 11,000 cardiac genes was screened by microarrays. More than 50 and 150 known genes were detected by differential expression analysis after 16- and 48-h-fasts, respectively. Genes for fatty acid oxidation and gluconeogenesis were increased, and genes for glycolysis were decreased. Many other genes for metabolism, signaling/cell cycle, cytoskeleton, and tissue antigens were affected by fasting. These data provide a broad perspective of the molecular events occurring physiologically in the heart in response to starvation.

    View details for DOI 10.1152/ajpendo.00017.2002

    View details for Web of Science ID 000176191000013

    View details for PubMedID 12067848

  • Effectiveness of diabetes management: Is improvement feasible? AMERICAN JOURNAL OF MEDICINE Basina, M., Kraemer, F. B. 2002; 112 (8): 670-672

    View details for Web of Science ID 000175917700012

    View details for PubMedID 12034419

  • Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system 60th Scientific Sessions of the American-Diabetes-Association Souza, S. C., Muliro, K. V., Liscum, L., Lien, P., Yamamoto, M. T., Schaffer, J. E., Dallal, G. E., Wang, X. Z., Kraemer, F. B., Obin, M., Greenberg, A. S. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. 2002: 8267–72


    Perilipin (Peri) A is a phosphoprotein located at the surface of intracellular lipid droplets in adipocytes. Activation of cyclic AMP-dependent protein kinase (PKA) results in the phosphorylation of Peri A and hormone-sensitive lipase (HSL), the predominant lipase in adipocytes, with concurrent stimulation of adipocyte lipolysis. To investigate the relative contributions of Peri A and HSL in basal and PKA-mediated lipolysis, we utilized NIH 3T3 fibroblasts lacking Peri A and HSL but stably overexpressing acyl-CoA synthetase 1 (ACS1) and fatty acid transport protein 1 (FATP1). When incubated with exogenous fatty acids, ACS1/FATP1 cells accumulated 5 times more triacylglycerol (TG) as compared with NIH 3T3 fibroblasts. Adenoviral-mediated expression of Peri A in ACS1/FATP1 cells enhanced TG accumulation and inhibited lipolysis, whereas expression of HSL fused to green fluorescent protein (GFPHSL) reduced TG accumulation and enhanced lipolysis. Forskolin treatment induced Peri A hyperphosphorylation and abrogated the inhibitory effect of Peri A on lipolysis. Expression of a mutated Peri A Delta 3 (Ser to Ala substitutions at PKA consensus sites Ser-81, Ser-222, and Ser-276) reduced Peri A hyperphosphorylation and blocked constitutive and forskolin-stimulated lipolysis. Thus, perilipin expression and phosphorylation state are critical regulators of lipid storage and hydrolysis in ACS1/FATP1 cells.

    View details for DOI 10.1074/jbc.M108329200

    View details for Web of Science ID 000174268000083

    View details for PubMedID 11751901

  • Adrenal neutral cholesteryl ester hydrolase: Identification, subcellular distribution, and sex differences ENDOCRINOLOGY Kraemer, F. B., Shen, W. J., Natu, V., Patel, S., Osuga, J., Ishibashi, S., Azhar, S. 2002; 143 (3): 801-806


    Adrenals express a high level of neutral cholesteryl ester hydrolase (CEH) activity, and male rats have greater activity than females; however, the identity of the enzyme(s) responsible for this activity and the basis for the sex differences are unknown. Using mice in which hormone-sensitive lipase (HSL) was inactivated by homologous recombination (HSL -/-), neutral CEH activity was reduced more than 98% compared with controls. Female HSL -/- mice showed a reduction in stimulated corticosterone values. Mechanical separation of rat adrenals revealed less HSL in the outer than the inner cortex. Examination of subfractions of rat adrenals showed that immunoreactive HSL was prominently expressed in microsomes, with lesser amounts in the cytosol and little to no HSL in mitochondrial and nuclear fractions or the lipid droplet. Four- to 10-fold more neutral CEH activity was in the microsomal fraction than any other fraction. No sex differences in the expression or subcellular distribution of HSL protein were found; however, neutral CEH activity was lower in the microsomal fraction of females, and female adrenals contained more cholesteryl esters. Thus, HSL appears to be responsible for most, if not all, of adrenal neutral CEH activity, is prominently expressed in microsomes, and its activity is influenced by sex.

    View details for Web of Science ID 000174034800009

    View details for PubMedID 11861500

  • Characterization of the functional interaction of adipocyte lipid-binding protein with hormone-sensitive lipase JOURNAL OF BIOLOGICAL CHEMISTRY Shen, W. J., Liang, Y., Hong, R., Patel, S., Natu, V., Sridhar, K., Jenkins, A., Bernlohr, D. A., Kraemer, F. B. 2001; 276 (52): 49443-49448


    Hormone-sensitive lipase (HSL) is an intracellular lipase that plays an important role in the hydrolysis of triacylglycerol in adipose tissue. HSL has been shown to interact with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that bind fatty acids and other hydrophobic ligands. The current studies have addressed the functional significance of the association and mapped the site of interaction between HSL and ALBP. Incubation of homogeneous ALBP with purified, recombinant HSL in vitro resulted in a 2-fold increase in substrate hydrolysis. Moreover, the ability of oleate to inhibit HSL hydrolytic activity was attenuated by co-incubation with ALBP. Co-transfection of Chinese hamster ovary cells with HSL and ALBP resulted in greater hydrolytic activity than transfection of cells with HSL and vector alone. Deletional mutations of HSL localized the region of HSL that interacts with ALBP to amino acids 192-200, and site-directed mutagenesis of individual amino acids in this region identified His-194 and Glu-199 as critical for mediating the interaction of HSL with ALBP. Interestingly, HSL mutants H194L and E199A, each of which retained normal basal hydrolytic activity, failed to display an increase in hydrolytic activity when co-transfected with wild type ALBP. Therefore, ALBP increases the hydrolytic activity of HSL through its ability to bind and sequester fatty acids and via specific protein-protein interaction. Thus, HSL and ALBP constitute a functionally important lipolytic complex.

    View details for DOI 10.1074/jbc.M104095200

    View details for Web of Science ID 000173922100106

    View details for PubMedID 11682468

  • Stimulation of lipolysis and hormone-sensitive lipase via the extracellular signal-regulated kinase pathway JOURNAL OF BIOLOGICAL CHEMISTRY Greenberg, A. S., Shen, W. J., Muliro, K., Patel, S., Souza, S. C., Roth, R. A., Kraemer, F. B. 2001; 276 (48): 45456-45461


    Hormonally stimulated lipolysis occurs by activation of cyclic AMP-dependent protein kinase (PKA) which phosphorylates hormone-sensitive lipase (HSL) and increases adipocyte lipolysis. Evidence suggests that catecholamines not only can activate PKA, but also the mitogen-activated protein kinase pathway and extracellular signal-regulated kinase (ERK). We now demonstrate that two different inhibitors of MEK, the upstream activator of ERK, block catecholamine- and beta(3)-stimulated lipolysis by approximately 30%. Furthermore, treatment of adipocytes with dioctanoylglycerol, which activates ERK, increases lipolysis, although MEK inhibitors decrease dioctanoylglycerol-stimulated activation of lipolysis. Using a tamoxifen regulatable Raf system expressed in 3T3-L1 preadipocytes, exposure to tamoxifen causes a 14-fold activation of ERK within 15-30 min and results in approximately 2-fold increase in HSL activity. In addition, when differentiated 3T3-L1 cells expressing the regulatable Raf were exposed to tamoxifen, a 2-fold increase in lipolysis is observed. HSL is a substrate of activated ERK and site-directed mutagenesis of putative ERK consensus phosphorylation sites in HSL identified Ser(600) as the site phosphorylated by active ERK. When S600A HSL was expressed in 3T3-L1 cells expressing the regulatable Raf, tamoxifen treatment fails to increase its activity. Thus, activation of the ERK pathway appears to be able to regulate adipocyte lipolysis by phosphorylating HSL on Ser(600) and increasing the activity of HSL.

    View details for Web of Science ID 000172406700143

    View details for PubMedID 11581251

  • Mechanism of intracellular calcium ([Ca2+]i) inhibition of lipolysis in human adipocytes. FASEB journal Xue, B., Greenberg, A. G., Kraemer, F. B., Zemel, M. B. 2001; 15 (13): 2527-2529


    We investigated the mechanisms responsible for the anti-lipolytic effect of intracellular Ca2+ ([Ca2+]i) in human adipocytes. Increasing [Ca2+]i inhibited lipolysis induced by b-adrenergic receptor activation, A1 adenosine receptor inhibition, adenylate cyclase activation, and phosphodiesterase (PDE) inhibition, as well as by a hydrolyzable cAMP analog, but not by a nonhydrolyzable cAMP analog. This finding indicates that the anti-lipolytic effect of [Ca2+]i may be mediated by the activation of adipocyte PDE. Consistent with this theory, [Ca2+]i inhibition of isoproterenol-stimulated lipolysis was reversed completely by the nonselective PDE inhibitor isobutyl methylxanthine and also by the selective PDE 3B inhibitor cilostamide, but not by selective PDE 1 and 4 inhibitors. In addition, phosphatidylinositol-3 kinase inhibition with wortmannin completely prevented insulin's anti-lipolytic effect but only minimally blocked [Ca2+]i's effect, which suggests that [Ca2+]i and insulin may activate PDE 3B via different mechanisms. In contrast, the antilipolytic effect of [Ca2+]i was not affected by inhibitors of calmodulin, Ca2+/calmodulin-dependent kinase, protein phosphatase 2B, and protein kinase C. Finally, [Ca2+]i inhibited significantly isoproterenol-stimulated increases in cAMP levels and hormone-sensitive lipase phosphorylation in human adipocytes. In conclusion, increasing [Ca2+]i exerts an antilipolytic effect mainly by activation of PDE, leading to a decrease in cAMP and HSL phosphorylation and, consequently, inhibition of lipolysis.

    View details for PubMedID 11641262

  • Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation during the lactation cycle of the rat METABOLISM-CLINICAL AND EXPERIMENTAL Clifford, G. M., Kraemer, F. B., Yeaman, S. J., Vernon, R. G. 2001; 50 (11): 1264-1269


    The removal of the litter from lactating rats results in a decrease in the lipolytic response to catecholamines in maternal adipocytes; this effect can be prevented by concomitant treatment of the rats with growth hormone. The decrease in response to catecholamines following litter removal was not due to a change in the amount of either hormone-sensitive lipase (HSL) or perilipin per adipocyte or in the proportion of either of these proteins associated with the fat droplet. Incubation in vitro with isoproterenol did not cause any apparent net translocation of HSL to the fat droplet in adipocytes from the mature female rats in any state used in this study, but isoproterenol did cause a movement of perlipin away from the fat droplet. This translocation of perilipin was not altered by litter removal. Thus, the decrease in response to catecholamines found on litter removal from lactating rats appears to be due to a diminished ability to activate HSL associated with fat droplet.

    View details for Web of Science ID 000172078300002

    View details for PubMedID 11699042

  • Absence of cardiac lipid accumulation in transgenic mice with heart-specific HSL overexpression AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Suzuki, J., Shen, W. J., Nelson, B. D., Patel, S., Veerkamp, J. H., Selwood, S. P., Murphy, G. M., Reaven, E., Kraemer, F. B. 2001; 281 (4): E857-E866


    Hormone-sensitive lipase (HSL) hydrolyzes triglyceride (TG) in adipose tissue. HSL is also expressed in heart. To explore the actions of cardiac HSL, heart-specific, tetracycline (Tc)-controlled HSL-overexpressing mice were generated. Tc-responsive element-HSL transgenic (Tg) mice were generated and crossed with myosin heavy chain (MHC)alpha-tTA Tg mice, which express the Tc-responsive transactivator (tTA) in the heart. The double-Tg mice (MHC-HSL) were maintained with doxycycline (Dox) to suppress Tg HSL. Upon removal of Dox, cardiac HSL activity and protein increased 12- and 8-fold, respectively, and the expression was heart specific. Although cardiac TG content increased twofold in control mice after an overnight fast, it did not increase in HSL-induced mice. Electron microscopy showed numerous lipid droplets in the myocardium of fasted control mice, whereas fasted HSL-induced mice showed virtually no droplets. Microarray analysis showed altered expression of cardiac genes for fatty acid oxidation, transcription factors, signaling molecules, cytoskeletal proteins, and histocompatibility antigens in HSL-induced mice. Thus cardiac HSL plays a role in controlling accumulation of triglyceride droplets and can affect the expression of a number of cardiac genes.

    View details for Web of Science ID 000171583200026

    View details for PubMedID 11551864

  • Mechanism of intracellular calcium ([Ca2+](i)) inhibition of lipolysis in human adipocytes FASEB JOURNAL Xue, B. Z., Greenberg, A. G., Kraemer, F. B., Zemel, M. B. 2001; 15 (11): 2527-?
  • Subcellular localization of insulin receptor substrate family proteins associated with phosphatidylinositol 3-kinase activity and alterations in lipolysis in primary mouse adipocytes from IRS-1 null mice DIABETES Tsuji, Y., Kaburagi, Y., Terauchi, Y., Satoh, S., Kubota, N., Tamemoto, H., Kraemer, F. B., Sekihara, H., Aizawa, S., Akanuma, Y., Tobe, K., Kimura, S., Kadowaki, T. 2001; 50 (6): 1455-1463


    To clarify the roles of insulin receptor substrate (IRS) family proteins in phosphatidylinositol (PI) 3-kinase activation and insulin actions in adipocytes, we investigated the intracellular localization of IRS family proteins and PI 3-kinase activation in response to insulin by fractionation of mouse adipocytes from wild-type and IRS-1 null mice. In adipocytes from wild-type mice, tyrosine-phosphorylated IRS-1 and IRS-2, which were found to associate with PI 3-kinase in response to insulin, were detected in the plasma membrane (PM) and low-density microsome (LDM) fractions. By contrast, tyrosine-phosphorylated IRS-3 (pp60), which was found to associate with PI 3-kinase, was predominantly localized in the PM fraction. In adipocytes from IRS-1-null mice, insulin-stimulated PI 3-kinase activity in anti-phosphotyrosine (alphaPY) immunoprecipitates in the LDM fraction was almost exclusively mediated via IRS-2 and was reduced to 25%; however, insulin-stimulated PI 3-kinase activity in the PM fraction was primarily mediated via IRS-3 and was reduced to 60%. To determine the potential functional impact of the distinct subcellular localization of IRSs and associating PI 3-kinase activity on adipocyte-specific metabolic actions, we examined lipolysis in IRS-1 null mice. The level of isoproterenol-induced lipolysis was increased 5.1-fold in adipocytes from IRS-1 null mice as compared with wild-type mice. Moreover, hormone-sensitive lipase (HSL) protein was increased 4.3-fold in adipocytes from IRS-1-null mice compared with wild-type mice, and HSL mRNA expression was also increased. The antilipolytic effect of insulin in IRS-1 null adipocytes, however, was comparable to that in wild-type mice. Thus, discordance between these two insulin actions as well as the transcriptional and translational effect (HSL mRNA and protein regulation) and the PM effect (antilipolysis) of insulin may be explained by distinct roles of both PI 3-kinase activity associated with IRS-1/IRS-2 and PI 3-kinase activity associated with IRS-3 in insulin actions related to their subcellular localization.

