Enhanced acyl-CoA:cholesterol acyltransferase activity increases cholesterol levels on the lipid droplet surface and impairs adipocyte function.
ABSTRACT: Cholesterol plays essential structural and signaling roles in mammalian cells, but too much cholesterol can cause cytotoxicity. Acyl-CoA:cholesterol acyltransferases 1 and 2 (ACAT1/2) convert cholesterol into its storage form, cholesteryl esters, regulating a key step in cellular cholesterol homeostasis. Adipose tissue can store >50% of whole-body cholesterol. Interestingly, however, almost no ACAT activity is present in adipose tissue, and most adipose cholesterol is stored in its free form. We therefore hypothesized that increased cholesterol esterification may have detrimental effects on adipose tissue function. Here, using several approaches, including protein overexpression, quantitative RT-PCR, immunofluorescence, and various biochemical assays, we found that ACAT1 expression is significantly increased in the adipose tissue of the ob/ob mice. We further demonstrated that ACAT1/2 overexpression partially inhibited the differentiation of 3T3-L1 preadipocytes. In mature adipocytes, increased ACAT activity reduced the size of lipid droplets (LDs) and inhibited lipolysis and insulin signaling. Paradoxically, the amount of free cholesterol increased on the surface of LDs in ACAT1/2-overexpressing adipocytes, accompanied by increased LD localization of caveolin-1. Moreover, cholesterol depletion in adipocytes by treating the cells with cholesterol-deficient media or ?-cyclodextrins induced changes in cholesterol distribution that were similar to those caused by ACAT1/2 overexpression. Our results suggest that ACAT1/2 overexpression increases the level of free cholesterol on the LD surface, thereby impeding adipocyte function. These findings provide detailed insights into the role of free cholesterol in LD and adipocyte function and suggest that ACAT inhibitors have potential utility for managing disorders associated with extreme obesity.
Project description:As adipose tissue is the major cholesterol storage organ and most of the intracellular cholesterol is distributed to lipid droplets (LDs), cholesterol homeostasis may have a role in the regulation of adipocyte size and function. ACATs catalyze the formation of cholesteryl ester (CE) from free cholesterol to modulate the cholesterol balance. Despite the well-documented role of ACATs in hypercholesterolemia, their role in LD development during adipogenesis remains elusive. Here, we identify ACATs as regulators of de novo lipogenesis and LD formation in murine 3T3-L1 adipocytes. Pharmacological inhibition of ACAT activity suppressed intracellular cholesterol and CE levels, and reduced expression of genes involved in cholesterol uptake and efflux. ACAT inhibition resulted in decreased de novo lipogenesis, as demonstrated by reduced maturation of SREBP1 and SREBP1-downstream lipogenic gene expression. Consistent with this observation, knockdown of either ACAT isoform reduced total adipocyte lipid content by approximately 40%. These results demonstrate that ACATs are required for storage ability of lipids and cholesterol in adipocytes.
Project description:Sterol regulatory element-binding protein-1 (SREBP-1) has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor ? (PPAR?) enhances perilipin (plin) gene expression, resulting in generating lipid droplets (LDs) to store triacylglycerol (TAG) in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT) of plin-/- mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin-/- mouse embryonic fibroblasts (MEFs) differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER), alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin-/- WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.
Project description:AIMS/INTRODUCTION:Fat-specific protein 27 (FSP27) α is the major isoform of FSP27 in white adipose tissue (WAT), and is essential for large unilocular lipid droplet (LD) formation in white adipocytes. In contrast, FSP27β is abundantly expressed in brown adipose tissue (BAT), and plays an important role in small multilocular LD formation. In FSP27 KO mice in which FSP27α and β are both depleted, WAT is characterized by multilocular LD formation, and by increased mitochondrial abundance and energy expenditure, whereas BAT conversely manifests large oligolocular LDs and reduced energy expenditure. MATERIALS AND METHODS:We investigated the effects of autophagy in WAT and BAT of wild type (WT) and FSP27 knockout (KO) mice. In addition, we examined the effects of FSP27α and FSP27β to the induction of autophagy in COS cells. RESULTS:Food deprivation induced autophagy in BAT of WT mice, as well as in WAT of FSP27 KO mice, suggesting that enhanced autophagy is characteristic of adipocytes with small multilocular LDs. Pharmacological inhibition of autophagy attenuated the fasting-induced loss of LD area in adipocytes with small multilocular LDs (BAT of WT mice and WAT of FSP27 KO mice), without affecting that in adipocytes with large unilocular or oligolocular LDs (WAT of WT mice or in BAT of FSP27 KO mice). Overexpression of FSP27α inhibited autophagy induction by serum deprivation in COS cells, whereas that of FSP27β had no such effect. CONCLUSIONS:The present results thus showed that FSP27α inhibits autophagy and might thereby contribute to the energy-storage function of WAT.
