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Lipid droplets are ubiquitous organdies found in almost all living organisms for the storage of energy, sterols and precursors of membrane phospholipids. Many metabolic diseases including obesity, diabetes, fatty liver, cardiovascular disease and neutral lipid.-storage diseases are associated with abnormalities in intracellular lipid storage. It is becoming increasingly clear that, apart from a core of neutral lipids and a surface monolayer of polar lipids, many proteins are found in the lipid droplet, and lipid droplet associated proteins are functionally involved in the biology of lipid homeostasis and lipid metabolism. To investigate the relative expression of various lipid droplet proteins and the potential alterations in the expression of them in obesity and diabetes in vivo, we have examined the relative expression levels of 10 lipid droplet -associated proteins in white adipose tissue (WAT) of mice fed a normal chow or high fat diet using RT-taqman quantitative PCR analysis. They axe perilipin (Plin), adipocyte differentiation-related protein (ADRP, also known as adipophilin), tail-interacting protein of 47 kDa (Tip47), S3-12, lipid storage droplet protein 5 (LSDP-5), mouse homolog of protein encoded by PSO4/PRP 19 gene in yeast (prp 19P), vimentin (Vim), caveolin-1 (Cave-1), OXPAT, and associated with lipid droplet protein-1 (ALD-1). The following are the relative expression levels (normalized to control gene 36B4) in normal mouse WAT in decreasing order of abundance: Plin, Cave-1, S3-12, Van, ADRP, prp19P, LSDP-5, OXPAT, Tip47 and ALD-1. High fat feeding changed the expression level of several droplet associated proteins; Plin and Cave-1 were decreased about 30%, S3-12 was decreased almost 50%, whereas the expression of ADRP increased 25%. The others did not show significant changes with high fat feeding. We conclude that multiple lipid droplet associated proteins are expressed in white adipose tissue and are differentially regulated by high fat diet feeding. [ABSTRACT FROM AUTHOR]

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. How can progenitor cells attain a distinct differentiated cell identity, given the fluctuating signaling environment and redundancy of critical transcription factors? This study identifies a control principle whereby progenitor cells induced to differentiate can reach the desired distinct differentiated cell fate despite the possibility of many alternative fates en route. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Presents a study which evaluated automated telephone disease management with telephone nurse follow-up for improving diabetes treatment processes in the United States Department of Veterans Affairs clinics. Significance of telephone care programs; Research design and methodology; Results and conclusions.

Abstract 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 β-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 for cardiovascular disease beyond their direct anti-atherosclerosis properties previously described. Highlights • ABCA1 agonist peptides, CS6253 and T6991-2, improve glucose tolerance in two mouse models. • In vitro, CS6253 promotes insulin secretion, glucose transport and inhibits glucose production. • An intact leptin receptor signaling is required for the maximal glycemic actions of CS6253. • The peptides have the clinical potential to simultaneously treat atherosclerosis and diabetes. [ABSTRACT FROM AUTHOR]

Hormone-sensitive lipase (HSL) is an intracellular neutral lipase capable of hydrolyzing acylglycerols, as well as cholesteryl and retinyl esters. In mice, HSL deficiency results in male sterility. Lipid rafts, a plasma membrane microdomain enriched in cholesterol, sphingolipids and saturated glycerophospholipids, comprise a highly dynamic clustering of proteins and lipids that play a central role in signal transduction and intercellular communication. In the present work, we examined the effect of HSL deficiency in the lipid composition of plasma membrane microdomains in mouse testis. The lack of HSL affected the density of lipid rafts, as indicated by the shifting of caveolin 1 to denser fractions in the sucrose-gradient fractionation, and altered the sterol and phospholipid composition of both lipid raft and non-raft fractions, the biochemical differences among them being less obvious in HSL −/− than in HSL +/+ mice. Compared to HSL +/+ , the lipid rafts from HSL −/− mice had significantly less desmosterol and T-MAS, while the non-raft domain had increased cholesterol content. Lipid rafts from HSL −/− mice had reduced PUFA-containing phospholipid species but increased phosphatidylcholine and phosphatidylethanolamine species with monounsaturated fatty acid moieties, while the non-raft domain was enriched in phosphatidylethanolamine and phosphatidylserine species having four double bonds. The changes in both the sterols and phospholipid composition of lipid raft and non-raft microdomains may have consequences in signal transduction and contribute to the sterility observed in HSL-deficient male mice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • Mice with HSL deficiency have accelerated bone healing • HSL deficiency was associated with increased osteocalcin and Runx2 expression • Greater numbers of MSCs were found at the injury site in HSL deficient mice • There is increased propensity for MSC differentiation with HSL inhibition • Single cell RNAseq showed pre-osteoclasts having the most differentially expressed genes with HSL deficiency [ABSTRACT FROM AUTHOR]