    View details for Web of Science ID 000168961900028

    View details for PubMedID 11375348

  • Requirement of sp1 and estrogen receptor alpha interaction in 17 beta-estradiol-mediated transcriptional activation of the low density lipoprotein receptor gene expression ENDOCRINOLOGY Li, C., Briggs, M. R., Ahlborn, T. E., Kraemer, F. B., Liu, J. W. 2001; 142 (4): 1546-1553


    Estrogen is one of the most important physiological regulators of low density lipoprotein receptor (LDLR) expression. Despite many studies conducted in animals and humans showing increased expressions of LDLR messenger RNA by hormone treatment, the molecular basis of the effect of estrogen on LDLR transcription has not been clearly elucidated. By using HepG2 cells that transiently express functional estrogen receptor alpha (ERalpha) and LDLR promoter constructs, we show that the specific interaction of ERalpha with the transcription factor Sp1 bound to the LDLR promoter is responsible for the activation of LDLR transcription by estrogen. We demonstrate that 1) mutations to abrogate the binding of Sp1 to its recognition sequences present in repeat 1 and repeat 3 elements of the LDLR promoter completely abolish the ERalpha-mediated activation of the LDLR promoter activity; 2) mutations that abolish the selective DNA-binding activity or inactivate the C-terminal transcription activation function (AF2) of ERalpha had no effect on the ability of ERalpha to activate LDLR transcription; however, transcriptional activation was completely lost by deletion of the N-terminal transcription activation region (AF1); 3) a subregion of AF1 (amino acids 67-139) was further identified to be important for ERalpha to activate the LDLR promoter; and 4) ERalpha enhanced the formation of Sp1-repeat 3 DNA complexes. We also show that mutation at the sterol-responsive element-1 site diminishes the activity of ERalpha on LDLR transcription, thereby suggesting that the sterol-responsive element-1-binding protein may interact with the Sp1-ERalpha complex to trans-activate LDLR gene transcription. This study for the first time provides a molecular basis for an understanding of the regulation of LDLR transcription by estrogens.

    View details for Web of Science ID 000167845900022

    View details for PubMedID 11250935

  • Study of different hormone-sensitive lipase concentrations using a surface plasmon resonance sensor SENSORS AND ACTUATORS B-CHEMICAL Meriaudeau, F., Ferrell, T. L., Arakawa, E. T., Wig, A., Passian, A., Thundat, T., Shen, W. J., Patel, S., Kraemer, F. B. 2001; 73 (2-3): 192-198
  • Regulation of hormone-sensitive lipase Shen, W. J., Hong, R., Liang, Y., Patel, S., Natu, V., Bernhlor, D., Kraemer, F. B. FEDERATION AMER SOC EXP BIOL. 2001: A19–A19
  • Impact of automated calls with nurse follow-up on diabetes treatment outcomes in a Department of Veterans Affairs Health Care System - A randomized controlled trial DIABETES CARE Piette, J. D., Weinberger, M., Kraemer, F. B., McPhee, S. J. 2001; 24 (2): 202-208


    We evaluated automated telephone disease management (ATDM) with telephone nurse follow-up as a strategy for improving diabetes treatment processes and outcomes in Department of Veterans Affairs (VA) clinics. We also compared the results with those of a prior ATDM trial conducted in a county health care system.A total of 272 VA patients with diabetes using hypoglycemic medications were randomized. During the 1-year study period, intervention patients received biweekly ATDM health assessment and self-care education calls, and a nurse educator followed up with patients based on their ATDM assessment reports. Telephone surveys were used to measure patients' self-care, symptoms, and satisfaction with care. Outpatient service use was evaluated using electronic databases and self-reports, and glycemic control was measured by HbA1c and serum glucose testing.At 12 months, intervention patients reported more frequent glucose self-monitoring and foot inspections than patients receiving usual care and were more likely to be seen in podiatry and diabetes specialty clinics. Intervention patients also were more likely than control patients to have had a cholesterol test. Among patients with baseline HbA1c levels > or =8%, mean end-point values were lower among intervention patients than control patients (8.7 vs. 9.2%, respectively; P = 0.04). Among intervention and control patients with baseline values > or =9%, mean end-point values were 9.1 and 10.2%, respectively (P = 0.04). At follow-up, intervention patients reported fewer symptoms of poor glycemic control than control patients and greater satisfaction with their health care.This intervention improved the quality of VA diabetes care. Intervention effects for most end points replicated findings from the prior county clinic trial, although intervention-control differences in the current study were smaller because of the relatively good self-care and health status among the current study's enrollees.

    View details for Web of Science ID 000166681000003

    View details for PubMedID 11213866

  • Oncostatin M-induced growth inhibition and morphological changes of MDA-MB231 breast cancer cells are abolished by blocking the MEK/ERK signaling pathway BREAST CANCER RESEARCH AND TREATMENT Li, C., Ahlborn, T. E., Kraemer, F. B., Liu, J. W. 2001; 66 (2): 111-121


    Cytokine oncostatin M (OM) has profound effects on proliferation and differentiation of breast cancer cells. OM treated cells show reduced growth rate and differentiated phenotypes. The mechanisms underlying the OM growth-inhibitory activity in breast cancer cells have not been fully elucidated. In this study, we investigated the OM-elicited signaling pathways in breast cancer cell lines MDA-MB231 and MCF-7. We show that OM rapidly activates the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription (STAT) 1 and 3 in both cell lines. Intriguingly, OM-induced growth inhibition and morphological changes in MDA-MB231 cells are completely abolished by inhibitors to ERK upstream kinase MEK (nitrogen/extracellular-regulated protein kinase kinase), but the MEK inhibitors have little effects on OM growth-inhibitory activity in MCF-7 cells. In addition, expressions of the cyclin kinase inhibitors p21 and p27 are strongly induced by OM in MCF-7 cells, but their expression is only slightly increased by OM in MDA-MB231 cells. These data together demonstrate that the growth-inhibitory activity of OM can be mediated by different signaling pathways in a cell line-specific manner. While the MEK/ERK pathway is the predominant signaling pathway that leads to the growth inhibition of MDA-MB231 cells, activation of additional signaling pathways are necessary for OM to exert its growth-inhibitory activity in MCF-7 cells.

    View details for Web of Science ID 000168531800003

    View details for PubMedID 11437097

  • Human BMP-7/OP-1 induces the growth and differentiation of adipocytes and osteoblasts in bone marrow stromal cell cultures 21st Annual Meeting of the American-Society-for-Bone-and-Mineral-Research Chen, T. L., Shen, W. J., Kraemer, F. B. WILEY-LISS. 2001: 187–99


    We studied the effects of BMP-7/OP-1 on growth and differentiation of bone marrow stromal cells. BMS2, a mouse bone marrow stromal cell line capable of differentiating into adipocytes and osteoblasts, were treated in a serum-free medium containing differentiation agents that favor the expression of both lineages. BMP-7/OP-1 stimulated cell proliferation and differentiation concomitantly. These effects were dose- and growth phase-dependent. Cells were more sensitive to the treatment early in the culture (30-40% confluence) with a significant increase in cell proliferation and markers of differentiation at low concentrations. When treated later in the growth phase (90-100% confluence), no significant increase in cell proliferation was seen. The concentration requirement for cells later in the culture to reach an equivalent degree of differentiation was 3-10- fold higher than for cells treated early. In both cases, the effects on adipocyte differentiation were biphasic; low concentrations stimulated adipocyte differentiation which was inhibited at higher concentrations where stimulation of osteoblast markers were observed. We conclude that cell proliferation and cell differentiation into adipocyte/osteoblast can occur simultaneously under BMP-7/OP-1 treatment.

    View details for Web of Science ID 000169581200001

    View details for PubMedID 11527145

  • Lipoprotein receptors, macrophages, and sphingomyelinase ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Kraemer, F. B., Suzuki, J. 2000; 20 (12): 2509-2510

    View details for Web of Science ID 000165869900003

    View details for PubMedID 11116045

  • Masoprocol decreases rat lipolytic activity by decreasing the phosphorylation of HSL AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Gowri, M. S., Azhar, R. K., Kraemer, F. B., Reaven, G. M., Azhar, S. 2000; 279 (3): E593-E600


    Masoprocol (nordihydroguaiaretic acid), a lipoxygenase inhibitor isolated from the creosote bush, has been shown to decrease adipose tissue lipolytic activity both in vivo and in vitro. The present study was initiated to test the hypothesis that the decrease in lipolytic activity by masoprocol resulted from modulation of adipose tissue hormone-sensitive lipase (HSL) activity. The results indicate that oral administration of masoprocol to rats with fructose-induced hypertriglyceridemia significantly decreased their serum free fatty acid (FFA; P < 0.05), triglyceride (TG; P < 0.001), and insulin (P < 0.05) concentrations. In addition, isoproterenol-induced lipolytic rate and HSL activity were significantly lower (P < 0.001) in adipocytes isolated from masoprocol compared with vehicle-treated rats and was associated with a decrease in HSL protein. Incubation of masoprocol with adipocytes from chow-fed rats significantly inhibited isoproterenol-induced lipolytic activity and HSL activity, associated with a decrease in the ability of isoproterenol to phosphorylate HSL. Masoprocol had no apparent effect on adipose tissue phosphatidylinositol 3-kinase activity, but okadaic acid, a serine/threonine phosphatase inhibitor, blocked the antilipolytic effect of masoprocol. The results of these in vitro and in vivo experiments suggest that the antilipolytic activity of masoprocol is secondary to its ability to inhibit HSL phosphorylation, possibly by increasing phosphatase activity. As a consequence, masoprocol administration results in lower serum FFA and TG concentrations in hypertriglyceridemic rodents.

    View details for Web of Science ID 000088830300015

    View details for PubMedID 10950827

  • Hormone-sensitive lipase functions as an oligomer BIOCHEMISTRY Shen, W. J., Patel, S., Hong, R., Kraemer, F. B. 2000; 39 (9): 2392-2398


    Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase whose activity is regulated by reversible phosphorylation and which is thought to be the rate-limiting enzyme for the mobilization of FFA from adipose tissue. In the current studies the subunit structure of HSL has been explored using sucrose gradient centrifugation and in vivo and in vitro protein-protein interactions. Evidence is provided to demonstrate that HSL exists as a functional dimer composed of homologous subunits. Dimeric HSL displayed approximately 40-fold greater activity against cholesteryl ester substrate when compared with monomeric HSL without any differences in affinity for the substrate. Truncations of HSL identified the importance of the N-terminal 300 amino acids, as well as other regions, in participating in the oligomerization of HSL. These studies support the notion that the N-terminal region of HSL represents a docking domain for protein-protein interactions and provide an additional mechanism for the posttranslational control of HSL activity in the cell via oligomerization.

    View details for Web of Science ID 000085769700031

    View details for PubMedID 10694408

  • Identification of a novel sterol-independent regulatory element in the human low density lipoprotein receptor promoter JOURNAL OF BIOLOGICAL CHEMISTRY Liu, J. W., Ahlborn, T. E., Briggs, M. R., Kraemer, F. B. 2000; 275 (7): 5214-5221


    The cytokine oncostatin M (OM) activates human low density lipoprotein receptor (LDLR) gene transcription through a sterol-independent mechanism. Previous studies conducted in our laboratory have narrowed the OM-responsive element to promoter region -52 to +13, which contains the repeat 3 and two TATA-like sequences. We now identify LDLR promoter region -17 to -1 as a sterol-independent regulatory element (SIRE) that is critically involved in OM-, transcription factor CCAAT/enhancer-binding protein (C/EBP)-, and second messenger cAMP-mediated activation of LDLR transcription. The SIRE sequence overlaps the previously described TATA-like element and consists of an active C/EBP-binding site (-17 to -9) and a functional cAMP-responsive element (CRE) (-8 to -1). We demonstrate that (a) mutations within either the C/EBP or CRE site have no impact on basal or cholesterol-mediated repression of LDLR transcription, but they completely abolish OM-mediated activation of LDLR transcription; (b) replacing the repeat 3 sequence that contains the Sp1-binding site with a yeast transcription factor GAL4-binding site in the LDLR promoter construct does not affect OM inducibility, thereby demonstrating that OM induction is mediated through the SIRE sequence in conjunction with a strong activator bound to the repeat 3 sequence; (c) electrophoretic mobility shift and supershift assays confirm the specific binding of transcription factors C/EBP and cAMP-responsive element-binding protein to the SIRE; (d) cotransfection of a human C/EBPbeta expression vector (pEF-NFIL6) with the LDLR promoter construct pLDLR234 increases LDLR promoter activity; and (e) OM and dibutyryl cAMP synergistically activate LDLR transcription through this regulatory element. This study identifies, for the first time, a cis-acting regulatory element in the LDLR promoter that is responsible for sterol-independent regulation of LDLR transcription.

    View details for Web of Science ID 000085378200095

    View details for PubMedID 10671569

  • Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation of rat adipocytes JOURNAL OF BIOLOGICAL CHEMISTRY Clifford, G. M., Londos, C., Kraemer, F. B., Vernon, R. G., Yeaman, S. J. 2000; 275 (7): 5011-5015


    Adipocyte lipolysis was compared with hormone-sensitive lipase (HSL)/perilipin subcellular distribution and perilipin phosphorylation using Western blot analysis. Under basal conditions, HSL resided predominantly in the cytosol and unphosphorylated perilipin upon the lipid droplet. Upon lipolytic stimulation of adipocytes isolated from young rats with the beta-adrenergic agonist, isoproterenol, HSL translocated from the cytosol to the lipid droplet, but there was no movement of perilipin from the droplet to the cytosol; however, perilipin phosphorylation was observed. By contrast, upon lipolytic stimulation and perilipin phosphorylation in cells from more mature rats, there was no HSL translocation but a significant movement of perilipin away from the lipid droplet. Adipocytes from younger rats had markedly greater rates of lipolysis than those from the older rats. Thus high rates of lipolysis require translocation of HSL to the lipid droplet and translocation of HSL and perilipin can occur independently of each other. A loss of the ability to translocate HSL to the lipid droplet probably contributes to the diminished lipolytic response to catecholamines with age.

    View details for Web of Science ID 000085378200067

    View details for PubMedID 10671541

  • Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Osuga, J., Ishibashi, S., Oka, T., Yagyu, H., Tozawa, R., Fujimoto, A., Shionoiri, F., Yahagi, N., Kraemer, F. B., Tsutsumi, O., Yamada, N. 2000; 97 (2): 787-792


    Hormone-sensitive lipase (HSL) is known to mediate the hydrolysis not only of triacylglycerol stored in adipose tissue but also of cholesterol esters in the adrenals, ovaries, testes, and macrophages. To elucidate its precise role in the development of obesity and steroidogenesis, we generated HSL knockout mice by homologous recombination in embryonic stem cells. Mice homozygous for the mutant HSL allele (HSL-/-) were superficially normal except that the males were sterile because of oligospermia. HSL-/- mice did not have hypogonadism or adrenal insufficiency. Instead, the testes completely lacked neutral cholesterol ester hydrolase (NCEH) activities and contained increased amounts of cholesterol ester. Many epithelial cells in the seminiferous tubules were vacuolated. NCEH activities were completely absent from both brown adipose tissue (BAT) and white adipose tissue (WAT) in HSL-/- mice. Consistently, adipocytes were significantly enlarged in the BAT (5-fold) and, to a lesser extent in the WAT (2-fold), supporting the concept that the hydrolysis of triacylglycerol was, at least in part, impaired in HSL-/- mice. The BAT mass was increased by 1.65-fold, but the WAT mass remained unchanged. Discrepancy of the size differences between cell and tissue suggests the heterogeneity of adipocytes. Despite these morphological changes, HSL-/- mice were neither obese nor cold sensitive. Furthermore, WAT from HSL-/- mice retained 40% of triacylglycerol lipase activities compared with the wild-type WAT. In conclusion, HSL is required for spermatogenesis but is not the only enzyme that mediates the hydrolysis of triacylglycerol stored in adipocytes.

