Suppression of preadipocyte differentiation and promotion of adipocyte death by HIV protease inhibitors.
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Many human immunodeficiency virus (HIV)-infected patients taking combination antiretroviral therapy that includes HIV protease inhibitors experience atrophy of peripheral subcutaneous adipose tissue. We investigated the effects of HIV protease inhibitors on adipogenesis and adipocyte survival using the 3T3-L1 preadipocyte cell line. Several HIV protease inhibitors were found either to inhibit preadipocyte differentiation or to promote adipocyte cell death. One protease inhibitor, nelfinavir, elicited both of these effects strongly. When induced to differentiate in the presence of nelfinavir, 3T3-L1 preadipocytes failed to accumulate cytoplasmic triacylglycerol and failed to express normal levels of the adipogenic transcription factors CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. The level of the proteolytically processed, active 68-kDa form of sterol regulatory element-binding protein-1, a transcription factor known to promote lipogenic gene expression, also was reduced markedly in nelfinavir-treated cells, whereas the level of the 125-kDa precursor form of this protein was unaffected. The inhibitory effect of nelfinavir occurred subsequent to critical early events in preadipocyte differentiation, expression of CCAAT/enhancer-binding protein beta and completion of the mitotic clonal expansion phase, because these events were unaffected by nelfinavir treatment. In addition, nelfinavir treatment of fully differentiated 3T3-L1 adipocytes resulted in DNA strand cleavage and severe loss of cell viability. In contrast, cell proliferation and viability of preadipocytes were unaffected by nelfinavir treatment. Thus, molecular or cellular changes that occur during acquisition of the adipocyte phenotype promote susceptibility to nelfinavir-induced cell death. When considered together, these results suggest that nelfinavir may promote adipose tissue atrophy by compromising adipocyte viability and preventing replacement of lost adipocytes by inhibiting preadipocyte differentiation. (+info)
Stimulation of tumor-associated fatty acid synthase expression by growth factor activation of the sterol regulatory element-binding protein pathway.
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Increased expression of fatty acid synthase (FAS) is observed in a clinically aggressive subset of various common cancers and interference with FAS offers promising opportunities for selective chemotherapeutic intervention. The mechanisms by which FAS expression is (up)-regulated in these tumors remain, however, largely unknown. Recently we demonstrated that in LNCaP prostate cancer cells FAS expression is markedly elevated by androgens via an indirect pathway involving sterol regulatory element-binding proteins (SREBPs). Here, we also show that growth factors such as EGF are able to stimulate FAS mRNA, protein and activity. Several observations also indicate that the effects of EGF on FAS expression are ultimately mediated by SREBPs. EGF stimulates SREBP-1c mRNA expression and induces an increase in mature nuclear SREBP-1. Moreover, in transient transfection studies EGF stimulates the transcriptional activity of a 178 bp FAS promoter fragment harboring a complex SREBP-binding site. Deletion or mutation of this binding site abolishes these effects and ectopic expression of dominant negative SREBP-1 inhibits FAS expression and induction in intact LNCaP cells. Given the frequent dysregulation of growth factor signaling in cancer and the key role of SREBP-1 in lipid homeostasis, growth factor-induced activation of the SREBP pathway is proposed as one of the mechanisms responsible for up-regulation of lipogenic gene expression in a subset of cancer cells. (+info)
Unsaturated fatty acids down-regulate srebp isoforms 1a and 1c by two mechanisms in HEK-293 cells.