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 NHERF2mRNAlevels 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 theMERMdomains 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. [ABSTRACT FROM AUTHOR]

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 kinaseA(PKA) pathway, by acAMPanalog 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 theLXRtarget 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. [ABSTRACT FROM AUTHOR]

Summary: Introduction: 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). Methods: 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. Results: 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). Conclusions: 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. [Copyright &y& Elsevier]

Abstract: 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 (200mg/kg) for 16weeks. 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. [ABSTRACT FROM AUTHOR]

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 (KU) 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 KU 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 KU mice. Compared with Langendorff-perfused hearts isolated from fasted WT mice, the isolated KU hearts also displayed higher contractile function and a blunted response to FA. Although cardiac glucose uptake in KU mice was comparable with WT mice under all conditions tested, cardiac VLDL uptake and lipoprotein lipase (LPL) activity were twofold higher in KU mice during fasting. The KU 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. [ABSTRACT FROM AUTHOR]

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. Here we explored aspects of metabolism, steroid hormone production and neurobehavior of DN-SNAP25mut mice. 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. 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. • Dominant negative SNAP-25 results in enhanced insulin action in mice • Dominant negative SNAP-25 results in increased adrenal and hormonal production • Mice with dominant negative SNAP-25 display increased anxiety • Dominant negative SNAP-25 causes increased energy expenditure and beigeing of fat [ABSTRACT FROM AUTHOR]

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 ~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-α, 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-β, 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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 (fonskolin)-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 Pen 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 Pen A regulates ATGL-dependent lipolysis and identify serine 517 as the Pen A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphonylated or phosphomimetic serine 517. [ABSTRACT FROM AUTHOR]

Abstract: An unique isoform of hormone-sensitive lipase (HSL) is expressed in β-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 β-cell HSL and increased HSL hydrolytic activity in clonal β-cells. Short-term treatment with GLP-1 increased HSL activity without changing the expression of the β-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 β-cell isoform of HSL is involved in maintaining lipid homeostasis in islets and contributes to the proper control of GSIS. [Copyright &y& Elsevier]

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 periipin null mice (Pen-/- 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 AΔ1-6). PKA-stimulated HSL translocation was confirmed in differentiated brown adipocytes from perilipin null mice expressing an adipose-specific Peri AΔ1-6 transgene. Thus, PKA-induced HSL translocation was independent of perilipin phosphorylation. However, Peri AΔ1-6 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 AΔ1-6. 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. [ABSTRACT FROM AUTHOR]

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 AFABPHSL 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 µM 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. [ABSTRACT FROM AUTHOR]

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). [14C]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. [14C]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)-α and -#x03B5; 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. [Copyright &y& Elsevier]

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 AMPdependent 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 (Ser[sup 81], Ser[sup 222], and Ser[sup 276]). PKAstimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301-517 (Ser[sup 433], Ser[sup 492], and Ser[sup 517]). Peri 301-517 prorooted 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 nonHSL, 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. [ABSTRACT FROM AUTHOR]

To elucidate the role of hormone-sensitive lipase (HSL) in diet-induced obesity, HSLdeficient (HSL[sup -/-]) and wild-type mice were fed normal chow or high-fat diets. HSL[sup -/-] 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[sup -/-] mice. Serum insulin levels in the fed state and tumor necrosis factor-α 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[sup -/-] mice than in controls. Expression of transcription factors associated with adipogenesis (peroxisome proliferator-activated receptor-γ CAAT/enhancer-binding protein-α) 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[sup -/-] 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[sup -/-] mice. Our results suggest that impaired lipolysis in HSL deficiency affects lipid metabolism through alterations of adipose differentiation and adipose-derived hormone levels. [ABSTRACT FROM AUTHOR]