    View details for Web of Science ID 000084873100050

    View details for PubMedID 10639158

    View details for PubMedCentralID PMC15409

  • Do automated calls with nurse follow-up improve self-care and glycemic control among vulnerable patients with diabetes? AMERICAN JOURNAL OF MEDICINE Piette, J. D., Weinberger, M., McPhee, S. J., Mah, C. A., Kraemer, F. B., Crapo, L. M. 2000; 108 (1): 20-27


    We sought to evaluate the effect of automated telephone assessment and self-care education calls with nurse follow-up on the management of diabetes.We enrolled 280 English- or Spanish-speaking adults with diabetes who were using hypoglycemic medications and who were treated in a county health care system. Patients were randomly assigned to usual care or to receive an intervention that consisted of usual care plus bi-weekly automated assessment and self-care education calls with telephone follow-up by a nurse educator. Outcomes measured at 12 months included survey-reported self-care, perceived glycemic control, and symptoms, as well as glycosylated hemoglobin (Hb A1c) and serum glucose levels.We collected follow-up data for 89% of enrollees (248 patients). Compared with usual care patients, intervention patients reported more frequent glucose monitoring, foot inspection, and weight monitoring, and fewer problems with medication adherence (all P -0.03). Follow-up Hb A,, levels were 0.3% lower in the intervention group (P = 0.1), and about twice as many intervention patients had Hb A1c levels within the normal range (P = 0.04). Serum glucose levels were 41 mg/dL lower among intervention patients than usual care patients (P = 0.002). Intervention patients also reported better glycemic control (P = 0.005) and fewer diabetic symptoms (P <0.0001 ), including fewer symptoms of hyperglycemia and hypoglycemia.Automated calls with telephone nurse follow-up may be an effective strategy for improving self-care behavior and glycemic control, and for decreasing symptoms among vulnerable patients with diabetes.

    View details for Web of Science ID 000084710800005

    View details for PubMedID 11059437

  • Use of automated telephone disease management calls in an ethnically diverse sample of low-income patients with diabetes 15th Annual Meeting of the Association-for-Health-Services-Research Piette, J. D., McPhee, S. J., Weinberger, M., Mah, C. A., Kraemer, F. B. AMER DIABETES ASSOC. 1999: 1302–9


    We examined whether low-income patients with diabetes were able and willing to use automated telephone disease management (ATDM) calls to provide health status information that could improve the quality of their care.A total of 252 adults with diabetes, 30 of whom spoke Spanish as their primary language, were enrolled at the time of clinic visits in a Department of Veterans Affairs health care system (n = 132) or a county health care system (n = 120). Patients received ATDM calls for 12 months and responded to queries using their touch-tone telephones. We examined 1) whether patients completed ATDM assessments consistently over the year and used the calls to report their self-monitored blood glucose (SMBG) levels, 2) the characteristics of patients most likely to use the system frequently, 3) whether patients responded consistently within ATDM assessments, and 4) whether ATDM assessments differentiated among groups of patients with different clinical profiles at baseline.Half of all patients completed at least 77% of their attempted assessments, and one-fourth completed at least 91%. Half of all patients reported SMBG levels during at least 86% of their assessments. Patients completed assessments and reported glucose levels consistently over the year. Health status indicators were the most important determinants of assessment completion rates, while socioeconomic factors were more strongly associated with patients' likelihood of reporting SMBG data during assessments. Patients' responses within assessments were consistent, and the information they provided during their initial assessments identified groups with poor glycemic control and other health problems.Most low-income patients with diabetes can and will use ATDM calls as part of their care. The information they provide is reliable and has clinical significance. ATDM calls could improve the information base for diabetes management while relieving some of the pressures of delivering diabetes care under cost constraints.

    View details for Web of Science ID 000081562600013

    View details for PubMedID 10480775

  • A unique mechanism of desensitization to lipolysis mediated by beta(3)-adrenoceptor in rats with thermal injury AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Ikezu, T., Yasuhara, S., Granneman, J. G., Kraemer, F. B., Okamoto, T., Tompkins, R. G., Martyn, J. A. 1999; 277 (2): E316-E324


    Thermal injury causes a hypermetabolic state associated with increased levels of catabolic hormones, but the molecular bases for the metabolic abnormalities are poorly understood. We investigated the lipolytic responses after beta(3)-adrenoceptor (beta(3)-AR) agonists and evaluated the associated changes in beta-AR and its downstream signaling molecules in adipocytes isolated from rats with thermal injury. Maximal lipolytic responses to a specific beta(3)-AR agonist, BRL-37344, were significantly attenuated at post burn days (PBD) 3 and 7. Despite significant reduction of the cell surface beta(3)-AR number and its mRNA at PBD 3 and 7, BRL-37344 and forskolin-stimulated cAMP levels were not decreased. Glycerol production in response to dibutyryl cAMP, a direct stimulant of hormone-sensitive lipase (HSL) via protein kinase A (PKA), was significantly attenuated. Although immunoblot analysis indicated no differences in the expression and activity of PKA or in the expression of HSL, HSL activity showed significant reductions. Finally, beta(3)-AR-induced insulin secretion was indeed attenuated in vivo. These studies indicate that the beta(3)-AR system is desensitized after burns, both in the adipocytes and in beta(3)-AR-induced secretion of insulin. Furthermore, these data suggest a complex and unique mechanism underlying the altered signaling of lipolysis at the level of HSL in animals after burns.

    View details for Web of Science ID 000081809200017

    View details for PubMedID 10444428

  • Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shen, W. J., Sridhar, K., Bernlohr, D. A., Kraemer, F. B. 1999; 96 (10): 5528-5532


    Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue. By using the yeast two-hybrid system to examine the potential interaction of HSL with other cellular proteins, evidence is provided to demonstrate a direct interaction of HSL with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that binds fatty acids, retinoids, and other hydrophobic ligands. The interaction was demonstrated in vitro by the binding of ALBP to HSL translated in vitro, to HSL in extracts of HSL overexpressing Chinese hamster ovary (CHO) cells, and to HSL in extracts of rat adipose tissue. Finally, the presence of ALBP was documented in immune complexes from rat adipose tissue immunoprecipitated with anti-HSL antibodies. The HSL-ALBP interaction was mapped to an N-terminal 300-aa region of HSL that is distinct from the C-terminal catalytic domain. These results suggest that HSL-derived fatty acids are bound by ALBP to facilitate intracellular trafficking of hydrophobic lipids.

    View details for Web of Science ID 000080246500039

    View details for PubMedID 10318917

  • Heart-specific, tetracycline-controlled overexpression of hormone-sensitive lipase in vivo Suzuki, J., Shen, W. J., Patel, S., Kraemer, F. B. FEDERATION AMER SOC EXP BIOL. 1999: A1382–A1382
  • Induction of low density lipoprotein receptor (LDLR) transcription by oncostatin M is mediated by the extracellular signal-regulated kinase signaling pathway and the repeat 3 element of the LDLR promoter JOURNAL OF BIOLOGICAL CHEMISTRY Li, C., Kraemer, F. B., Ahlborn, T. E., Liu, J. W. 1999; 274 (10): 6747-6753


    Oncostatin M (OM) activates the transcription of the human low density lipoprotein receptor (LDLR) in HepG2 cells through a sterol-independent mechanism. Our previous studies showed that mutations within the repeat 3 sequence of the LDLR promoter significantly decreased OM activity on LDLR promoter luciferase reporter constructs that contain the sterol responsive element-1 (repeat 2) and Sp1 binding sites (repeats 1 and 3). In this study, we investigated the signal transduction pathways that are involved in OM-induced LDLR transcription. In HepG2 cells, OM induced a rapid increase in LDLR mRNA expression, with increases detected at 30 min and maximal induction at 1 h. This OM effect was not blocked by protein synthesis inhibitors, inhibitors of p38 kinase, phosphatidylinositol 3-kinase, or c-Jun N-terminal kinase, but OM activity was completely abolished by pretreating cells with inhibitors of the extracellular signal-regulated kinase (ERK) kinase (mitogen/ERK kinase (MEK)). To investigate whether the repeat 3 sequence of the LDLR promoter is the OM-responsive element that converts ERK activation at the promoter level, three luciferase reporters, pLDLR-TATA containing only the TATA-like elements of the promoter, pLDLR-R3 containing repeat 3 and the TATA-like elements, and pLDLR-234 containing repeats 1, 2, 3 and the TATA-like elements were constructed and transiently transfected into HepG2 cells. OM had no effect on the basal promoter construct pLDLR-TATA; however, including a single copy of repeat 3 sequence in the TATA vector (pLDLR-R3) resulted in a full OM response. The activity of OM on pLDLR-R3 was identical to that of pLDLR-234. Importantly, the ability of OM to increase luciferase activities in both pLDLR-R3- and pLDLR-234-transfected cells was blocked in a dose-dependent manner by inhibition of MEK. These results demonstrate that the mitogen-activated protein kinase MEK/ERK cascade is the essential signaling pathway by which OM activates LDLR gene transcription and provide the first evidence that the repeat 3 element is a new downstream target of ERK activation.

    View details for Web of Science ID 000078902800096

    View details for PubMedID 10037774

  • Mutational analysis of structural features of rat hormone-sensitive lipase BIOCHEMISTRY Shen, W. J., Patel, S., Natu, V., Kraemer, F. B. 1998; 37 (25): 8973-8979


    Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triacylglycerols and cholesteryl esters. HSL activity is regulated via phosphorylation-dephosphorylation, with cyclic AMP-dependent protein kinase increasing activity following phosphorylation of a single serine and Ca2+/calmodulin-dependent protein kinase II phosphorylating another serine at a basal site. The current studies used site-directed mutagenesis to show that Ser-563 of rat HSL is phosphorylated by cyclic AMP-dependent protein kinase and that Ser-565 is phosphorylated by Ca2+/calmodulin-dependent protein kinase II. Mutation of Ser-563-->Ala eliminated HSL hydrolytic activity against cholesteryl ester, triacylglycerol, and diacylglycerol substrates to the same extent as mutation of Ser-423-->Ala, the presumed catalytic site. Mutation of Ser-565-->Ala modestly decreased HSL activity. In contrast, mutation of Ser-563-->Asp preserved HSL hydrolytic activity and even increased activity 20% above the control wild-type enzyme. Molecular modeling of the catalytic pocket of HSL suggested the involvement of Val-710. Mutation of Val-710-->Ala resulted in an 85% loss of HSL hydrolytic activity. The results of these studies illustrate the importance of the presence of a hydroxyl group or negative charge at residue 563, either for proper conformation of rat HSL or for proper stabilization of substrate to allow maintenance of hydrolytic activity, as well as the importance of the involvement of additional amino acids in the catalytic pocket of the enzyme.

    View details for Web of Science ID 000074462100019

    View details for PubMedID 9636039

  • Insulin regulates lipoprotein lipase activity in rat adipose cells via wortmannin- and rapamycin-sensitive pathways METABOLISM-CLINICAL AND EXPERIMENTAL Kraemer, F. B., Takeda, D., Natu, V., Sztalryd, C. 1998; 47 (5): 555-559


    Lipoprotein lipase (LPL) hydrolyzes the triacylglycerol component of circulating lipoprotein particles, mediating the uptake of fatty acids into adipose tissue and muscle. Insulin is the principal factor responsible for regulating LPL activity in adipose tissue, yet the mechanisms whereby insulin controls LPL expression are unknown. The current studies used wortmannin, a specific inhibitor of phosphatidylinositol (PI) 3-kinase, and rapamycin, a specific inhibitor of activation of phosphoprotein 70 ribosomal protein S6 kinase (p70s6k), to explore some of the components of the insulin signaling pathway controlling LPL activity in adipose cells. Preincubation of isolated rat adipose cells with wortmannin completely abrogated the stimulation of LPL activity by insulin, while preincubation with rapamycin caused approximately a 60% inhibition of insulin-stimulated LPL activity. Thus, the current studies show that the regulation of adipose tissue LPL by insulin is mediated via a wortmannin-sensitive pathway, most likely PI 3-kinase, and that a rapamycin-sensitive pathway, most likely p705s6k, constitutes an important downstream component in the insulin signaling pathway through which LPL is regulated.

    View details for Web of Science ID 000073506000011

    View details for PubMedID 9591746

  • Dietary regulation of the very low density lipoprotein receptor in mouse heart and fat HORMONE AND METABOLIC RESEARCH Kwok, S., SINGHBIST, A., Natu, V., Kraemer, F. B. 1997; 29 (10): 524-529


    The very low density lipoprotein (VLDL) receptor is a member of the LDL receptor family. As opposed to the LDL receptor, the VLDL receptor is expressed primarily in muscle and adipose tissue. Although the VLDL receptor is capable of binding lipoproteins, its functional role is still unclear. Previous studies found that VLDL receptor expression is unaffected by fasting in the rat. The current studies examined whether VLDL receptor expression is altered with fasting in the mouse. Balb/c mice were fasted for periods up to 48 hours, killed, hearts and epididymal fat obtained, and total membranes prepared. To detect the VLDL receptor a portion of the rat VLDL receptor was expressed as a bacterial fusion protein, purified and used to immunize rabbits. The antibodies raised specifically recognized intact VLDL receptor. When cardiac membranes were immunoblotted, VLDL receptor expression increased progressively with fasting, doubling at 36 hours. In contrast, VLDL receptor expression decreased progressively with fasting in membranes from epididymal fat, being reduced 70% by 48 hours. Thus, VLDL receptor expression appears to be regulated in mouse heart and fat by nutritional perturbation, supporting a potential role for the VLDL receptor in the delivery of triglycerides/fatty acids as fuel.

    View details for Web of Science ID A1997YH13700011

    View details for PubMedID 9405982

  • Overexpression of hormone-sensitive lipase in Chinese hamster ovary cells leads to abnormalities in cholesterol homeostasis JOURNAL OF LIPID RESEARCH Kraemer, F. B., Fong, L., Patel, S., Natu, V., KOMAROMY, M. C. 1997; 38 (8): 1553-1561


    Hormone-sensitive lipase (HSL) is an intracellular enzyme that functions as both a neutral triglyceride and cholesteryl ester hydrolase. In order to explore the effects of HSL on cholesterol homeostasis, Chinese hamster ovary (CHO) cells were transfected with rat HSL and several different stable cell lines that overexpress HSL mRNA, HSL protein, and HSL activity approximately 600-fold were isolated. Cells transfected with HSL contained less cholesteryl esters and unesterified cholesterol than control cells. HSL transfectants expressed 20-60% fewer LDL receptors than control cells when grown in lipid-depleted media or in the presence of mevinolin, as assessed by binding and degradation of LDL and immunoblotting of LDL receptors. In contrast, the rate of cholesterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were increased 3- to 14-fold in HSL transfectants grown in sterol replete media. The rate of cholesterol synthesis and the activity of HMG-CoA reductase increased when cells were grown in lipid-depleted media, and remained markedly elevated compared to control cells. These results show that the regulation of LDL receptor expression and cholesterol synthesis can be dissociated through the actions of HSL and suggest multiple control mechanisms for sterol-responsive genes.

