Ceramide Transporter CERT Is Involved in Muscle Insulin Signaling Defects Under Lipotoxic Conditions.

One main mechanism of insulin resistance (IR), a key feature of type 2 diabetes, is the accumulation of saturated fatty acids (FAs) in the muscles of obese patients with type 2 diabetes. Understanding the mechanism that underlies lipid-induced IR is an important challenge. Saturated FAs are metabolized into lipid derivatives called ceramides, and their accumulation plays a central role in the development of muscle IR.

Caveolin-1 expression and cavin stability regulate caveolae dynamics in adipocyte lipid store fluctuation.

Adipocytes specialized in the storage of energy as fat are among the most caveolae-enriched cell types. Loss of caveolae produces lipodystrophic diabetes in humans, which cannot be reversed by endothelial rescue of caveolin expression in mice, indicating major importance of adipocyte caveolae. However, how caveolae participate in fat cell functions is poorly understood.

The lipoatrophic caveolin-1 deficient mouse model reveals autophagy in mature adipocytes.

Adipose tissue lipoatrophy caused by caveolin gene deletion in mice is not linked to defective adipocyte differentiation. We show that adipose tissue development cannot be rescued by endothelial specific caveolin-1 re-expression, indicating primordial role of caveolin in mature adipocytes. Partial or total caveolin deficiency in adipocytes induced broad protein expression defects, including but not limited to previously described downregulation of insulin receptor.

Lipid droplet analysis in caveolin-deficient adipocytes: alterations in surface phospholipid composition and maturation defects.

Caveolins form plasmalemnal invaginated caveolae. They also locate around intracellular lipid droplets but their role in this location remains unclear. By studying primary adipocytes that highly express caveolin-1, we characterized the impact of caveolin-1 deficiency on lipid droplet proteome and lipidome.

Targeting of PKCzeta and PKB to caveolin-enriched microdomains represents a crucial step underpinning the disruption in PKB-directed signalling by ceramide.

Elevated ceramide concentrations in adipocytes and skeletal muscle impair PKB (protein kinase B; also known as Akt)-directed insulin signalling to key hormonal end points. An important feature of this inhibition involves the ceramide-induced activation of atypical PKCzeta (protein kinase C-zeta), which associates with and negatively regulates PKB. In the present study, we demonstrate that this inhibition is critically dependent on the targeting and subsequent retention of PKCzeta-PKB within CEM (caveolin-enriched microdomains), which is facilitated by kinase interactions with caveolin.

DnaJA4 is a SREBP-regulated chaperone involved in the cholesterol biosynthesis pathway.

Using subtractive hybridization technique in 3T3-L1 adipocytes overexpressing constitutively active SREBP2, we have identified a DnaJ/Hsp40 chaperone, DnaJA4, as a new SREBP-responsive gene. SREBP2 regulation was demonstrated by changes in DnaJA4 mRNA under conditions of altered sterol status that were strictly parallel to that of well-characterized SREBP targets (LDL receptor and HMG-CoA reductase). The role of SREBP2 was further established using adenoviral overexpression of a dominant negative SREBP2, which abolished cholesterol-regulated changes in DnaJA4 expression.

Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: a role for caveolar endocytosis.

We have investigated the targeting of caveolin to lipid bodies in adipocytes that express high levels of caveolins and contain well-developed lipid droplets. We observed that the lipid droplets isolated from adipocytes of caveolin-1 knock out mice contained dramatically reduced levels of cholesterol, indicating that caveolin is required for maintaining the cholesterol content of this organelle. Analysis of caveolin distribution by cell fractionation and fluorescent light microscopy in 3T3-L1 adipocytes indicated that addition of cholesterol rapidly stimulated translocation of caveolin to lipid droplets.

Insulin and angiotensin II induce the translocation of scavenger receptor class B, type I from intracellular sites to the plasma membrane of adipocytes.

Scavenger receptor class B, type I (SR-BI) mediates the selective uptake of lipids from high density lipoproteins and is expressed in several types of tissues. However, to date little is known about its role in adipocytes. In this study, we investigated the cellular distribution of SR-BI in 3T3-L1 adipocytes and its regulation by hormones known to increase lipid storage such as angiotensin II (Ang II) and insulin.

New insights into how adipocytes sense their triglyceride stores. Is cholesterol a signal?

In recent years, our view of adipose tissue has evolved from a passive sink for energy storage to an active tissue producing multiple molecules acting on various tissues in different aspects of energy homeostasis. The production of adipose-derived secretory products is tightly regulated as a function of adipocyte lipid accumulation, but the mechanisms by which fat cells are able to sense the levels of their triglyceride stores still remains largely unknown. This paper reviews new insights into this question taking cholesterol as a potential intracellular signaling molecule.

Regulation of ABCA1 expression and cholesterol efflux during adipose differentiation of 3T3-L1 cells.