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Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that increase the synthesis of fatty acids as well as cholesterol in animal cells. All three SREBP isoforms (SREBP-1a, -1c, and -2) are subject to feedback regulation by cholesterol, which blocks their proteolytic release from membranes. Previous data indicate that the SREBPs are also negatively regulated by unsaturated fatty acids, but the mechanism is uncertain. In the current experiments, unsaturated fatty acids decreased the nuclear content of SREBP-1, but not SREBP-2, in cultured human embryonic kidney (HEK)-293 cells. The potency of unsaturated fatty acids increased with increasing chain length and degree of unsaturation. Oleate, linoleate, and arachidonate were all effective, but the saturated fatty acids palmitate and stearate were not effective. Down-regulation occurred at two levels. The mRNAs encoding SREBP-1a and SREBP-1c were markedly reduced, and the proteolytic processing of these SREBPs was inhibited. When SREBP-1a was produced by a cDNA expressed from an independent promoter, unsaturated fatty acids reduced nuclear SREBP-1a without affecting the mRNA level. There was no effect when the cDNA encoded a truncated version that was not membrane-bound. When administered together, sterols and unsaturated fatty acids potentiated each other in reducing nuclear SREBP-1. In the absence of fatty acids, sterols did not cause a sustained reduction of nuclear SREBP-1, but they did reduce nuclear SREBP-2. We conclude that unsaturated fatty acids, as well as sterols, can down-regulate nuclear SREBPs and that unsaturated fatty acids have their greatest inhibitory effects on SREBP-1a and SREBP-1c, whereas sterols have their greatest inhibitory effects on SREBP-2. (+info)
Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta.
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The liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily that are bound and activated by oxysterols. These receptors serve as sterol sensors to regulate the transcription of gene products that control intracellular cholesterol homeostasis through catabolism and transport. In this report, we describe a novel LXR target, the sterol regulatory element-binding protein-1c gene (SREBP-1c), which encodes a membrane-bound transcription factor of the basic helix-loop-helix-leucine zipper family. SREBP-1c expression was markedly increased in mouse tissues in an LXR-dependent manner by dietary cholesterol and synthetic agonists for both LXR and its heterodimer partner, the retinoid X receptor (RXR). Expression of the related gene products, SREBP-1a and SREBP-2, were not increased. Analysis of the mouse SREBP-1c gene promoter revealed an RXR/LXR DNA-binding site that is essential for this regulation. The transcriptional increase in SREBP-1c mRNA by RXR/LXR was accompanied by a similar increase in the level of the nuclear, active form of the SREBP-1c protein and an increase in fatty acid synthesis. Because this active form of SREBP-1c controls the transcription of genes involved in fatty acid biosynthesis, our results reveal a unique regulatory interplay between cholesterol and fatty acid metabolism. (+info)
Role of LXRs in control of lipogenesis.
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The discovery of oxysterols as the endogenous liver X receptor (LXR) ligands and subsequent gene targeting studies in mice provided strong evidence that LXR plays a central role in cholesterol metabolism. The identification here of a synthetic, nonsteroidal LXR-selective agonist series represented by T0314407 and T0901317 revealed a novel physiological role of LXR. Oral administration of T0901317 to mice and hamsters showed that LXR activated the coordinate expression of major fatty acid biosynthetic genes (lipogenesis) and increased plasma triglyceride and phospholipid levels in both species. Complementary studies in cell culture and animals suggested that the increase in plasma lipids occurs via LXR-mediated induction of the sterol regulatory element-binding protein 1 (SREBP-1) lipogenic program. (+info)
Interferon-gamma-induced regulation of peroxisome proliferator-activated receptor gamma and STATs in adipocytes.
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Interferon-gamma (IFN-gamma) is known primarily for its roles in immunological responses but also has been shown to affect fat metabolism and adipocyte gene expression. To further investigate the effects of IFN-gamma on fat cells, we examined the effects of this cytokine on the expression of adipocyte transcription factors in 3T3-L1 adipocytes. Although IFN-gamma regulated the expression of several adipocyte transcription factors, IFN-gamma treatment resulted in a rapid reduction of both peroxisome proliferator-activated receptor (PPAR) protein and mRNA. A 48-h exposure to IFN-gamma also resulted in a decrease of both CCAAT/enhancer-binding alpha and sterol regulatory element binding protein (SREBP-1) expression. The short half-life of both the PPARgamma mRNA and protein likely contributed to the rapid decline of both cytosolic and nuclear PPARgamma in the presence of IFN-gamma. Our studies clearly demonstrated that the IFN-gamma-induced loss of PPARgamma protein is partially inhibited in the presence of two distinct proteasome inhibitors. Moreover, IFN-gamma also inhibited the transcription of PPARgamma, which was accompanied by a decrease in PPARgamma mRNA accumulation. In addition, exposure to IFN-gamma resulted in a substantial increase in STAT 1 expression and a small increase in STAT 3 expression. IFN-gamma treatment of 3T3-L1 adipocytes (48-96 h) resulted in a substantial inhibition of insulin-sensitive glucose uptake. These data clearly demonstrate that IFN-gamma treatment results in the development of insulin resistance, which is accompanied by the regulation of various adipocyte transcription factors, in particular the synthesis and degradation of PPARgamma. (+info)
Copper deficiency induces hepatic fatty acid synthase gene transcription in rats by increasing the nuclear content of mature sterol regulatory element binding protein 1.