    View details for Web of Science ID A1997XU91100005

    View details for PubMedID 9300777

  • Retinyl ester hydrolysis and retinol efflux from BFC-1 beta adipocytes JOURNAL OF BIOLOGICAL CHEMISTRY Wei, S. H., Lai, K., Patel, S., PIANTEDOSI, R., Shen, H., Colantuoni, V., Kraemer, F. B., Blaner, W. S. 1997; 272 (22): 14159-14165


    Adipose tissue is an important storage depot for retinol, but there are no data regarding retinol mobilization from adipose stores. To address this, dibutyryl cAMP was provided to murine BFC-1beta adipocytes and its effects on retinol efflux assessed. High performance liquid chromatography analysis of retinol and retinyl esters in adipocytes and media indicated that cAMP stimulated, in a time- and dose-dependent manner, retinol accumulation in the culture media and decreased cellular retinyl ester concentrations. Study of adipocyte retinol-binding protein synthesis and secretion indicated that cAMP-stimulated retinol efflux into the media did not result from increased retinol-retinol-binding protein secretion but was dependent on the presence of fetal bovine serum in the culture media. Since our data suggested that retinyl esters can be hydrolyzed by a cAMP-dependent enzyme like hormone-sensitive lipase (HSL), in separate studies, we purified a HSL-containing fraction from BFC-1beta adipocytes and demonstrated that it catalyzed retinyl palmitate hydrolysis. Homogenates of Chinese hamster ovary cells overexpressing HSL catalyzed retinyl palmitate hydrolysis in a time-, protein-, and substrate-dependent manner, with an apparent Km for retinyl palmitate of 161 microM, whereas homogenates from control Chinese hamster ovary cells did not.

    View details for Web of Science ID A1997XB49200032

    View details for PubMedID 9162045

  • Down-regulation of hormone-sensitive lipase in sterol ester-laden J774.2 macrophages BIOCHEMICAL JOURNAL JEPSON, C. A., Harrison, J. A., Kraemer, F. B., Yeaman, S. J. 1996; 318: 173-177


    The development of atherosclerotic plaques in arteries is a key step in atherogenesis, with cholesterol ester accumulation in macrophage-derived foam cells being recognized as a major pathogenic event in this process. In this study, the mouse macrophage cell line J774.2 was induced to accumulate intracellular sterol esters by incubation with 25-hydroxycholesterol in the presence of oleic acid. The accumulation of sterol esters in these cells was found to be accompanied by a marked decrease in the activity of the enzyme responsible for their hydrolysis, namely hormone-sensitive lipase (HSL); Western blotting studies revealed a corresponding decrease in the levels of the HSL polypeptide. Similar findings were obtained after incubation with oxidized low-density lipoprotein or very-low-density lipoprotein. These findings suggest that down-regulation of the expression of HSL is important in cholesterol ester accumulation in macrophages.

    View details for Web of Science ID A1996VE10100025

    View details for PubMedID 8761468

  • Alterations of lipolysis and lipoprotein lipase in chronically nicotine-treated rats AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Sztalryd, C., Hamilton, J., Horwitz, B. A., Johnson, P., Kraemer, F. B. 1996; 270 (2): E215-E223


    These studies examined the cellular mechanisms for lower adiposity seen with nicotine ingestion. Rats were infused with nicotine or saline for 1 wk and adipocytes isolated from epididymal fat pads. Nicotine-infused rats gained 37% less weight and had 21% smaller fat pads. Basal lipolysis was 78% higher, whereas the maximal lipolytic response to isoproterenol was blunted in adipocytes from nicotine-infused rats. The antilipolytic actions of adenosine and the levels of serum catecholamines were unaffected by nicotine. The nicotine-induced alteration in lipolysis was not associated with any changes in hormone-sensitive lipase. Nicotine caused a 30% decrease in lipoprotein lipase (LPL) activity, without any changes in LPL mass or mRNA levels, in epididymal fat in the fed state. In contrast, LPL activity, mass, and mRNA levels in heart were increased by nicotine whether animals were fed or fasted. These studies provide evidence for multiple mechanistic events underlying nicotine-induced alterations in weight and suggest that nicotine diverts fat storage away from adipose tissue and toward utilization by muscle.

    View details for Web of Science ID A1996TW10000003

    View details for PubMedID 8779941

  • Effects of streptozotocin-induced diabetes on low density lipoprotein receptor expression in rat adipose tissue JOURNAL OF LIPID RESEARCH Swami, S., Sztalryd, C., Kraemer, F. B. 1996; 37 (2): 229-236


    Low density lipoprotein (LDL) receptors are found in most cells, including adipose cells. LDL receptors are primarily regulated by cellular cholesterol content. Insulin and insulin deficiency have been reported to have varying effects on LDL receptors in various tissues. The present study was undertaken to assess the in vivo effects of streptozotocin-induced diabetes on LDL receptor expression and cholesterol content in adipose tissue and liver, Diabetes was induced by a single dose of streptozotocin. After 3 days, some animals were treated with insulin, and all animals were killed 10 days after induction of diabetes. Compared to control rats, 10 days of diabetes caused a decrease in adipose cell size and cellular unesterified cholesterol and cholesteryl esters, and insulin treatment returned these towards normal. No changes were observed in hepatic lipid content with diabetes or insulin treatment. Diabetes was associated with an approximately 50% reduction in immunoreactive LDL receptors in adipose cells (P < 0.01) that was returned to normal with insulin treatment. The levels of LDL receptor mRNA decreased approximately 80% (P < 0.001) in adipose cells isolated from streptozotocin-induced diabetic rats and returned to normal with insulin treatment. Hepatic LDL receptors and mRNA levels were unaffected by diabetes or insulin treatment. In conclusion, diabetes decreased LDL receptor expression in adipose cells while total cellular cholesterol content also declined.

    View details for Web of Science ID A1996TZ91000002

    View details for PubMedID 9026522

  • Isoproterenol decreases LDL receptor expression in rat adipose cells: Activation of cyclic AMP-dependent proteolysis JOURNAL OF LIPID RESEARCH Kraemer, F. B., Natu, V., SINGHBIST, A., Patel, S., KOMAROMY, M. C., Medicherla, S., Azhar, S., Sztalryd, C. 1996; 37 (2): 237-249


    The low density lipoprotein (LDL) receptor is part of a family of proteins that mediate the uptake of lipoproteins into cells. In this paper we have demonstrated the over-expression in E. coli of a rat LDL receptor fusion protein that contains the region of the receptor sharing homology with the EGF precursor. The fusion protein was utilized to immunize rabbits and successfully generate antibodies that recognize the intact LDL receptor. These anti-LDL receptor/fusion protein antibodies were used to examine the effects of cyclic AMP on the expression of LDL receptors in isolated rat adipocytes. Incubation of adipocytes with isoproterenol caused a dose-dependent diminution in intact LDL receptors in the plasma membrane with the concomitant appearance of smaller immunoreactive proteins. Pulse-chase experiments demonstrated that isoproterenol rapidly shortened the initial half-life of intact, immunoprecipitable LDL receptors in the plasma membrane. The effects of isoproterenol on LDL receptor expression were mimicked by forskolin, by an analog of cyclic AMP, and by ACTH. In contrast, incubation with propranolol blocked the effects of isoproterenol on LDL receptor expression. While antioxidants and several different protease inhibitors had no effects, N-acetyl-leucine-leucine-methionine (ALLM) was able to prevent the isoproterenol-induced effects on LDL receptors. Thus, it appears that agents acting via cyclic AMP cause a rapid decrease in LDL receptors in the plasma membranes of isolated adipose cells due to the apparent stimulation of an ALLM-sensitive protease that degrades the LDL receptor. These results suggest a novel mechanism for the posttranscriptional regulation of LDL receptor expression in adipocytes.

    View details for Web of Science ID A1996TZ91000003

    View details for PubMedID 9026523

  • Aberrations in normal systemic lipid metabolism in ovarian cancer patients GYNECOLOGIC ONCOLOGY GERCELTAYLOR, C., Doering, D. L., Kraemer, F. B., Taylor, D. D. 1996; 60 (1): 35-41


    Numerous investigations have demonstrated altered systemic lipid metabolism in cancer patients, as well as aberrant lipid utilization by tumor cells. The most common measure of altered systemic lipid metabolism in these individuals has been hyperlipidemia. Although cachexia is not generally considered to be associated with gynecologic cancers, this study demonstrates the presence of lipolysis-promoting activity, detectable in sera and ascites of ovarian cancer patients and indices of altered systemic lipid metabolism. Elevated lipolysis promoting activity was detectable in the sera of 7/9 patients and in the ascites of 5/5 patients. Since previous studies have indicated that cancer patients exhibit a 2.5-fold enhancement in hormone-sensitive lipase (HSL) versus normal controls, as a potential mechanism for elevated lipolysis, the ability of ascites-derived factors to induce HSl was examined. The addition of three of four ascites fluids increased the level of HSL in normal adipocytes. All of the patients' samples exhibited elevated lipid levels versus normal peritoneal fluid. Isolation and analysis of lipids from three ovarian cancer patients revealed four consistent altered lipid parameters compared to normal peritoneal fluid: elevated monoglycerides, diglycerides, and free fatty acids and decreased triacylglycerides. While "classical" cachexia is not a common feature of ovarian cancer, the presence of circulating lipolysis-promoting activity and altered lipid metabolism, generally observed in cachectic individuals, can be demonstrated in these ovarian cancer patients. Based on recent evidence indicating a role of lipids in carcinogenic initiation or promotion, the presence of tumor-derived lipolysis-promoting factor and lipid metabolism alterations may provide a mechanism for the epidemiologically observed association between lipids and certain cancers, including ovarian cancer.

    View details for Web of Science ID A1996TP79100008

    View details for PubMedID 8557225

  • Mechanism of anti-lipolytic action of acipimox in isolated rat adipocytes DIABETOLOGIA CHRISTIE, A. W., McCormick, D. K., Emmison, N., Kraemer, F. B., Alberti, K. G., Yeaman, S. J. 1996; 39 (1): 45-53


    Acipimox is commonly used to treat hypertriglyceridaemia in non-insulin-dependent diabetic patients, but its precise mechanism of action has yet to be elucidated. We examined the in vitro effects of acipimox on the lipolytic regulatory cascade in epididymal adipocytes isolated from Wistar rats. Acipimox inhibited the lipolytic rate stimulated by adenosine deaminase (1 U/ml) in a concentration-dependent manner, reaching a near-basal value at 10 mumol/l acipimox. Lipolysis activated by sub-maximal levels of isoproterenol in combination with adenosine deaminase (20 mU/ml) was significantly (p < 0.05) decreased by 100 mumol/l acipimox, whereas, in the absence of adenosine deaminase, 100 mumol/l acipimox showed no significant (p > 0.05) inhibition. These findings suggested that the anti-lipolytic mechanism regulated by adenosine may also be regulated by acipimox. Acipimox diminished the intracellular cyclic AMP level produced by 25 nmol/l isoproterenol in the presence of adenosine deaminase (20 mU/ml) in a concentration-dependent manner. At the same level of stimulation, acipimox inhibited the cyclic AMP-dependent protein kinase activity ratio and lipolytic rate over the same concentration range, with significant (p < 0.05) reductions occurring at and above, 0.5 mumol/l and 10 mumol/l acipimox, respectively. Western blotting showed that upon lipolytic stimulation (1 U/ml adenosine deaminase; 100 nmol/l isoproterenol) a threefold increase in the lipolytic rate was accompanied by a significant (p < 0.05) rise in hormone-sensitive lipase associated with the lipid fraction. Acipimox (1 mmol/l) and insulin (1 nmol/l) re-distributed hormone-sensitive lipase back to the cytosol, with a corresponding significant (p < 0.05) loss from the fat cake fraction of adipocyte homogenates. In conclusion, the anti-lipolytic action of acipimox is mediated through suppression of intracellular cyclic AMP levels, with the subsequent decrease in cyclic AMP-dependent protein kinase activity, leading to the reduced association of hormone-sensitive lipase with triacylglycerol substrate in the lipid droplet of adipocytes.

    View details for Web of Science ID A1996TR70900007

    View details for PubMedID 8720602



    Insulin deficiency as seen in insulin-dependent diabetes mellitus causes an activation of lipolysis in adipose tissue that results in hydrolysis of stored triglycerides and release of large amounts of fatty acids into the plasma, leading to diabetic ketoacidosis (DKA). Hormone-sensitive lipase (HSL) is thought to be the rate-limiting enzyme of lipolysis in adipose tissue. This study was designed to examine the effects of insulin deficiency on the regulation of HSL in isolated adipocytes. Insulin deficiency was induced by a single dose of streptozotocin. After 8 days, some animals were treated with insulin, and all animals were killed 10 days after induction of insulin deficiency. Compared with levels in control rats, 10 days of insulin deficiency increased HSL activity twofold (P < .05), as assayed for neutral cholesterol esterase activity, and insulin treatment returned HSL activity to normal. HSL protein was increased twofold (P < .05) in streptozotocin-induced diabetic rats, as estimated by immunoblotting, but remained elevated after insulin treatment. Levels of HSL mRNA assessed by Northern blot analysis also increased twofold (P < .01) in adipose cells isolated from streptozotocin-induced diabetic rats, and remained elevated after insulin treatment. In conclusion, our studies suggest that 10 days of insulin deficiency increases HSL expression via pretranslational mechanisms and short-term insulin treatment returns HSL activity to normal via posttranslational mechanisms in adipose tissue.

    View details for Web of Science ID A1995TD89100004

    View details for PubMedID 7476323



    Antibodies generated against specific proteins are useful tools for studying the physiology and cell biology of the protein of interest. Although antibodies have been successfully generated against lipoprotein lipase (LPL) and used to elucidate many aspects of its biology, there have been problems with the specificity, affinity and availability of these antibodies. To circumvent these problems, we have expressed a portion of human LPL as a bacterial fusion protein. The human LPL bacterial fusion protein was utilized to generate polyclonal antibodies in rabbits that recognize intact human, rat and bovine LPL. Using these antibodies, it was possible to demonstrate a direct correlation between LPL mass and LPL activity from different samples of human post-heparin plasma. In addition, these antibodies were used to develop an ELISA for the measurement of LPL in tissue or plasma. This is a useful means for obtaining polyclonal antibodies to LPL in sufficient quantity and without contaminating mammalian proteins.

    View details for Web of Science ID A1995RW88500009

    View details for PubMedID 7475911



    Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triglycerides within adipocytes and is thought to be the rate limiting enzyme in lipolysis; however, direct evidence to prove this concept has been lacking. The present study was designed to establish the function of HSL in adipocytes. A 2360-bp fragment containing the entire HSL coding region was cloned into the vector pCEP4 and was used to transfect the 3T3-F442A adipogenic cell line. Nondifferentiated, transfected cells were screened for HSL overexpression by indirect immunofluorescence microscopy and confirmed by immunoblotting cell extracts with anti-HSL/fusion protein antibodies and by Northern blots for HSL mRNA. Stable transfectants overexpressing HSL were obtained and cloned. Compared with undifferentiated 3T3-F442A cells transfected with pCEP4 not containing the insert (vector alone) where HSL expression was very low, undifferentiated HSL transfectants had up to a 100-fold increase in HSL activity. Likewise, immunoreactive HSL protein and HSL mRNA levels were increased up to 100-fold in HSL transfectants. When confluent cells were allowed to differentiate by exposure to insulin, HSL expression increased in vector alone transfected cells, but remained below that observed in HSL transfectants. A similar degree of differentiation was seen in both vector alone and HSL transfectants when based on the induction of lipoprotein lipase. Cellular triglyceride content increased dramatically in the vector alone transfected cells while triglyceride content was markedly reduced in the HSL transfectants. The expression of late markers of adipocyte differentiation, such as aP2 and GPDH, was diminished and appeared to vary with the degree to which HSL was overexpressed and the cellular triglyceride content was reduced. Thus, the overexpression of HSL in 3T3-F442A cells prevents differentiated adipocytes from taking on the appearance of fat cells, i.e., accumulating triglyceride. Furthermore, the overexpression of HSL directly or indirectly attenuates the expression of several genes that appear during late adipocyte differentiation.

