Early enforcement of cell identity by a functional component of the terminally differentiated state.

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.

Hormone-sensitive lipase protects adipose triglyceride lipase-deficient mice from lethal lipotoxic cardiomyopathy.

Lipid droplets (LDs) are multifunctional organelles that regulate energy storage and cellular homeostasis. The first step of triacylglycerol 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 lipase in the heart, we hypothesized that activation of HSL might compensate for ATGL deficiency. To test this hypothesis, we crossed ATGL-KO (AKO) mice and cardiac-specific HSL-overexpressing mice (cHSL) to establish homozygous AKO mice and AKO mice with cardiac-specific HSL overexpression (AKO+cHSL). We found that cardiac triacylglycerol content was 160-fold higher in AKO relative to Wt mice, whereas that of AKO+cHSL mice was comparable to the latter. In addition, AKO cardiac tissues exhibited reduced mRNA expression of PPARα-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 diacylglycerol contents, whereas 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 life span of AKO+cHSL mice was comparable to that of Wt mice. HSL overexpression normalizes lipotoxic cardiomyopathy in AKO mice and the findings highlight the applicability of cardiac HSL activation as a therapeutic strategy for ATGL deficiency-associated lipotoxic cardiomyopathies.

Mice deficient in ER protein seipin have reduced adrenal cholesteryl ester lipid droplet formation and utilization.

Cholesteryl ester (CE)-rich lipid droplets (LDs) 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 CE-rich LD formation have not been directly studied and are assumed by most to occur in a fashion analogous to triacylglycerol-rich LDs. Seipin is an endoplasmic reticulum protein that forms oligomeric complexes at endoplasmic reticulum-LD contact sites, and seipin deficiency results in severe alterations in LD maturation and morphology as seen in Berardinelli-Seip congenital lipodystrophy type 2. While seipin is critical for triacylglycerol-rich LD formation, no studies have directly addressed whether seipin is important for CE-rich LD biogenesis. To address this issue, mice with deficient expression of seipin specifically in adrenal, testis, and ovary, steroidogenic tissues that accumulate CE-rich LDs under normal physiological conditions, were generated. We found that the steroidogenic-specific seipin-deficient mice displayed a marked reduction in LD and CE accumulation in the adrenals, demonstrating the pivotal role of seipin in CE-rich LD accumulation/formation. Moreover, the reduction in CE-rich LDs 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.

Chemerin regulates formation and function of brown adipose tissue: Ablation results in increased insulin resistance with high fat challenge and aging.

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.

SR-B1: A Unique Multifunctional Receptor for Cholesterol Influx and Efflux.

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.

Slc43a3 is a regulator of free fatty acid flux.

Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA 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 FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake.

Plasma membrane cholesterol trafficking in steroidogenesis.

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 α-soluble N-ethylmaleimide-sensitive factor attachment protein (α-SNAP) are enriched in adrenal PMs, and adrenocorticotropic hormone treatment of rats recruited STX5 and α-SNAP to adrenal PMs and mitochondria. Immunodepletion of STX5 and α-SNAP from PMs decreased steroidogenesis supported by PMs in vitro. Protease digestion of PMs decreased, whereas recombinant STX5 or α-SNAP restored, the PMs' ability to support steroidogenesis. Knockdown of either STX5 or α-SNAP in Y1 cells decreased stimulated steroidogenesis. These results indicate that STX5 and α-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.

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.

Long-chain acyl-CoA synthetase 4 (ACSL4) has a unique substrate specificity for arachidonic acid. Hepatic ACSL4 is coregulated with the phospholipid (PL)-remodeling enzyme lysophosphatidylcholine (LPC) acyltransferase 3 by peroxisome proliferator-activated receptor δ to modulate the plasma triglyceride (TG) metabolism. In this study, we investigated the acute effects of hepatic ACSL4 deficiency on lipid metabolism in adult mice fed a high-fat diet (HFD). Adenovirus-mediated expression of a mouse ACSL4 shRNA (Ad-shAcsl4) in the liver of HFD-fed mice led to a 43% reduction of hepatic arachidonoyl-CoA synthetase activity and a 53% decrease in ACSL4 protein levels compared with mice receiving control adenovirus (Ad-shLacZ). Attenuated ACSL4 expression resulted in a substantial decrease in circulating VLDL-TG levels without affecting plasma cholesterol. Lipidomics profiling revealed that knocking down ACSL4 altered liver PL compositions, with the greatest impact on accumulation of abundant LPC species (LPC 16:0 and LPC 18:0) and lysophosphatidylethanolamine (LPE) species (LPE 16:0 and LPE 18:0). In addition, fasting glucose and insulin levels were higher in Ad-shAcsl4-transduced mice versus control (Ad-shLacZ). Glucose tolerance testing further indicated an insulin-resistant phenotype upon knockdown of ACSL4. These results provide the first in vivo evidence that ACSL4 plays a role in plasma TG and glucose metabolism and hepatic PL synthesis of hyperlipidemic mice.