Project description:OBJECTIVE:Brown adipose tissue (BAT) thermogenesis depends on the mobilization and oxidation of fatty acids from intracellular lipid droplets (LD) within brown adipocytes (BAs); however, the identity and function of LD proteins that control BAT lipolysis remain incomplete. Proteomic analysis of mouse BAT subcellular fractions identified vacuolar protein sorting 13C (VPS13C) as a novel LD protein. The aim of this work was to investigate the role of VPS13C on BA LDs. METHODS:Biochemical fractionation and high resolution confocal and immuno-transmission electron microscopy (TEM) were used to determine the subcellular distribution of VPS13C in mouse BAT, white adipose tissue, and BA cell culture. Lentivirus-delivered shRNA was used to determine the role of VPS13C in regulating lipolysis and gene expression in cultured BA cells. RESULTS:We found that VPS13C is highly expressed in mouse BAT where it is targeted to multilocular LDs in a subspherical subdomain. In inguinal white adipocytes, VPS13C was mainly observed on small LDs and β3-adrenergic stimulation increased VPS13C in this depot. Silencing of VPS13C in cultured BAs decreased LD size and triglyceride content, increased basal free fatty acid release, augmented the expression of thermogenic genes, and enhanced the lipolytic potency and efficacy of isoproterenol. Mechanistically, we found that BA lipolysis required activation of adipose tissue triglyceride lipase (ATGL) and that loss of VPS13C greatly increased the association of ATGL to LDs. CONCLUSIONS:VPS13C is present on BA LDs where is targeted to a distinct subdomain. VPS13C limits the access of ATGL to LD and loss of VPS13C elevates lipolysis and promotes oxidative gene expression.
Project description:Lipid bodies are eukaryotic structures for temporary storage of neutral lipids such as acylglycerols and steryl esters. Fatty acyl-CoA and cholesterol are two substrates for cholesteryl ester (CE) synthesis via the ACAT reaction. The intracellular parasite Toxoplasma gondii is incapable of sterol synthesis and unremittingly scavenges cholesterol from mammalian host cells. We previously demonstrated that the parasite expresses a cholesteryl ester-synthesizing enzyme, TgACAT1. In this article, we identified and characterized a second ACAT-like enzyme, TgACAT2, which shares 56% identity with TgACAT1. Both enzymes are endoplasmic reticulum-associated and contribute to CE formation for storage in lipid bodies. While TgACAT1 preferentially utilizes palmitoyl-CoA, TgACAT2 has broader fatty acid specificity and produces more CE. Genetic ablation of each individual ACAT results in parasite growth impairment whereas dual ablation of ACAT1 and ACAT2 is not tolerated by Toxoplasma. ?ACAT1 and ?ACAT2 parasites have reduced CE levels, fewer lipid bodies, and accumulate free cholesterol, which causes injurious membrane effects. Mutant parasites are particularly vulnerable to ACAT inhibitors. This study underlines the important physiological role of ACAT enzymes to store cholesterol in a sterol-auxotrophic organism such as Toxoplasma, and furthermore opens up possibilities of exploiting TgACAT as targets for the development of antitoxoplasmosis drugs.
Project description:We previously showed that adipose differentiation related protein (Adfp)-deficient mice display a 60% reduction in hepatic triglyceride (TG) content. In this study, we investigated the role of ADFP in lipid and glucose homeostasis in a genetic obesity model, Lep(ob/ob) mice. We bred Adfp(-/-) mice with Lep(ob/ob) mice to create Lep(ob/ob)/Adfp(-/-) and Lep(ob/ob)/Adfp(+/+) mice and analyzed the hepatic lipids, lipid droplet (LD) morphology, LD protein composition and distribution, lipogenic gene expression, and VLDL secretion, as well as insulin sensitivity of the two groups of mice. Compared with Lep(ob/ob)/Adfp(+/+) mice, Lep(ob/ob)/Adfp(-/-) mice displayed an increased VLDL secretion rate, a 25% reduction in hepatic TG associated with improvement in fatty liver grossly and microscopically with a change of the size of LDs in a proportion of the hepatocytes and a redistribution of major LD-associated proteins from the cytoplasmic compartment to the LD surface. There was no detectable change in lipogenic gene expression. Lep(ob/ob)/Adfp(-/-) mice also had improved glucose tolerance and insulin sensitivity in both liver and muscle. The alteration of LD size in the liver of Lep(ob/ob)/Adfp(-/-) mice despite the relocation of other LDPs to the LD indicates a nonredundant role for ADFP in determining the size and distribution of hepatic LDs.