    View details for Web of Science ID A1995RB21200030

    View details for PubMedID 7769105



    A partially-purified diacylglycerol (DG) lipase from bovine aorta has been characterized with respect to the effects of lipid metabolites and two lipase inhibitors, phenylboronic acid and tetrahydrolipstatin (THL). DG lipase activity was determined by the hydrolysis of the sn-1 position of 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol. The products of the lipase reaction, 2-monoacylglycerol (2-monoolein) and non-esterified fatty acids (oleate, archidonate) produced a concentration-dependent (20-200 microM) inhibition of DG lipase activity. Oleoyl-CoA and dioleoylphosphatidic acid also inhibited aortic DG lipase activity, but lysophosphatidylcholine had little or no effect. The inhibition of aortic DG lipase by phenylboronic acid was competitive, with a Ki of approx. 4 mM. THL was a very potent inhibitor of aortic DG lipase; the concentration required for inhibition to 50% of control was 2-6 nM. THL inhibition was reduced when the concentration of substrate in the assay was increased. Attempts to identify the aortic DG lipase by covalent-labelling with [14C]THL were unsuccessful. Immunoblotting experiments revealed that hormone-sensitive triacylglycerol lipase (HSL) could not be detected in bovine aorta.

    View details for Web of Science ID A1995QG27700009

    View details for PubMedID 7857971

  • Knowledge-based temporal abstraction in diabetes therapy. Medinfo. MEDINFO Shahar, Y., Das, A. K., Tu, S. W., Kraemer, F. B., Basso, L. V., Musen, M. A. 1995; 8: 852-856


    We suggest a general framework for solving the task of creating abstract, interval-based concepts from time-stamped clinical data. We refer to this problem-solving framework as the knowledge-based temporal-abstraction (KBTA) method. The KBTA method emphasizes explicit representation, acquisition, maintenance, reuse, and the sharing of knowledge required for abstraction of time-oriented clinical data. We describe the subtasks into which the KBTA method decomposes its task, the problem-solving mechanisms that solve these subtasks, and the knowledge necessary for instantiating these mechanisms in a particular clinical domain. We have implemented the KBTA method in the RESUME system and have applied it to the task of monitoring the care of insulin-dependent diabetics.

    View details for PubMedID 8591345

  • LOW-DENSITY-LIPOPROTEIN RECEPTORS IN RAT ADIPOSE-CELLS - SUBCELLULAR-LOCALIZATION AND REGULATION BY INSULIN JOURNAL OF LIPID RESEARCH Kraemer, F. B., SATHER, S. A., Park, B., Sztalryd, C., Natu, V., May, K., Nishimura, H., Simpson, I., Cooper, A. D., Cushman, S. W. 1994; 35 (10): 1760-1772


    The distribution of LDL receptors within subcellular compartments of isolated rat adipose cells and the effects of insulin on their expression have been assessed. By immunoblotting with specific anti-rat LDL receptor antibodies, LDL receptors were 2.3- and 4.5-fold enriched in endoplasmic reticulum-rich high-density microsomes (HDM) and Golgi complex-rich low-density microsomes (LDM), respectively, compared to plasma membranes (PM). This distribution was similar in cultured cells in which total receptors were increased 2.5-fold compared to freshly isolated cells. After correction for enzyme recoveries, LDL receptors were distributed approximately 4% in HDM, approximately 73% in LDM, and approximately 23% in PM. Insulin decreased total LDL receptors in adipose cells approximately 44%, with a 48% and 49% decrease in HDM and LDM, respectively, without any changes in PM. In contrast, insulin caused an increase of glucose transporters in PM while also decreasing glucose transporters in LDM. When adipose cells were depleted of potassium to inhibit receptor-mediated endocytosis, insulin again caused a decrease of LDL receptors in LDM but now increased LDL receptors in PM. Insulin increased the rate of LDL receptor synthesis approximately 24%, but decreased their half life approximately 40%. Thus, in isolated adipose cells the majority of LDL receptors appear to be located in an intracellular compartment that co-sediments with the Golgi complex rather than located in the PM. The LDL receptors localized in intracellular compartments seem to be functionally regulated as insulin acutely diminishes the number of receptors by apparently accelerating their rate of degradation through, as yet, incompletely determined mechanisms.

    View details for Web of Science ID A1994PM25300004

    View details for PubMedID 7852853



    Lipoprotein lipase (LPL) is differentially expressed in fat and heart under a variety of physiological conditions, particularly during development. LPL activity, protein and mRNA levels have been shown to rise dramatically during the first three weeks of life. The mechanism responsible for these changes in LPL in the heart during development are not fully understood. In order to address the changes in LPL in the heart during development, we measured steady state levels of LPL mRNA and the rate of LPL transcription during development. Northern blot analysis of LPL mRNA in the heart of rats from 5-120 days of age showed no changes in transcript size; however, steady state levels of LPL mRNA increased approximately 10-fold by day 20 and remained elevated thereafter. The rate of LPL transcription increased dramatically between day 5-20 and remained elevated thereafter. Thus, as opposed to other settings where posttranscriptional and posttranslational mechanisms can be important, the increased expression of LPL in rat heart that occurs during development appears to be largely due to the transcriptional activation of the gene.

    View details for Web of Science ID A1994NZ24000028

    View details for PubMedID 8048956



    Regional differences in lipolytic activity of isolated fat cells have been observed in rats, with internal fat depots displaying greater rates of lipolysis than subcutaneous fat. These differences in lipolysis have been attributed to a variety of mechanisms, including regional differences in adrenergic receptors, in adenosine sensitivity, and in regional blood flow. In the present study, we have explored whether differences in hormone-sensitive lipase (HSL), the rate-limiting enzyme in lipolysis, might contribute to regional differences in lipolysis in the rat. Adipocytes were isolated from epididymal, retroperitoneal, and dorsal-subcutaneous fat depots, and HSL activity, HSL immunoreactive protein, the rate of HSL synthesis, and the steady-state levels of HSL mRNA were assessed. HSL activity was similar in epididymal and retroperitoneal adipocytes, but was twofold to 2.5-fold greater in these cells than in subcutaneous adipocytes (P < .01). The amount of immunoreactive HSL protein was also similar in epididymal and retroperitoneal adipocytes, but was twofold greater in these cells than in subcutaneous adipocytes (P < .001). The rate of incorporation of 35S-methionine into immunoprecipitable HSL was approximately 2.5-fold greater in epididymal than in subcutaneous adipocytes (P < .01). Finally, HSL mRNA levels were similar in epididymal and retroperitoneal fat depots, and were approximately 1.4-fold greater in these cells than in subcutaneous adipocytes (P < .001). These results demonstrate that site-related differences in the rate of basal lipolysis among various fat depots in the rat are in part due to variations in the expression of HSL.

    View details for Web of Science ID A1994MY29300020

    View details for PubMedID 8121309



    Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. The activity of HSL is thought to be primarily regulated by phosphorylation-dephosphorylation reactions. Although FFA levels are elevated during fasting, it has been difficult to demonstrate an increase in HSL activity with fasting. The current studies were undertaken to explore directly the regulation of HSL expression in adipose tissue in the rat during fasting. Rats were fasted for periods up to 5 days and HSL activity, HSL immunoreactive protein, and HSL mRNA levels were measured both in intact epididymal adipose tissue and in isolated adipose cells. Fasting caused a progressive decline in total body weight and the weight of epididymal fat pads, whereas adipose cell size decreased approximately 50% after 2 days of fasting. Serum FFA levels approximately doubled within 1 day of fasting and remained elevated thereafter. Basal lipolysis, measured as glycerol release, did not increase until 2 days of fasting. HSL activity remained relatively unchanged until 3 days of fasting when it was increased twofold after 3-5 days of fasting. Likewise, HSL immunoreactive protein and HSL mRNA levels increased twofold after 3-5 days of fasting. Thus HSL activity appears to be regulated by pretranslational mechanisms during prolonged fasting. However, increases in FFA flux during short-term fasting appear to involve either post-translational control of HSL or the regulation of other enzymes.

    View details for Web of Science ID A1994NA80100035

    View details for PubMedID 8141275



    Adipose tissue metabolism is exquisitely sensitive to caloric intake. With increasing adiposity more triglyceride and cholesterol are stored within increasingly large adipocytes, whereas less triglyceride and cholesterol are stored as the size of the fat cell decreases. A portion of the uptake of cholesterol by adipocytes is mediated by low-density lipoprotein (LDL) receptors. The present studies addressed whether LDL receptors are differentially regulated in adipose tissue and the liver during fasting in the rat. Two days of fasting caused a reduction in body weight with an approximately 40% decrease in the epididymal fat depot and fat cell size. No changes in serum cholesterol were noted, but serum triglycerides fell approximately 55% with fasting. LDL receptors detected by immunoblotting decreased progressively with fasting to levels that were 95% below controls in adipocytes isolated from epididymal fat pads by 2-3 days. In contrast, hepatic LDL receptor expression was unaltered by fasting. After 2 days of fasting, the rate of synthesis of LDL receptors in isolated adipose cells was decreased approximately 35%, whereas levels of LDL receptor mRNA were diminished approximately 55%. It is concluded that the expression of LDL receptors in rat adipocytes is markedly downregulated during fasting through transcriptional and posttranscriptional mechanisms. Furthermore, LDL receptor expression is differentially regulated in adipose tissue and liver during fasting in the rat.

    View details for Web of Science ID A1994NA59100038

    View details for PubMedID 8304441

  • KNOWLEDGE-BASED TEMPORAL ABSTRACTION FOR DIABETIC MONITORING 18th Annual Symposium on Computer Applications in Medical Care - Transforming Information, Changing Health Care Shahar, Y., Das, A. K., Tu, S. W., Kraemer, F. B., Musen, M. A. BMJ PUBLISHING GROUP. 1994: 697–701


    We have developed a general method that solves the task of creating abstract, interval-based concepts from time-stamped clinical data. We refer to this method as knowledge-based temporal-abstraction (KBTA). In this paper, we focus on the knowledge representation, acquisition, maintenance, reuse and sharing aspects of the KBTA method. We describe five problem-solving mechanisms that solve the five subtasks into which the KBTA method decomposes its task, and four types of knowledge necessary for instantiating these mechanisms in a particular domain. We present an example of instantiating the KBTA method in the clinical area of monitoring insulin-dependent-diabetes patients.

    View details for Web of Science ID A1994QF21600123

    View details for PubMedID 7950015



    It has been hypothesized that the renin-angiotensin system plays a pathophysiologic role in the renal hemodynamic abnormalities that occur in diabetes mellitus and thereby contributes to the development of diabetic nephropathy. In this study, the tissue-specific regulation of renin and angiotensinogen mRNA levels and the abundance of glomerular angiotensin II receptors were examined in male Sprague-Dawley rats (160 to 240 g) made diabetic with streptozotocin. One subgroup of diabetic rats remained untreated, whereas a second diabetic subgroup received twice-daily doses of insulin to ameliorate hyperglycemia. Animals were euthanized 2 wk after the induction of diabetes. Mean plasma glucose levels at the time of euthanasia were significantly elevated in the untreated diabetic animals when compared with controls or insulin-treated diabetic rats. Weight gain was similar in control and insulin-treated diabetic rats, whereas the untreated diabetic rats gained significantly less. Plasma renin concentration did not differ between control, diabetic, and insulin-treated diabetic groups. In the kidney, no significant differences were found in either angiotensinogen or renin mRNA levels in diabetic animals, whereas glomerular angiotensin II receptors were significantly less abundant in untreated rats as compared with control or insulin-treated diabetic subgroups. Angiotensinogen mRNA levels were significantly lower in the livers and adrenals of diabetic rats in comparison to those in controls and insulin-treated diabetic rats, whereas angiotensinogen mRNA levels in the brain remained unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1993MN38700010

    View details for PubMedID 8130360



    Hormone-sensitive lipase (HSL) is an intracellular neutral lipase found in a variety of tissues, primarily in adipose and steroidogenic tissues, that hydrolyzes triglycerides and cholesteryl esters. In the rat testis steady-state levels of HSL mRNA increase dramatically during sexual maturation. In addition, HSL-like immunoreactive proteins of 84, -89, and -102 kD have been observed in sexually immature rats with additional -113 and -127 kD immunoreactive proteins expressed in mature animals. In the present studies the ability of human chorionic gonadotropin (hCG) to regulate the expression of HSL and HSL-like immunoreactive proteins in rat testes has been examined. Treatment of sexually immature rats with daily injections of hCG caused a gradual increase in HSL activity that reached an 80% rise (P < 0.01) after 5 days. This was paralleled by a 3-fold increase (P < 0.01) in the 84 kD protein representing the active HSL enzyme. The -89 kD immunoreactive protein was also increased -5-fold (P < 0.01) in parallel to the 84 kD protein and HSL activity. The -102 kD immunoreactive protein was increased by hCG treatment (P < 0.01); however, its expression did not follow changes in HSL activity or in the 84 and -89 kD immunoreactive proteins, peaking within 12 h and declining thereafter. The -113 and -127 kD immunoreactive proteins did not appear during the 5 days of hCG treatment. Steady-state levels of HSL mRNA increased 60-100% (P < 0.02) in parallel to the changes in HSL activity and in the 84 and -89 kD immunoreactive proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1993KX50100009

    View details for PubMedID 8496666



    Hormone-sensitive lipase (HSL) is an intracellular neutral lipase found in a variety of tissues, but primarily in adipose and steroidogenic tissues, that hydrolyzes triglycerides and cholesteryl esters. In the present studies, a portion of rat HSL cDNA was subcloned into a pET expression system and the resulting recombinant fusion protein was over-expressed in E. coli. The approximately 26 kD HSL/fusion protein was used to generate polyclonal antibodies in rabbits that recognize intact HSL (84 kD) in rat adipose tissue, ovary, adrenal, testis, heart, and lung, as well as in human adipose tissue. In addition, there was an approximately 89 kD protein observed in all rat tissues expressing the 84 kD protein. Unique to testes, there was an immunoreactive protein of approximately 102 kD in sexually immature rats, and additional immunoreactive proteins of approximately 113 kD and approximately 127 kD in sexually mature rats. The anti-HSL/fusion protein antibodies could remove approximately 60-80% of total neutral cholesterol esterase activity from extracts of rat adipose tissue and immunoprecipitated a single 84 kD protein after labeling of adipocytes with [32P]orthophosphate. The incorporation of 32P into the 84 kD HSL protein was stimulated 4-fold by incubation of adipocytes with isoproterenol. The half life of [35S]methionine-labeled HSL was approximately 4 h in normal rat adipocytes. The production of an HSL/fusion protein and generation of antibodies that recognize native HSL should be valuable tools in exploring the mechanisms regulating the expression of HSL activity and the function and localization of its immunoreactive proteins.