Cardiac overexpression of perilipin 2 induces atrial steatosis, connexin 43 remodeling, and atrial fibrillation in aged mice.

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 mo before the study. Electron microscopy of the atria of PLIN2-Tg mice showed accumulation of small lipid droplets around mitochondrial chains, and five- to ninefold greater atrial triacylglycerol (TAG) content compared with wild-type (WT) mice. Electrocardiography showed significantly longer RR intervals in PLIN2-Tg mice than in WT mice. Transesophageal electrical burst pacing resulted in significantly higher prevalence of sustained (>5 min) AF (69%) in PLIN2-Tg mice than in WT mice (24%), although it was comparable in younger (4-mo-old) mice. Connexin 43 (Cx43), a gap junction protein, was localized at the intercalated disks 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.

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.

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.

Scavenger receptor B type 1: expression, molecular regulation, and cholesterol transport function.

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 posttranscriptional and posttranslational regulation. SR-B1 operates in several metabolic processes and contributes to pathogenesis of atherosclerosis, inflammation, hepatitis C virus 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, posttranscriptional, and posttranslational 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 SR-B1's role may yield improved therapies for a wide variety of conditions.

Nordihydroguaiaretic Acid, a Lignan from Larrea tridentata (Creosote Bush), Protects Against American Lifestyle-Induced Obesity Syndrome Diet-Induced Metabolic Dysfunction in Mice.

To determine the effects of nordihydroguaiaretic acid (NDGA) on metabolic and molecular changes in response to feeding a typical American fast food or Western diet, mice were fed an American lifestyle-induced obesity syndrome (ALIOS) diet and subjected to metabolic analysis. Male C57BL/6J mice were randomly assigned to the ALIOS diet, the ALIOS diet supplemented with NDGA (NDGA+ALIOS), or a control diet and were maintained on the specific diet for 8 weeks. Mice fed the ALIOS diet showed increased body, liver, and epididymal fat pad weight as well as increased plasma alanine transaminase (ALT) and aspartate aminotransferase (AST) levels (a measure of liver injury) and liver triglyceride content. Coadministration of NDGA normalized body and epididymal fat pad weight, ALT and AST levels, and liver triglycerides. NDGA treatment also improved insulin sensitivity but not glucose intolerance in mice fed the ALIOS diet. In mice fed the NDGA+ALIOS diet, NDGA supplementation induced peroxisome proliferator-activated receptor α (PPARα; the master regulator of fatty acid oxidation) and mRNA levels of carnitine palmitoyltransferases Cpt1c and Cpt2, key genes involved in fatty acid oxidation, compared with the ALIOS diet. NDGA significantly reduced liver endoplasmic reticulum (ER) stress response C/EBP homologous protein, compared with chow or the ALIOS diet, and also ameliorated ALIOS diet-induced elevation of apoptosis signaling protein, caspase 3. Likewise, NDGA downregulated the ALIOS diet-induced mRNA levels of Pparg, fatty acid synthase Fasn, and diacylglycerol acyltransferase Dgat2 NDGA treatment of ALIOS-fed mice upregulated the hepatic expression of antioxidant enzymes, glutathione peroxidase 4, and peroxiredoxin 3 proteins. In conclusion, we provide evidence that NDGA improves metabolic dysregulation by simultaneously modulating the PPARα transcription factor and key genes involved in fatty acid oxidation, key antioxidant and lipogenic enzymes, and apoptosis and ER stress signaling pathways.

Cardiac overexpression of perilipin 2 induces dynamic steatosis: prevention by hormone-sensitive lipase.