Project description:Lipid droplets (LDs), also called adiposomes, are found in many eukaryotic cells, and are highly upregulated in lipid-storage cells, such as adipocytes. The mechanism by which adiposomes and their component neutral lipids are degraded is an important health issue with the rapidly spreading epidemic of obesity. Recently, a novel triglyceride lipase (adipose triglyceride lipase (ATGL)) that catalyses the initial step in triglyceride hydrolysis in adipocyte LDs was identified. Here, we show that ATGL also functions in non-adipocyte cells, and has an important role in LD degradation in these cells. Overexpression of wild-type ATGL causes a marked decrease in LD size, whereas a catalytically inactive mutant retains the ability to localize to LDs, but is unable to decrease their size. Depletion of ATGL by RNA interference leads to a significant increase in the size of LDs. These results show that ATGL has an important role in LD/adiposome turnover in mammalian cells.
Project description:The total contribution of the acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, to mammalian triacylglycerol (TG) synthesis has not been determined. Similarly, whether DGAT enzymes are required for lipid droplet (LD) formation is unknown. In this study, we examined the requirement for DGAT enzymes in TG synthesis and LDs in differentiated adipocytes with genetic deletions of DGAT1 and DGAT2. Adipocytes with a single deletion of either enzyme were capable of TG synthesis and LD formation. In contrast, adipocytes with deletions of both DGATs were severely lacking in TG and did not have LDs, indicating that DGAT1 and DGAT2 account for nearly all TG synthesis in adipocytes and appear to be required for LD formation during adipogenesis. DGAT enzymes were not absolutely required for LD formation in mammalian cells, however; macrophages deficient in both DGAT enzymes were able to form LDs when incubated with cholesterol-rich lipoproteins. Although adipocytes lacking both DGATs had no TG or LDs, they were fully differentiated by multiple criteria. Our findings show that DGAT1 and DGAT2 account for the vast majority of TG synthesis in mice, and DGAT function is required for LDs in adipocytes, but not in all cell types.
Project description:Cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative diseases, including the abnormal accumulation of amyloid-beta, one of the pathological hallmarks of Alzheimer disease (AD). Acyl-CoA:cholesterol acyltransferases (ACAT1 and ACAT2) are two enzymes that convert free cholesterol to cholesteryl esters. ACAT inhibitors have recently emerged as promising drug candidates for AD therapy. However, how ACAT inhibitors act in the brain has so far remained unclear. Here we show that ACAT1 is the major functional isoenzyme in the mouse brain. ACAT1 gene ablation (A1-) in triple transgenic (i.e., 3XTg-AD) mice leads to more than 60% reduction in full-length human APPswe as well as its proteolytic fragments, and ameliorates cognitive deficits. At 4 months of age, A1- causes a 32% content increase in 24-hydroxycholesterol (24SOH), the major oxysterol in the brain. It also causes a 65% protein content decrease in HMG-CoA reductase (HMGR) and a 28% decrease in sterol synthesis rate in AD mouse brains. In hippocampal neurons, A1- causes an increase in the 24SOH synthesis rate; treating hippocampal neuronal cells with 24SOH causes rapid declines in hAPP and in HMGR protein levels. A model is provided to explain our findings: in neurons, A1- causes increases in cholesterol and 24SOH contents in the endoplasmic reticulum, which cause reductions in hAPP and HMGR protein contents and lead to amelioration of amyloid pathology. Our study supports the potential of ACAT1 as a therapeutic target for treating certain forms of AD.
Project description:Interventions on macrophages/foam cells to redirect intracellular cholesterol towards efflux pathways could become a very valuable addition to our therapeutic arsenal against atherosclerosis. However, certain manipulations of the cholesteryl ester cycle, such as the inhibition of ACAT1, an ER-resident enzyme that re-esterifies cholesterol, are not well tolerated. Previously we showed that targeting perilipin-2 (PLIN2), a major lipid droplet (LD)-associated protein in macrophages, prevents foam cell formation and protects against atherosclerosis. Here we have assessed the tolerance of PLIN2-deficient bone marrow derived macrophages (BMM) to several lipid loading conditions similar to the found during atherosclerosis development, including exposure to modified low-density lipoprotein (mLDL) and 7-ketocholesterol (7-KC), a free cholesterol (FC) metabolite, in media with or without cholesterol acceptors. BMM isolated from mice that do or do not express PLIN2 were tested for apoptosis (TUNEL and cleaved caspase-3), ER stress (CHOP induction and XBP-1 splicing), and inflammation (TNF-? and IL-6 mRNA levels). Like in other cell types, PLIN2 deficiency impairs LD buildup in BMM. However, while most stress parameters were elevated in macrophages under ACAT inhibition and 7-KC loading, PLIN2 inactivation was well tolerated. The data support the safety of targeting PLIN2 to prevent foam cell formation and atherosclerosis.