    View details for Web of Science ID A1993KX50100015

    View details for PubMedID 8496671

  • REGULATION OF LIPOPROTEIN-LIPASE IN THE DIABETIC RAT JOURNAL OF CLINICAL INVESTIGATION TAVANGAR, K., Murata, Y., Pedersen, M. E., Goers, J. F., Hoffman, A. R., Kraemer, F. B. 1992; 90 (5): 1672-1678


    Diabetes mellitus is associated with a reduction of lipoprotein lipase (LPL) activity and development of hypertriglyceridemia. In the current experiments the mechanisms involved in the regulation of LPL have been examined in control rats, streptozocin-induced diabetic rats, and diabetic rats treated chronically or with a single injection of insulin. Diabetes decreased adipose tissue LPL activity partially by decreasing immunoreactive LPL protein and the steady-state levels of LPL mRNA, but primarily by reducing the catalytic activity of LPL. Both chronic and acute insulin increased adipose tissue LPL activity by correcting the defect in the catalytic activity of LPL and increasing immunoreactive LPL protein; however, only chronic insulin restored LPL mRNA levels to normal. In the heart, LPL activity tended to be elevated with diabetes in parallel to an increase in immunoreactive LPL protein even though levels of LPL mRNA declined. Both chronic and acute insulin normalized LPL activity and immunoreactive LPL protein, while only chronic insulin corrected the levels of LPL mRNA. No changes in the catalytic activity of LPL in heart were detected among the groups. Thus, diabetes and insulin treatment regulate LPL expression pretranslationally, translationally, and post-translationally, with tissue-specific differences apparent in the mechanisms involved.

    View details for Web of Science ID A1992JZ47500006

    View details for PubMedID 1430198



    Because the accumulation of lipid in macrophages is a characteristic feature of atherosclerosis, the mechanisms by which this lipid accumulation occurs have been intensively studied. This paper reviews the receptor- and non-receptor-mediated pathways that promote lipid accumulation in macrophages. Particular emphasis is placed on the contributions of two secretory products of macrophages, lipoprotein lipase and apolipoprotein E, to both receptor- and non-receptor-mediated uptake of triglyceride-rich lipoproteins by macrophages. The hormonal, lipid, and immunological factors that regulate the secretion of LpL and apoE by macrophages are discussed, as are how changes in the secretion of apoE and LpL that can modulate the uptake of triglyceride-rich lipoproteins by macrophages might influence the atherosclerotic process in people with diabetes.

    View details for Web of Science ID A1992JQ02000015

    View details for PubMedID 1526341

  • DEVELOPMENTAL REGULATION OF LIPOPROTEIN-LIPASE IN RATS AMERICAN JOURNAL OF PHYSIOLOGY TAVANGAR, K., Murata, Y., Patel, S., Kalinyak, J. E., Pedersen, M. E., Goers, J. F., Hoffman, A. R., Kraemer, F. B. 1992; 262 (3): E330-E337


    To evaluate changes in lipoprotein lipase (LPL) expression during development, levels of LPL mRNA, protein, and enzyme activity were measured in heart, epididymal fat, kidney, and brain of rats, from late gestation through 24 mo. LPL mRNA, protein, and enzyme activity were low in fetal and neonatal hearts. LPL mRNA increased 11-fold by 60 days and remained at this level thereafter; LPL protein and enzyme activity increased 10-fold by weaning, before declining to low values by 3 mo. LPL mRNA levels, protein, and enzyme activity did not change in epididymal fat from 3 wk to 21 mo. In the kidney, LPL mRNA levels were high at the end of gestation but fluctuated during the first month. LPL protein and activity were low at day 1 and rose eightfold to peak values by day 7 before decreasing to low levels by weaning. LPL mRNA levels were relatively high in fetal brains and then fell 60% during the neonatal period. LPL protein peaked at day 7 before falling 95% by weaning. Thus LPL is under complex tissue-specific regulation involving transcriptional and posttranscriptional mechanisms.

    View details for Web of Science ID A1992HK36200046

    View details for PubMedID 1550225



    1. Diabetic autonomic neuropathy causes loss of sympathetic cardiovascular control and is associated with increased vascular sensitivity to catecholamines. Supersensitivity to catecholamines could be due to either a postsynaptic increase in vascular sensitivity or to decreased catecholamine uptake into peripheral sympathetic nerve endings. 2. To differentiate between these possible mechanisms we have measured the responsiveness in vivo to noradrenaline and phenylephrine with local infusions into peripheral veins of diabetic patients with and without symptomatic autonomic neuropathy and of healthy control subjects. The dorsal hand vein compliance technique was used. 3. Symptomatic diabetic patients required significantly lower doses of noradrenaline for half-maximal venoconstriction (ED50) (geometric mean 2.14 ng/min) than control subjects (geometric mean 6.61 ng/min, P = 0.032), but there was no difference in the results from the phenylephrine dose-response curves between the groups. There were no differences in venous responsiveness to noradrenaline or phenylephrine between the asymptomatic diabetic group and the control group. However, in the asymptomatic diabetic group, postural blood pressure change (an index of loss of sympathetic control) was correlated with the ED50 for noradrenaline (r = 0.74, P = 0.014), but not with the ED50 for phenylephrine. In the control group the ED50 values for noradrenaline and phenylephrine were correlated with each other (r = 0.81, P = 0.0005). 4. Both vasopressor drugs act on vascular alpha-adrenoceptors, but only noradrenaline is taken up into peripheral sympathetic nerve endings. Our results suggest that, in diabetic patients, vascular supersensitivity to catecholamines is primarily determined by decreased neuronal catecholamine uptake. A postsynaptic increase in vascular alpha-adrenoceptor stimulation does not appear to be prominent in diabetic autonomic neuropathy.

    View details for Web of Science ID A1992HD88500007

    View details for PubMedID 1347259



    The hydrolysis of triglycerides and cholesteryl esters stored within cells is mediated by the enzyme, hormone-sensitive lipase. In adipose tissue and heart, hormone-sensitive lipase primarily hydrolyzes stored triglycerides to free fatty acids, while in steroidogenic tissues, it principally converts cholesteryl esters to free cholesterol for steroid hormone production. To determine whether hormone-sensitive lipase is under tissue-specific, developmental regulation, the steady state levels of hormone-sensitive lipase mRNA were determined in normal rats from late fetal life through 2 years of age. Hormone-sensitive lipase mRNA levels did not appear to vary in adipose tissue from epididymal fat pads obtained from animals between 3 weeks and 2 years of age. In heart, hormone-sensitive lipase mRNA levels were lowest in the fetus increased rapidly within the first day postnatally, and then gradually increased to stable adult levels by 2 months that were 3-fold higher than observed in fetal rats. Steady state mRNA levels of hormone-sensitive lipase in the adrenals were lowest in fetal rats, increased 4-fold during the first day and peaked at levels that were 9-fold higher by the end of the first week. Thereafter, levels fell and remained 3- to 4-fold higher than at birth throughout adult life. Hormone-sensitive lipase mRNA was undetectable in testes before 4 weeks of age and increased 25-fold to stable adult levels between 4 and 12 weeks. Thus, hormone-sensitive lipase is differentially expressed and regulated in a tissue-specific fashion during development and aging.

    View details for Web of Science ID A1991GC37200007

    View details for PubMedID 1770312

  • ELISA MEASUREMENT OF LDL RECEPTORS JOURNAL OF LIPID RESEARCH May, K., Kraemer, F. B., Chen, J., Cooper, A. D. 1990; 31 (9): 1683-1691


    An enzyme-linked immunosorbent assay was developed for measurement of low density lipoprotein (LDL) receptors. A monospecific polyclonal antibody to LDL receptor purified from rat liver that reacted with rat, mouse, canine, and human LDL receptor was used. With this assay, LDL receptors could be measured on 2-4 x 10(5) adherent cells and 1.0 x 10(5) cells in suspension, although results were more variable with cell suspensions. Membranes from a variety of receptor-rich and receptor-poor tissues could be assayed directly after adherence of the membranes to the ELISA plate by an overnight incubation. In some instances, the quality of the assay was improved by first solubilizing the membranes. The sensitivity of the assay is such that between 0.15 and 2 micrograms of membrane protein is required. This could be obtained from leukocytes in a modest (20-30 ml) quantity of human blood. The assay was used to demonstrate the rapid down-regulation of LDL receptors in human mononuclear leukocytes in response to a cholesterol-containing meal. Overall, the results support the use of ELISA technology to measure LDL receptors, particularly for physiologic studies.

    View details for Web of Science ID A1990EB10500016

    View details for PubMedID 2246618



    To gain a detailed understanding of those factors that govern the processing of dietary-derived lipoprotein remnants by macrophages we examined the uptake and degradation of rat triacylglycerol-rich chylomicron remnants and rat cholesterol-rich beta-very low density lipoprotein (beta-VLDL) by J774 cells and primary cultures of mouse peritoneal macrophages. The level of cell associated 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants reached a similar equilibrium level within 2 h of incubation at 37 degrees C. However, the degradation of 125I-labeled beta-VLDL was two to three times greater than the degradation of 125I-labeled chylomicron remnants at each time point examined, with rates of degradation of 161.0 +/- 36.0 and 60.1 +/- 6.6 ng degraded/h per mg cell protein, respectively. At similar extracellular concentrations of protein or cholesterol, the relative rate of cholesteryl ester hydrolysis from [3H]cholesteryl oleate/cholesteryl [14C]oleate-labeled chylomicron remnants was one-third to one-half that of similarly labeled beta-VLDL. The reduction in the relative rate of chylomicron remnant degradation by macrophages occurred in the absence of chylomicron remnant-induced alterations in low density lipoprotein (LDL) receptor recycling or in retroendocytosis of either 125I-labeled lipoprotein. The rate of internalization of 125I-labeled beta-VLDL by J774 cells was greater than that of 125I-labeled chylomicron remnants, with initial rates of internalization of 0.21 ng/min per mg cell protein for 125I-labeled chylomicron remnants and 0.39 ng/min per mg cell protein for 125I-labeled beta-VLDL. The degradation of 125I-labeled chylomicron remnants and 125I-labeled beta-VLDL was dependent on lysosomal enzyme activity: preincubation of macrophages with the lysosomotropic agent monensin reduced the degradation of both lipoproteins by greater than 90%. However, the pH-dependent rate of degradation of 125I-labeled chylomicron remnants by lysosomal enzymes isolated from J774 cells was 50% that of 125I-labeled beta-VLDL. The difference in degradation rates was dependent on the ratio of lipoprotein to lysosomal protein used and was greatest at ratios greater than 50. The degradation of 125I-labeled beta-VLDL by isolated lysosomes was reduced 30-40% by preincubation of beta-VLDL with 25-50 micrograms oleic acid/ml, suggesting that released free fatty acids could cause the slower degradation of chylomicron remnants. Thus, differences in the rate of uptake and degradation of remnant lipoproteins of different compositions by macrophages are determined by at least two factors: 1) differences in the rates of lipoprotein internalization and 2) differences in the rate of lysosomal degradation.

    View details for Web of Science ID A1990DW71300007

    View details for PubMedID 2280181



    We have developed a simple and rapid procedure for the isolation of total RNA from small amounts of adipose tissue. Using this method, it is possible to obtain quantitative recovery of RNA from less than 300 mg of adipose tissue, with an average yield of 70 micrograms of RNA per gram of adipose tissue. Northern blot analysis of rat epididymal adipose tissue RNA samples was performed using a beta-actin probe and demonstrated that intact total RNA had been isolated. The procedure has been adapted for use in 1.5-ml microcentrifuge Eppendorf tubes, providing a convenient and inexpensive method for the reproducible recovery of intact RNA from sparse samples of adipose tissue.

    View details for Web of Science ID A1990CY74300011

    View details for PubMedID 1694062



    The effects of activation on lipid and lipoprotein metabolism were examined in resident murine macrophages, inflammatory cells elicited by thioglycolate, primed cells elicited by pyran copolymer, and activated cells elicited by Corynebacterium parvum. Low density lipoprotein receptors were reduced by 70%, while scavenger receptors were reduced 60% in activated cells. Basal cholesteryl ester and triglyceride synthesis were increased fourfold in activated cells, whereas the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase was high in resident cells and progressively declined by greater than 80% in activated cells. Activities of neutral cholesteryl esterase and neutral triglyceride lipase were increased two- to fourfold in inflammatory, primed, and activated macrophages. These results demonstrate the diverse changes in lipid and lipoprotein metabolism that occur with activation and emphasize how the behavior of macrophages in atherosclerotic lesions can be altered by activation.

    View details for Web of Science ID A1990CJ80300002

    View details for PubMedID 2297348



    A high plasma prorenin is a marker of microvascular complications of diabetes. We have followed 56 adults and 120 children with uncomplicated insulin-dependent (type 1) diabetes. When plasma prorenin rises above the normal range in an adolescent or adult with type 1 diabetes, signs of nephropathy, retinopathy, or neuropathy follow within one to two years. The earliest sign may be intermittent microalbuminuria, which can often be abolished by improved control of hyperglycemia. The association between increased plasma prorenin and complications of noninsulin-dependent (type 2) diabetes is less reliable in patients with hypertension and in those receiving medication that affects plasma prorenin. The oral hypoglycemic agent, glipizide, lowers plasma prorenin, but its effect on prognosis is unknown. Plasma prorenin and renin decline as blood pressure rises, whereas the prevalence of micro- and macroalbuminuria increases. Many drugs used to control hypertension affect the level of prorenin. In the majority of our patients with type 2 diabetes who are hypertensive or are taking a medication that affects plasma prorenin, microalbuminuria may prove to be a more reliable warning of vascular complications.

    View details for PubMedID 2655663



    The effects of ligand binding to the scavenger receptor on the secretion of lipoprotein lipase by murine macrophages were examined. Inflammatory macrophages exposed to acetylated low-density lipoprotein (AcLDL) exhibited a dose-dependent, 40-80% increase in lipoprotein lipase secretion. This stimulation appeared to be unrelated to intracellular cholesterol and triacylglycerol levels and to phagocytosis in general. Resident and inflammatory macrophages treated with maleylated bovine serum albumin (Mal-BSA) showed a 3-fold increase in lipoprotein lipase secretion in a dose-dependent and time-dependent fashion. In contrast, dextran sulfate, which is another ligand recognized by the scavenger receptor, caused a dose-dependent decrease in lipoprotein lipase secretion. Casein, a ligand recognized by the Mal-BSA receptor, did not affect lipoprotein lipase secretion nor the ability of Mal-BSA to stimulate the enzyme, while dextran sulfate abolished the stimulatory effects of Mal-BSA. Since ethylamine, an inhibitor of receptor-mediated endocytosis, attenuated the increase in lipoprotein lipase secretion induced by AcLDL and Mal-BSA, but did not affect the inhibition induced by dextran sulfate, it is suggested that receptor-mediated endocytosis of ligands via the scavenger receptor might play a key role in the stimulation of lipoprotein lipase secretion in macrophages. This study reveals another mechanism for regulation of macrophage lipoprotein lipase secretion.

    View details for Web of Science ID A1988Q834800003

    View details for PubMedID 3179335



    We investigated the effects of insulin deficiency and insulin treatment on the secretion of lipoprotein lipase (LPL) by murine macrophages. Streptozocin-induced insulin deficiency caused hyperglycemia and hypertriglyceridemia in mice. Peritoneal macrophages isolated from insulin-deficient mice secreted 70% less LPL activity than control mice. A 65% decrease in LPL activity in epididymal adipose tissue, without any changes in heart LPL activity, was also seen with insulin deficiency. One week of insulin treatment lowered plasma glucose and triglyceride levels in insulin-deficient mice. Additionally, 1 wk of insulin treatment increased LPL secretion by macrophages, but to only one-half of control, while normalizing adipose tissue LPL activity. One injection of insulin also increased LPL secretion by macrophages to one-half of control and normalized adipose tissue LPL activity, even though plasma glucose and triglyceride levels were not affected. In vitro insulin treatment of macrophages isolated from control or insulin-deficient mice had no effect on LPL secretion. The results suggest that insulin does not exert a direct effect on the LPL secretion by macrophages but that deficiency of insulin indirectly causes a profound decrease in macrophage LPL secretion. These changes in macrophage LPL secretion may contribute to the atherosclerotic process in diabetes mellitus.