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Dietary copper (Cu) deficiency results in an accelerated rate of hepatic fatty acid synthase gene transcription and an enhanced rate of hepatic lipid synthesis. Because the nuclear transcription factor sterol regulatory element binding protein-1 (SREBP-1) is a strong enhancer of fatty acid synthase promoter activity, it was hypothesized that Cu deficiency induces fatty acid synthase gene transcription by increasing the nuclear localization of mature SREBP-1. Male weanling rats were pair-fed a Cu-adequate (6.0 mg/kg) or Cu-deficient (0.6 mg/kg) diet (AIN-93) for 28 d. DNase I hypersensitivity site mapping of the hepatic fatty acid synthase gene revealed the presence of four major hypersensitivity sites located at -8700 to -8600, -7300 to -6900, -600 to -400 and -100 to +50. Although Cu deficiency did not change the hypersensitivity site pattern or intensity, in vitro footprinting of the region between -100 and +50 indicated that Cu deficiency enhanced DNA protein interactions within this region. The sequence between -68 and -58 contains the DNA recognition sequence for SREBP-1 and upstream stimulatory element-1 (USF-1). Western blot analysis revealed that the dietary Cu deficiency increased the hepatic nuclear content of mature SREBP-1 by 150% (P: < 0.05), and it concomitantly decreased the membrane content of precursor SREBP-1 by 45% (P: < 0.05). Changes in the hepatic distribution of SREBP-1 associated with Cu deficiency were not accompanied by changes in SREBP-1 mRNA. The nuclear content of USF-1 was unaffected by dietary Cu status. The hepatic increase in mature SREBP-1 of Cu-deficient rats was accompanied by a 400% increase and an 80% decrease in the abundance of fatty acid synthase and cholesterol 7-alpha hydroxylase mRNA, respectively. hepatic These data indicate that a Cu deficiency stimulates hepatic lipogenic gene expression by increasing the hepatic translocation of mature SREBP-1. (+info)
Structure and regulation of mammalian squalene synthase.
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Mammalian squalene synthase (SQS) catalyzes the first reaction of the branch of the isoprenoid metabolic pathway committed specifically to sterol biosynthesis. SQS produces squalene in an unusual two-step reaction in which two molecules of farnesyl diphosphate are condensed head-to-head. Recent studies have advanced understanding of the reaction mechanism, the functional domains of the enzyme, and transcriptional regulation of the gene. Site-directed mutagenesis has identified conserved Asp, Tyr, and Phe residues that are essential for SQS activity. The Asp residues are hypothesized to be required for substrate binding; the Tyr and Phe residues may stabilize carbocation reaction intermediates. The elucidation of SQS crystal structure will most likely direct future research on the relationship between enzyme structure and function. SQS activity, protein, and mRNA levels are regulated by cholesterol status and by the cytokines TNF-alpha and IL-1beta. Activation of the SQS promoter in response to cholesterol deficit is mediated by sterol regulatory element binding proteins SREBP-1a and SREBP-2. The precise contributions made by individual SREBPs and accessory transcription factors to SQS transcriptional control, and the mechanisms underlying cytokine regulation of SQS are major foci of current research. (+info)