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 µM oleate, A-FABP and E-FABP each bound to HSL with high affinity (K[sub d] of 0.5 and 3 nM, respectively) in a ∼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 ∼2-fold using cholesteryl oleate as substrate but exhibited no activation using p-nitrophenyl butyrate. The activation by AFABP 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Presents information on a study that investigated the effect of starvation on cardiac gene expression by use of oligonucleotide arrays. Methodology of the study; Results and discussion on the study.

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. [ABSTRACT FROM AUTHOR]

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 LXRβ, 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 LXRα (Nr1h3) and LXRβ (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. • HSL is a key lipase in mice testis, as HSL deficiency results in male sterility. • HSL deficiency or lack in mice testis induces lipid homeostasis alterations. • HSL lack affects the cellular localization and expression of SR-BI, LDRr, ABCA1. • Key receptors/transporters involved in cellular cholesterol uptake and efflux. [ABSTRACT FROM AUTHOR]

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. • Excessive ROS production leads to oxidative damage to DNA, proteins and lipids. • Mitochondrial SOD2 protects cells against mitochondria-generated ROS toxicity. • SOD2-deficiency leads to excessive ROS production and oxidative stress. • Knockdown of SOD2 inhibits hormone-stimulated granulosa cell steroidogenesis. • SOD2 deficiency attenuates the expression of StAR and key steroidogenic genes. [ABSTRACT FROM AUTHOR]

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. 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. Unlabelled Image • ACSL4 preferentially esterifies polyunsaturated fatty acids such as arachidonic acid. • AA induces ACSL4 degradation via the ubiquitin-proteasome pathway. • Using LC-MS/MS & IP, we identified p115 as a novel ACSL4 interacting protein. • We established and validated that AA enhances ACSL4 interaction with p115. • We demonstrated that ACSL4 and p115 protein levels in liver are inversely correlated. [ABSTRACT FROM AUTHOR]

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. • ZDSD rat is a polygenic model of obesity, MetS and T2D with an intact leptin pathway. • ZDSD rats develop prediabetes, MetS and T2D that mimic development in humans. • ZDSD rats exhibit altered expression of key hepatic metabolic enzymes/proteins. • ZDSD rats exhibit elevated levels of ER stress/UPR markers. • Enhanced ER stress/UPR contributes to IR and associated metabolic abnormalities. [ABSTRACT FROM AUTHOR]

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. • SR-B1 can facilitate fatty acid uptake. • BLT-1, a specific inhibitor of SR-B1-mediated HDL-CE uptake, does not block SR-B1-mediated FA uptake. • Primary adipocytes from SR-B1 knockout mice have attenuated FA uptake. • SSO, an inhibitor of CD36-mediated FA uptake, does not block SR-B1-mediated FA uptake. [ABSTRACT FROM AUTHOR]

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. • Mice when fed a novel NASH diet develop obesity, insulin resistance, steatosis, fibrosis, inflammation, ER/oxidative stress and liver injury. • NDGA ameliorates these metabolic abnormalities and improved hepatic steatosis and fibrosis and NASH pathology. • NDGA attenuated expression of key genes involved in fibrosis, progenitor and macrophage markers with little or no effect on lipogenic genes. • NDGA exerts anti-NASH/anti-fibrotic actions and has therapeutic potential for treatment of patients with NASH. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2024. Published by Elsevier Inc.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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.

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.

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 (PPAR α ; 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 PPAR α transcription factor and key genes involved in fatty acid oxidation, key antioxidant and lipogenic enzymes, and apoptosis and ER stress signaling pathways., (U.S. Government work not protected by U.S. copyright.)

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., (Published by Elsevier B.V.)

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.

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.

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., (Published by Elsevier Inc.)

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., (Published by Elsevier Ireland Ltd.)