    View details for Web of Science ID A1988P437000014

    View details for PubMedID 3292329



    Growth-stimulated synchronized cells exhibit a rapid increase in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC activity prior to the onset of DNA synthesis. Under normal culture conditions, HMG-CoA reductase activity exhibits wide variations among experiments. To determine whether this phenomenon is dependent on cell replication, we used J774 macrophage-like cells to compare changes in reductase activity in cells synchronized by serum deprivation and then growth-stimulated by fresh media containing serum to unsynchronized cells treated with fresh media and serum. Under these conditions, no increase in [3H]thymidine incorporation into cell DNA was seen in unsynchronized cells, but a large increase was observed in synchronized cells 10-12 h after media change. Although the growth characteristics differed between the cells, reductase activity was low at the time of media change and increased 10 to 20-fold 5-10 h after media change, returning to basal levels by 24 h in both synchronized and unsynchronized cells. This pattern of reductase activity was observed in unsynchronized cells from a variety of cell lineages, although the magnitude of the changes varied. Fluctuations of [14C]acetate incorporation into cholesterol were observed in parallel to alterations in reductase activity. LDL receptor expression also paralleled the changes in reductase activity, but scavenger receptor expression was not affected. Addition of lipoproteins at the time of media change inhibited the rise in reductase activity by 80-90%. The increase in reductase activity was not due to a stimulation of cholesterol efflux into the medium, but evidence for the secretion into the media of an inhibitory factor was obtained. These results suggest that cell requirements for cholesterol are not always directly related to replication, and that standard culture conditions induce transient fluctuations in reductase activity and lipoprotein receptor expression.

    View details for Web of Science ID A1988P640400003

    View details for PubMedID 3401509



    When urinary albumin excretion was measured by radioimmunoassay, most diabetics excreted more albumin than nondiabetic subjects. Microalbuminuria was defined as an albumin excretion greater than 30 mg/g of urinary creatinine, more than twice the upper limit of normal. Intermittent microalbuminuria was found in 20% of patients with insulin-dependent diabetes mellitus (IDDM) or non-insulin-dependent diabetes mellitus (NIDDM). Continuous microalbuminuria occurred in a similar percentage of patients with NIDDM, but less frequently in patients with IDDM. Rigorous control of glycemia was followed by cessation of microalbuminuria in nearly half of these patients. Microalbuminuria was associated with an increased incidence of other microvascular complications, as well as a distinctly higher plasma prorenin value in IDDM. Hypertension of 160/100 mm Hg or above was accompanied by increased albumin excretion and lower plasma prorenin values than in normotensive diabetics.

    View details for Web of Science ID A1988M903000024

    View details for PubMedID 3281621



    The effect of halothane on isoproterenol-stimulated lipolysis was determined in isolated rat epididymal fat cells. The maximal lipolytic response (Emax) activated by isoproterenol was 350 +/- 61 nmol of glycerol/10(5) cells/hr with an EC50 of 5.1 X 10(-9) M. When the adipocytes were simultaneously bubbled with 2.5% halothane, the Emax decreased to 158 +/- 43 nmol of glycerol/10(5) cells/hr and the dose response curve for isoproterenol was shifted to the right (EC50 3.5 X 10(-8) M, p less than 0.05). When lipolysis was maximally stimulated with (-)-isoproterenol (10(-6)M), the inhibitory effect of halothane was found to be both dose dependent (IC50 approximately 2.5%, v/v) and reversible following washout. Neither the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (2 X 10(-3)M), nor forskolin (10(-6) M) was able to normalize lipolysis in the presence of halothane. The activation of cAMP-dependent protein kinase (EC activity by isoproterenol was not different in halothane-exposed cells when compared to unexposed cells. When control adipocytes were exposed to isoproterenol (10(-6) M), there was a 2.5-fold increase in the activity of hormone-sensitive lipase (EC from 0.64 +/- 0.13 to 1.53 +/- 0.32 pkat (pmol/sec) per mg (p less than 0.005, n = 10). However, in the presence of halothane (2.5%, v/v) isoproterenol stimulation of hormone-sensitive lipase was attenuated by 50% to values of 1.06 +/- 0.23 pkat/mg (p less than 0.01, n = 10). Halothane had no direct inhibitory effect on hormone-sensitive lipase since this enzyme's activity was unaffected when homogenates of isoproterenol-stimulated control cells were incubated with halothane. These studies suggest that halothane impairs the activation of hormone-sensitive lipase by cAMP-dependent protein kinase and in this manner inhibits beta-adrenergic-stimulated lipolysis.

    View details for Web of Science ID A1988M717900014

    View details for PubMedID 3352597


    View details for Web of Science ID A1987J409800001

    View details for PubMedID 3038488



    The receptor-mediated uptake of rat hypercholesterolemic very low density lipoproteins (beta VLDL) and rat chylomicron remnants was studied in monolayer cultures of the J774 and P388D1 macrophage cell lines and in primary cultures of mouse peritoneal macrophages. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was reduced 80-90% in the presence of high concentrations of unlabeled human low density lipoproteins (LDL). Human acetyl-LDL did not significantly compete at any concentration tested. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was also competitively inhibited by specific polyclonal antibodies directed against the estrogen-induced LDL receptor of rat liver. Incubation in the presence of anti-LDL receptor IgG, but not nonimmune IgG, reduced specific uptake greater than 80%. Anti-LDL receptor IgG, 125I-beta VLDL, and 125I-chylomicron remnants bound to two protein components of apparent molecular weights 125,000 and 111,000 on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70-90% of 125I-lipoprotein binding was confined to the 125,000-Da peptide. Binding of 125I-beta VLDL and 125I-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. Comparison of anti-LDL receptor IgG immunoblot profiles of detergent-solubilized membranes from mouse macrophages, fibroblasts, and liver, and normal and estrogen-induced rat liver demonstrated that the immunoreactive LDL receptor of mouse cells is of a lower molecular weight than that of rat liver. Incubation of J774 cells with 1.0 micrograms of 25-hydroxycholesterol/ml plus 20 micrograms of cholesterol/ml for 48 h decreased 125I-beta VLDL uptake and immuno- and ligand blotting to the 125,000- and 111,000-Da peptides by only 25%. Taken together, these data demonstrate that uptake of beta VLDL and chylomicron remnants by macrophages is mediated by an LDL receptor that is immunologically related to the LDL receptor of rat liver.

    View details for Web of Science ID A1987G509800061

    View details for PubMedID 3546288



    The regulation of the secretion of lipoprotein lipase was studied in primary cultures of mouse peritoneal macrophages and in the murine macrophage cell line J774. As previously reported, both cell types secrete a lipase with the characteristics of lipoprotein lipase. Incubation of macrophages with insulin, insulin-like growth factor, and L-thyroxine had no effect on lipoprotein lipase secretion. Incubation with dexamethasone and with several agents which increase intracellular cyclic AMP led to a decrease in lipoprotein lipase secretion by mouse peritoneal macrophages. These results suggest that the hormonal regulation of lipoprotein lipase in macrophages is different from that in adipose tissue and heart muscle. Incubation of the macrophages with heparin caused a marked increase in the secretion of lipoprotein lipase. Short incubations with heparin (5 min) caused a release of the enzyme into the media, while longer incubations caused a 2-8-fold increase in net lipoprotein lipase secretion which was maximal after 2-16 h depending on cell type, and persisted for 24 h. The effect of heparin was dose-dependent and specific (it was not duplicated by other glycosaminoglycans). The mechanism of heparin-induced increase in lipoprotein lipase secretion was explored. The increase was not caused by the release of a presynthesized intracellular pool of lipoprotein lipase or by the stabilization of lipoprotein lipase by heparin after secretion. The heparin-induced increase in lipoprotein lipase secretion was dependent on protein synthesis. The secretion of lipoprotein lipase by macrophages in response to low levels of heparin may be a significant factor in the formation of atherosclerotic lesions.

    View details for Web of Science ID A1986F472000009

    View details for PubMedID 2431720



    Heart and heart-lung transplant recipients at Stanford (Calif) University Medical Center were routinely prescribed long-term calcium carbonate antacid therapy to aid in the prevention of peptic ulcer disease and osteoporosis associated with glucocorticoid immunosuppressive therapy. Patients consumed 4 to more than 10 g/d of elemental calcium. Since calcium carbonate also provides the essential ingredients for the development of the milk-alkali syndrome, the laboratory flow sheets of 297 heart and heart-lung transplant recipients were reviewed to examine the incidence of hypercalcemia. Sixty-five patients developed significant hypercalcemia after transplantation. Thirty-one patients were alkalotic at the time of hypercalcemia; 37 had impairment in renal function. It is likely that most of these patients had the milk-alkali syndrome. While most patients became eucalcemic by discontinuing calcium carbonate therapy, intravenous hydration and forced diuresis were used to treat severe cases. It is possible that the incidence of the milk-alkali syndrome will increase with the current popularity of prescribing calcium carbonate for the prevention and treatment of osteoporosis.

    View details for Web of Science ID A1986E259300014

    View details for PubMedID 3532984



    To characterize lipoprotein uptake by macrophages, we studied J774 murine macrophage-derived cells. Uptake of 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants was saturable, specific, and of high affinity. Maximal specific uptake and the concentration at which half-maximal uptake occurred were similar for both beta-VLDL and chylomicron remnants. Specific uptake of 125I-labeled chylomicrons was only 1/5 that of the other two lipoproteins. Cholesterol loading decreased 125I-labeled chylomicron remnant and 125I-labeled beta-VLDL uptake by 25%. Chylomicron remnants and beta-VLDL were equipotent in cross-competition studies; acetyl-LDL did not compete, and human LDL was a poor competitor. Although the amounts of cell-associated lipoproteins were similar, beta-VLDL and chylomicron remnants had different effects on cellular lipid metabolism. beta-VLDL produced a threefold stimulation while chylomicron remnants caused a decrease in [3H]oleate incorporation into cholesteryl ester. beta-VLDL had no effect while chylomicron remnants caused a threefold increase in [3H]oleate incorporation into triacylglycerol. beta-VLDL produced a 44% suppression and chylomicron remnants produced a 78% increase in HMG-CoA reductase activity. In summary, J774 macrophages express a receptor site that recognizes both beta-VLDL and chylomicron remnants; however, these lipoproteins exhibit strikingly different effects on intracellular lipid metabolism.

    View details for Web of Science ID A1986E941500005

    View details for PubMedID 3025323



    Lipoprotein lipase (LPL) activity was measured in the media of cultured mouse peritoneal macrophages that were isolated after the intraperitoneal injection of inflammatory agents in order to yield a variety of states of activation. Fully activated macrophages obtained from Corynebacterium parvum-injected mice secreted very low levels of LPL when compared to unstimulated macrophages, while inflammatory and primed macrophages had increased LPL secretion. When inflammatory macrophages were incubated with conditioned medium obtained from fully activated macrophages, LPL secretion decreased in a time- and dose-dependent fashion. The factor(s) secreted by fully activated macrophages that inhibited LPL secretion was shown to be thermolabile and distinct from tumor necrosis factor. These results demonstrate that activation dramatically alters macrophage LPL secretion.

    View details for Web of Science ID A1986E228000032

    View details for PubMedID 3760780



    Studies have demonstrated that ketoconazole and related imidazoles block gonadal and adrenal steroidogenesis in humans by inhibiting several cytochrome P-450-dependent enzymes. Moreover, recent evidence suggests that cholesterol production in humans is also affected by ketoconazole. In the present experiments cultured normal human fibroblasts have been used to explore the effects of ketoconazole on cholesterol synthesis. Ketoconazole inhibited cholesterol synthesis (greater than 90% suppression in 1 hr) rapidly by blocking the conversion of methyl sterols to cholesterol. Dihydrolanosterol was the major methyl sterol which accumulated with ketoconazole. At high concentrations of ketoconazole, the conversion of squalene to methyl sterols was also inhibited. The inhibition of cholesterol synthesis was dose-dependent with an IC50 approximately 2.8 X 10(-8) M. In parallel to the inhibition of cholesterol synthesis, there was a reciprocal increase in methyl sterol production. The related imidazole antimycotic, clotrimazole, had similar effects, whereas the imidazole anesthetic, etomidate, had little effect on cholesterol synthesis. Confluent cells exposed to ketoconazole had a 90% fall in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that declined with a T1/2 approximately 3.7 hr. In conclusion, ketoconazole has multiple effects on cholesterol synthesis, directly inhibiting late steps by blocking the conversion of methyl sterols to cholesterol and indirectly suppressing total sterol synthesis via feedback inhibition by sterol intermediates of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

    View details for Web of Science ID A1986E029900022

    View details for PubMedID 3746668



    Mice made insulin deficient by the injection of streptozocin develop hyperglycemia and hypertriglyceridemia with triglyceride-rich, very-low-density lipoproteins (VLDLs). Thioglycolate-elicited peritoneal macrophages freshly isolated from insulin-deficient mice have increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is reflected in a greater rate of cholesterol synthesis by these macrophages. In contrast, thioglycolate-elicited macrophages from control mice with diet-induced hypertriglyceridemia had normal levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Cell surface receptors responsible for VLDL uptake are decreased in macrophages isolated from insulin-deficient mice, although receptors for acetylated low-density lipoproteins are not altered. Insulin treatment of insulin-deficient mice lowers plasma glucose and triglyceride concentrations. Additionally, insulin treatment returns the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and the rate of cholesterol synthesis in thioglycolate-elicited macrophages to normal while increasing the number of receptors responsible for VLDL uptake. It is suggested that the increases in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and the rate of cholesterol synthesis in macrophages isolated from insulin-deficient mice are secondary to the reduction in the number of receptors responsible for VLDL uptake induced by insulin deficiency. These alterations in the cholesterol metabolism of macrophages occurring with insulin deficiency may have important implications for the atherosclerotic process in diabetes mellitus.

    View details for Web of Science ID A1986D116300006

    View details for PubMedID 3522317



    To determine if ketoconazole influences cholesterol metabolism in humans, plasma lipid levels were studied in seven men with advanced prostate cancer who were being treated with high-dose ketoconazole. Additionally, the effects of ketoconazole on cholesterol synthesis in cultured normal human fibroblasts were studied. High-dose ketoconazole therapy caused a 27 percent reduction in total serum cholesterol values without affecting serum triglyceride levels. The reduction in serum cholesterol levels was maintained for five months in six of seven patients. The fall in total cholesterol levels was due to a 38 percent reduction in low-density lipoprotein cholesterol levels without associated changes in high-density lipoprotein cholesterol levels. Serum lanosterol levels increased 46 percent during ketoconazole treatment. Studies in cultured normal human fibroblasts showed that ketoconazole inhibited cholesterol synthesis by blocking the conversion of lanosterol to cholesterol. These results establish that ketoconazole is a potent inhibitor of cholesterol production in vivo and in vitro.

