Wen-Jun Shen

Hormone-sensitive lipase control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis

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

Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes

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

IL-17 Regulates Adipogenesis, Glucose Homeostasis, and Obesity

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

Cellular cholesterol delivery, intracellular processing and utilization for biosynthesis of steroid hormones

Steroid hormones regulate diverse physiological functions such as reproduction, blood salt balance, maintenance of secondary sexual characteristics, response to stress, neuronal function and various metabolic processes. They are synthesized from cholesterol mainly in the adrenal gland and gonads in response to tissue-specific tropic hormones. These steroidogenic tissues are unique in that they require cholesterol not only for membrane biogenesis, maintenance of membrane fluidity and cell signaling, but also as the starting material for the biosynthesis of steroid hormones. It is not surprising, then, that cells of steroidogenic tissues have evolved with multiple pathways to assure the constant supply of cholesterol needed to maintain optimum steroid synthesis. The cholesterol utilized for steroidogenesis is derived from a combination of sources: 1) de novo synthesis in the endoplasmic reticulum (ER); 2) the mobilization of cholesteryl esters (CEs) stored in lipid droplets through cholesteryl ester hydrolase; 3) plasma lipoprotein-derived CEs obtained by either LDL receptor-mediated endocytic and/or SR-BI-mediated selective uptake; and 4) in some cultured cell systems from plasma membrane-associated free cholesterol. Here, we focus on recent insights into the molecules and cellular processes that mediate the uptake of plasma lipoprotein-derived cholesterol, events connected with the intracellular cholesterol processing and the role of crucial proteins that mediate cholesterol transport to mitochondria for its utilization for steroid hormone production. In particular, we discuss the structure and function of SR-BI, the importance of the selective cholesterol transport pathway in providing cholesterol substrate for steroid biosynthesis and the role of two key proteins, StAR and PBR/TSO in facilitating cholesterol delivery to inner mitochondrial membrane sites, where P450scc (CYP11A) is localized and where the conversion of cholesterol to pregnenolone (the common steroid precursor) takes place.

Functional interaction of hormone-sensitive lipase and perilipin in lipolysis

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

MicroRNAs 125a and 455 Repress Lipoprotein-Supported Steroidogenesis by Targeting Scavenger Receptor Class B Type I in Steroidogenic Cells

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

Human BMP-7/OP-1 induces the growth and differentiation of adipocytes and osteoblasts in bone marrow stromal cell cultures*

We studied the effects of BMP‐7/OP‐1 on growth and differentiation of bone marrow stromal cells. BMS2, a mouse bone marrow stromal cell line capable of differentiating into adipocytes and osteoblasts, were treated in a serum‐free medium containing differentiation agents that favor the expression of both lineages. BMP‐7/ OP‐1 stimulated cell proliferation and differentiation concomitantly. These effects were dose‐ and growth phase‐dependent. Cells were more sensitive to the treatment early in the culture (30–40% confluence) with a significant increase in cell proliferation and markers of differentiation at low concentrations. When treated later in the growth phase (90–100% confluence), no significant increase in cell proliferation was seen. The concentration requirement for cells later in the culture to reach an equivalent degree of differentiation was 3–10‐ fold higher than for cells treated early. In both cases, the effects on adipocyte differentiation were biphasic; low concentrations stimulated adipocyte differentiation which was inhibited at higher concentrations where stimulation of osteoblast markers were observed. We conclude that cell proliferation and cell differentiation into adipocyte/osteoblast can occur simultaneously under BMP‐7/OP‐1 treatment. J. Cell. Biochem. 82: 187–199, 2001. Published 2001 Wiley‐Liss, Inc.

Interaction of Hormone-sensitive Lipase with Steroidogeneic Acute Regulatory Protein FACILITATION OF CHOLESTEROL TRANSFER IN ADRENAL

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.

Characterization of age-related gene expression profiling in bone marrow and epididymal adipocytes.

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

Cardiac overexpression of hormone-sensitive lipase inhibits myocardial steatosis and fibrosis in streptozotocin diabetic mice.

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