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.

Lipid droplets and steroidogenic cells.

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.

A Novel Role of Salt-Inducible Kinase 1 (SIK1) in the Post-Translational Regulation of Scavenger Receptor Class B Type 1 Activity.

Salt-inducible kinase 1 (SIK1) is a serine/threonine kinase that belongs to the stress- and energy-sensing AMPK family of kinases. SIK1 expression is rapidly induced in Y1 adrenal cells in response to ACTH via the cAMP-PKA signaling cascade, and it has been suggested that an increased level of SIK1 expression inhibits adrenal steroidogenesis by repressing the cAMP-dependent transcription of steroidogenic proteins, CYP11A1 and StAR, by attenuating CREB transcriptional activity. Here we show that SIK1 stimulates adrenal steroidogenesis by modulating the selective HDL-CE transport activity of SR-B1. Overexpression of SIK1 increases cAMP-stimulated and SR-B1-mediated selective HDL-BODIPY-CE uptake in cell lines without impacting SR-B1 protein levels, whereas knockdown of SIK1 attenuated cAMP-stimulated selective HDL-BODIPY-CE uptake. SIK1 forms a complex with SR-B1 by interacting with its cytoplasmic C-terminal domain, and in vitro kinase activity measurements indicate that SIK1 can phosphorylate the C-terminal domain of SR-B1. Among potential phosphorylation sites, SIK1-catalyzed phosphorylation of Ser496 is critical for SIK1 stimulation of the selective CE transport activity of SR-B1. Mutational studies further demonstrated that both the intact catalytic activity of SIK1 and its PKA-catalyzed phosphorylation are essential for SIK1 stimulation of SR-B1 activity. Finally, overexpression of SIK1 caused time-dependent increases in SR-B1-mediated and HDL-supported steroid production in Y1 cells; however, these effects were lost with knockdown of SR-B1. Taken together, these studies establish a role for SIK1 in the positive regulation of selective HDL-CE transport function of SR-B1 and steroidogenesis and suggest a potential mechanism for SIK1 signaling in modulating SR-B1-mediated selective CE uptake and associated steroidogenesis.

Using SRM-MS to quantify nuclear protein abundance differences between adipose tissue depots of insulin-resistant mice.

Insulin resistance (IR) underlies metabolic disease. Visceral, but not subcutaneous, white adipose tissue (WAT) has been linked to the development of IR, potentially due to differences in regulatory protein abundance. Here we investigate how protein levels are changed in IR 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 (SVCs) was impaired. In an in vitro approach to understand the cause of this impaired differentiation, we compared insulin-resistant visceral SVCs 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.

Mechanisms of action of hormone-sensitive lipase in mouse Leydig cells: its role in the regulation of the steroidogenic acute regulatory protein.

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.

Regulation of expression and function of scavenger receptor class B, type I (SR-BI) by Na+/H+ exchanger regulatory factors (NHERFs).

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.

MicroRNA regulation of adrenal glucocorticoid and androgen biosynthesis.

Steroid hormones are derived from a common precursor molecule, cholesterol, and regulate a wide range of physiologic function including reproduction, salt balance, maintenance of secondary sexual characteristics, response to stress, neuronal function, and various metabolic processes. Among the steroids synthesized by the adrenal and gonadal tissues, adrenal mineralocorticoids, and glucocorticoids are essential for life. The process of steroidogenesis is regulated at multiple levels largely by transcriptional, posttranscriptional, translational, and posttranslational regulation of the steroidogenic enzymes (i.e., cytochrome P450s and hydroxysteroid dehydrogenases), cellular compartmentalization of the steroidogenic enzymes, and cholesterol processing and transport proteins. In recent years, small noncoding RNAs, termed microRNAs (miRNAs) have been recognized as major post-transcriptional regulators of gene expression with essential roles in numerous biological processes and disease pathologies. Although their role in the regulation of steroidogenesis is still emerging, several recent studies have contributed significantly to our understanding of the role miRNAs play in the regulation of the steroidogenic process. This chapter focuses on the recent developments in miRNA regulation of adrenal glucocorticoid and androgen production in humans and rodents.

Fat-specific protein 27 modulates nuclear factor of activated T cells 5 and the cellular response to stress.

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.