    View details for Web of Science ID A1986A879200014

    View details for PubMedID 3754384



    The lipid-laden foam cells from atherosclerotic lesions appear to be derived from macrophages which have accumulated lipids from plasma lipoproteins. When examined in vitro, thioglycolate-elicited mouse peritoneal macrophages do not accumulate lipids when exposed to normal low density lipoproteins (LDL), but take up very low density lipoproteins (VLDL) or chemically modified LDL with resultant lipid accumulation. Patients with noninsulin-dependent diabetes mellitus (NIDDM) have an increased incidence of atherosclerosis, due in part to disturbances in lipoprotein metabolism. We investigated the possibility that VLDL isolated from patients with NIDDM are taken up by mouse peritoneal macrophages more avidly than normal. Two groups of patients with NIDDM were studied; one group was normotriglyceridemic and the other group was hypertriglyceridemic. The VLDL from both normotriglyceridemic and hypertriglyceridemic patients were enriched in cholesterol and triglyceride compared to that from normal subjects. Thioglycolate-elicited mouse peritoneal macrophages bound and degraded greater amounts of VLDL isolated from patients with NIDDM (both normo- and hypertriglyceridemic) than of VLDL from normal subjects. While normal VLDL caused a marked increase in cellular triglyceride and cholesteryl ester contents in macrophages, VLDL isolated from patients with NIDDM resulted in an even greater cellular accumulation of lipids. These results suggest that the VLDL of patients with NIDDM have alterations in their composition and metabolic behavior. The increased uptake of VLDL by macrophages may contribute to the enhanced atherosclerosis present in NIDDM.

    View details for Web of Science ID A1985ALV2600021

    View details for PubMedID 4008610



    Plasma renin exists in an active form or as an inactive zymogen that resembles a prorenin present in homogenates of human kidneys. We examined the relation of diabetes and its microvascular complications with the level of plasma inactive renin activated by dialysis to pH 3.3. Plasma inactive renin was measured in 235 diabetic patients and 90 nondiabetic controls. In the controls, the level of plasma inactive renin increased slightly with age but was never above 50 ng per milliliter per hour. In young diabetic patients studied within three years of the onset of diabetes the concentration of inactive renin was normal, and in some older diabetics without complications it remained within the age-adjusted normal range for many years. However, in patients with retinopathy or albuminuria, plasma inactive renin was above the normal range with few exceptions, reaching levels 50 to 200 per cent above the upper limits of normal in patients with nephropathy. The frequency of neuropathy was also significantly higher among patients with levels above the normal range. In 37 per cent of the diabetics followed during one to three years of conventional treatment, plasma inactive renin increased significantly, but in another group of diabetics under intensive treatment, the level rose in only 7 per cent and fell in 43 per cent. We conclude that there is a close association between a high level of plasma inactive renin and the presence of microvascular complications, and that the level of inactive renin can be modified by intensive treatment of diabetes.

    View details for Web of Science ID A1985AHZ2600002

    View details for PubMedID 3887168



    A young woman with acromegaly and Zollinger-Ellison syndrome associated with a GH-releasing factor (GRF)- and gastrin-secreting metastatic islet cell carcinoma was studied by means of specific antisera which recognize various regions of the GRF molecule. Using specific immunohistochemical techniques, the tumor cells were shown to contain GRF, gastrin, and gastrin-releasing peptide, but not GH. During a 4-h period, plasma GRF levels averaged 5.6 +/- 1.4 ng/ml (+/- SD), while GH levels averaged 148 +/- 71 ng/ml. GH secretion was pulsatile and increased after TRH administration. GRF RIAs may be useful in establishing the diagnosis of acromegaly secondary to the ectopic secretion of GRF.

    View details for Web of Science ID A1984TP17300031

    View details for PubMedID 6090497

  • Characterization of the binding site on thioglycolate-stimulated mouse peritoneal macrophages that mediates the uptake of very low density lipoproteins. journal of biological chemistry Kraemer, F. B., Chen, Y. D., Lopez, R. D., Reaven, G. M. 1983; 258 (20): 12190-12197


    Isolated mouse peritoneal macrophages that had been stimulated with thioglycolate were shown to take up and degrade normal human 125I-very low density lipoproteins (VLDL). Uptake occurred via a specific cell surface receptor which was shown to be 1) temperature-dependent, 2) calcium-dependent, and 3) susceptible to proteolytic digestion. The receptor-mediated uptake and degradation of VLDL markedly stimulated the synthesis and accumulation of triglyceride and cholesteryl ester within macrophages. The degradation of the protein and lipid portions of VLDL occurred within lysosomes. Competition studies showed that the binding site for VLDL was different from the receptor for normal low density lipoproteins or for acetylated low density lipoproteins but that there was cross competition with beta-VLDL. In addition, positive charges appeared to play an important role in the recognition of VLDL by their receptors since polyamines were able to markedly inhibit VLDL binding, degradation, and lipid accumulation while negatively charged compounds were without effects. These studies indicate that 1) stimulated mouse peritoneal macrophages possess specific receptors which recognize normal human VLDL and 2) the receptor-mediated uptake of VLDL results in the accumulation of triglyceride and cholesteryl ester within macrophages.

    View details for PubMedID 6313642



    Studies in vitro have shown that glycosylation of low density lipoprotein (LDL) will decrease its ability to bind to its receptor. We have evaluated the possibility that such an event might occur in vivo in diabetes by comparing the binding and degradation by normal fibroblasts and mouse peritoneal macrophages of LDL obtained from normal control subjects and patients with Type 2 (non-insulin-dependent) diabetes mellitus. When compared with control subjects, Type 2 diabetic patients had elevated fasting glucose (increased by 160%), haemoglobin AIc (increased by 75%), triglyceride (increased by 550%), and cholesterol (increased by 48%) levels. LDL from Type 2 diabetic patients displayed populations of particles with more heterogeneous hydrated densities than LDL from control subjects, with enrichment in the triglyceride content of the lighter population. 125I-LDL from normal and Type 2 diabetic subjects bound to fibroblasts with similar binding affinities and binding capacities. The kinetics of degradation of LDL from normal and Type 2 diabetic subjects by fibroblasts were also similar. Furthermore, all populations of LDL particles from Type 2 diabetic patients were bound and degraded by normal fibroblasts in identical fashions. In addition, 125I-LDL from normal and Type 2 diabetic subjects were not bound or degraded by mouse peritoneal macrophages. It is concluded that the LDL of patients with Type 2 diabetes with moderate hyperglycaemia are not modified sufficiently to alter their normal binding and degradation by human fibroblasts or to cause their uptake by mouse peritoneal macrophages.

    View details for Web of Science ID A1982NX98800006

    View details for PubMedID 7117726



    The relationship between changes in tissue lipoprotein lipase (LPL) activity and triglyceride (TG) concentration has been studied in chronically uremic rats. Uremic rats had a 6-fold increase in BUN and 75% rise in TG levels associated with a 50% reduction in adipose tissue heparin-releasable LPL activity. However, total LPL activity of adipose tissue and muscle was not significantly decreased. Furthermore, there was no significant correlation between the rise in plasma TG concentration seen in uremic rats and the reduction in heparin-releasable adipose tissue LPL activity. These data suggest that the TG removal defect associated with chronic uremia is not a simple function of a decrease in LPL activity.

    View details for Web of Science ID A1982NJ49400017

    View details for PubMedID 7099342



    1. The effects of two isoenergetic diets differing only in the values for polyunsaturated: saturated fat (P:S values of 0.2 v. 2.0) were studied in twenty adult human volunteers. 2. A period of 14 d on the high P:S diet failed to produce significant changes in fasting triglyceride levels, though there were individual variations. On the other hand, fasting cholesterol levels dropped by 10% (P less than 0.005). High-density-lipoprotein-cholesterol concentrations were not influenced by changes in the P:S value. 3. Investigations into the mechanism by which changes in the P:S value might affect plasma triglyceride values revealed no consistent effects on very-low-density-lipoprotein kinetics, insulin secretion, insulin sensitivity or free fatty acid concentrations. 4. The results of this study suggest that the largest increase in dietary P:S values that is likely to be obtained on a long-term basis may have only a small effect on plasma triglyceride and cholesterol concentrations.

    View details for Web of Science ID A1982NH29300011

    View details for PubMedID 7066289



    Two test meals, varying only in type of carbohydrate foods, were given to 12 volunteers with impaired glucose tolerance. The carbohydrate content of one meal was composed of potato and gelatin, while the other meals contained an equivalent amount of carbohydrate as rice and corn. The two meals were otherwise identical, and the additional constituents were turkey, green salad, and salad dressing. Plasma glucose and insulin responses were significantly lower after the meal containing rice and corn, with the change in the insulin response being the most dramatic. These results raise the possibility that variations in source of dietary carbohydrate may have therapeutic utility in patients with impaired glucose tolerance.

    View details for Web of Science ID A1981MU80000018

    View details for PubMedID 7032274



    Insulin resistance was quantified with two different methods in 30 subjects with varying degrees of glucose tolerance. One method, the insulin suppression test, is performed by continuously infusing epinephrine, propranolol, insulin, and glucose. Epinephrine and propranolol suppress endogenous insulin release, and steady-state plasma levels of exogenous insulin and glucose are reached in all individuals. Because the steady-state insulin level is the same in all subjects, the height of the steady-state plasma glucose level provides a direct estimate of insulin resistance. The other method, the euglycemic clamp technique, produces a steady-state level of exogenous hyperinsulinemia by means of a primed and continuous insulin infusion. Glucose is also infused at a rate sufficient to prevent an insulin-induced fall in glucose concentration, and the amount of glucose required to maintain the basal plasma glucose level provides the estimates of insulin resistance. The results indicated that estimates of insulin resistance generated by the two methods were highly correlated (r = 0.93). Furthermore, both methods of assessing insulin resistance indicated that the greater the degree of glucose intolerance, the more severe the insulin resistance. These results serve to further emphasize the importance of insulin resistance in the pathogenesis of hyperglycemia in type II diabetes.

    View details for Web of Science ID A1981LP16100005

    View details for PubMedID 7014307



    We have previously postulated that resistance to insulin-mediated glucose uptake was the basic metabolic abnormality in patients with endogenous hypertriglyceridemia. In this situation, glucose tolerance would tend to deteriorate, and could only be maintained by the increased secretion of insulin. Although the ensuing hyperinsulinemia might prevent the development of glucose intolerance, we suggested that it would also lead to increased hepatic very low density (VLDL) triglyceride (TG) synthesis and secretion. In the current study we have quantified these four metabolic variables in 16 nonobese human subjects with plasma TG concentrations less than 175 mg/dl. The results demonstrate the following degree of correlation: insulin resistance (Formula: see text) insulin response to food (Formula: see text) VLDL-TG secretion rate (Formula: see text) plasma TG concentration. These data indicate that nonobese subjects with normal TG levels have the same relationship between degree of insulin sensitivity, insulin response to food, VLDL-TG secretion, and TG concentration previously described in patients with endogenous hypertriglyceridemia.

    View details for Web of Science ID A1981LE34800011

    View details for PubMedID 7007804



    Freshly prepared rat testicular membranes bind iodinated rat high density lipoprotein (HDL) with high affinity (Kd = 32 microgram of HDL protein/ml). This high density lipoprotein binding differs from low density lipoprotein binding by cultured human fibroblast cells in two ways; it is not affected by Ca2+ or ethylenediaminetetraacetic acid, and it is not sensitive to pronase and trypsin. Testicular binding activity is primarily found in interstitial tissue containing Leydig cells and can be modulated by human chorionic gonadotropin administration in vivo (2-fold increase of binding capacity, with no affinity change) with 250 units/kg of human chorionic gonadotropin injection daily for 4 days. The interstitial high density lipoprotein binding site also recognizes rat very low density lipoprotein, but not rat low density lipoprotein, as shown by displacement experiments. When membrane preparations of various other tissues were assessed for their high density lipoprotein binding, we found that the adrenal density lipoprotein binding, we found that the adrenal gland binds rat high density lipoprotein with similar affinity and capacity, while spleen, kidney, and heart showed no high affinity binding. In addition, when iodinated rat low density lipoprotein was tested for its ability to bind to testicular membranes, no high affinity saturable binding was observed. We conclude that there are specific high density lipoprotein-binding sites present in steroidogenic tissues; these binding sites are not found in the nonsteroidogenic tissues tested. Furthermore, no high affinity low density lipoprotein-binding sites can be demonstrated in the testis; thus it appears that high density lipoprotein, rather than low density lipoprotein, is the major cholesterol-carrying lipoprotein recognized by the rat testis.

    View details for Web of Science ID A1980KL96600043

    View details for PubMedID 7410418



    Plasma glucose and insulin responses were measured in 22 subjects after two meal tolerance tests that varied only in the food source of carbohydrate. Each meal contained 45% carbohydrate, 15% protein, and 40% fat and provided 40% of calculated daily caloric requirement. The meals elicited a similar glucose response; however, the insulin response was significantly lower when rice and corn supplied the carbohydrate as compared to potato and gelatin. The total insulin response, calculated as area under the response curve, was 60% (P less than 0.001) greater in the meal with potato and gelatin versus the rice and corn meal.

    View details for Web of Science ID A1980JW45400015

    View details for PubMedID 6992561



    Plasma triglyceride (TG) concentrations increase with advancing age. To determine if this phenomenon is due to age per se or to age-related changes in other metabolic variables, determination of fasting plasma TG, glucose, insulin, and free fatty acid (FFA) concentrations, as well as body mass index (BMI), were made on 167 normal subjects from 18 to 77 yr of age. Significant simple correlation coefficients (r) were found between TG concentrations and age (0.47), BMI (0.39) and fasting plasma glucose (0.40), insulin (0.24), and FFA (0.20) concentrations. Multiple regression analysis was used to determine the total amount of variability in TG concentration that could be accounted for by the combination of the examined metabolic parameters. A highly significant (p < 0.0001) total correlation of 0.57 was obtained, indicating that these variables could account for approximately one-third of the total variances. Partial correlation analysis (fixing four of the five variables) yielded a correlation coefficient of 0.35 (p < 0.001) between age and fasting TG concentration. Hence, age per se, or an age-dependent phenomenon, appears to be an independent factor with a role in determining plasma TG concentrations.

    View details for Web of Science ID A1980KR16400014

    View details for PubMedID 7001176



    Insulin deficiency was produced by streptozotocin in young (5-6 wk old) male rats, and measurements were made of plasma triglyceride and glucose concentrations and of lipoprotein lipase (LPL) activity of adipose tissue (epididymal) and muscle (gastrocnemius and soleus). Rats with streptozotocin-induced diabetes underwent a significant reduction in adipose tissue LPL activity (both total and heparin releasable), but the fall in LPL activity in these rats bore little relationship to their rise in plasma triglyceride concentration. Furthermore, muscle LPL activity was essentially unchanged in diabetic rats. Qualitatively similar changes were observed when measurements were made at either 8 a.m. (after the normal evening access to food) or 2 p.m. (6 h after food withdrawal). It is concluded that the hypertriglyceridemia that occurs secondary to insulin deficiency is not a simple function of decreased tissue LPL activity.

    View details for Web of Science ID A1980KJ84400009

    View details for PubMedID 6449394



    The unique combination of male hypogonadism with hypoparathyroidism, hypoadrenalism, hypothyroidism, diabetes mellitus, and alopecia totalis has been documented in a male patient who has been followed over the past 28 years. In this patient, first seen at the age of six for hypoparathyroidism alone, repeated clinical and laboratory endocrine evaluation detected the sequential development of the additional endocrine deficiencies. The presence of abnormal serum antibodies is consistent with an atuoimmune pathogenesis of this syndrome.

    View details for Web of Science ID A1976CT22800013

    View details for PubMedID 1008083