Adipose cells specialized in energy storage, contain large intracellular triglyceride-rich lipid droplets, are enriched with free cholesterol, and express sterol-regulated transcription factors such as liver X receptor (LXR). The recent identification of the LXR-dependent ATP binding cassette transporter A1 (ABCA1) pathway for cholesterol release from peripheral cells has led us to address the question of the expression and function of ABCA1 in adipocytes. In 3T3-L1 adipose cells, we observed a strong induction of ABCA1 mRNA during adipose differentiation, but only limited variations in ABCA1 protein.

Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene expression in adipocytes.

Enlarged fat cells exhibit modified metabolic capacities, which could be involved in the metabolic complications of obesity at the whole body level. We show here that sterol regulatory element-binding protein 2 (SREBP-2) and its target genes are induced in the adipose tissue of several models of rodent obesity, suggesting cholesterol imbalance in enlarged adipocytes. Within a particular fat pad, larger adipocytes have reduced membrane cholesterol concentrations compared with smaller fat cells, demonstrating that altered cholesterol distribution is characteristic of adipocyte hypertrophy per se.

Progesterone stimulates adipocyte determination and differentiation 1/sterol regulatory element-binding protein 1c gene expression. potential mechanism for the lipogenic effect of progesterone in adipose tissue.

Fatty acid synthase (FAS), a nutritionally regulated lipogenic enzyme, is transcriptionally controlled by ADD1/SREBP1c (adipocyte determination and differentiation 1/sterol regulatory element-binding protein 1c), through insulin-mediated stimulation of ADD1/SREBP1c expression. Progesterone exerts lipogenic effects on adipocytes, and FAS is highly induced in breast tumor cell lines upon progesterone treatment. We show here that progesterone up-regulates ADD1/SREBP1c expression in the MCF7 breast cancer cell line and the primary cultured preadipocyte from rat parametrial adipose tissue.

Functional antagonism between inhibitor of DNA binding (Id) and adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1c (ADD1/SREBP-1c) trans-factors for the regulation of fatty acid synthase promoter in adipocytes.

We show that Id (inhibitor of DNA binding) 2 and Id3, dominant negative members of the helix-loop-helix (HLH) family, interact with the adipocyte determination and differentiation factor 1 (ADD1)/sterol regulatory element-binding protein (SREBP) 1c, a transcription factor of the basic HLH-leucine zipper family that controls the expression of several key genes of adipose metabolism. Gel mobility-shift assays performed with in vitro-translated ADD1, Id2 or Id3 proteins and a fatty acid synthase (FAS) promoter oligonucleotide showed evidence for a marked inhibition of the formation of DNA-ADD1 complexes by Id2 or Id3 proteins. Co-immunoprecipitation studies using in vitro-translated proteins demonstrated further the physical interaction of Id and ADD1/SREBP-1c proteins in the absence of DNA.

ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose.

The transcription of genes encoding proteins involved in the hepatic synthesis of lipids from glucose is strongly stimulated by carbohydrate feeding. It is now well established that in the liver, glucose is the main activator of the expression of this group of genes, with insulin having only a permissive role. While ADD1/SREBP-1 has been implicated in lipogenic gene expression through temporal association with food intake and ectopic gain-of-function experiments, no genetic evidence for a requirement for this factor in glucose-mediated gene expression has been established.

Obesity-related overexpression of fatty-acid synthase gene in adipose tissue involves sterol regulatory element-binding protein transcription factors.

Elevated lipogenesis is a key determinant of exaggerated fat deposition in adipose tissue of obese Zucker rats. We previously delineated a region in the fatty-acid synthase promoter, which was responsible for obesity-related overexpression of the fatty-acid synthase (FAS) gene, by negatively regulating the activity of the downstream promoter in lean but not obese rat fat cells. The present study aimed to identify the transcriptional factors acting on this target region.

A GC-rich region containing Sp1 and Sp1-like binding sites is a crucial regulatory motif for fatty acid synthase gene promoter activity in adipocytes. Implication In the overactivity of FAS promoter in obese Zucker rats.

We have previously shown that the proximal 2-kb sequence of the fatty acid synthase (FAS) promoter transfected into rat adipocytes was highly sensitive to the cellular context, displaying an overactivity in obese (fa/fa) versus lean Zucker rat adipocytes. Using deletional analysis, we show here that FAS promoter activity mainly depends on a region from -200 to -126. This sequence exerts a strong negative effect on FAS promoter in adipocytes from lean rats but not in those from obese rats, resulting in a marked overtranscriptional activity in the latter cells.

C/EBP alpha expression in adipose tissue of genetically obese Zucker rats.

The adipose tissue of genetically obese Zucker rats is characterized by coordinated tissue specific overtranscription of a subset of genes related to lipid storage such as Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). We show that CCAAT/Enhancer Binding Protein alpha (C/EBP alpha) is an activator of GAPDH proximal promoter in transiently transfected mature rat adipocytes. C/EBP alpha mRNA levels were increased in adipose tissue but not in liver of obese as compared to lean rats at 30 days of age, i.

Evidence of increased glyceraldehyde-3-phosphate dehydrogenase and fatty acid synthetase promoter activities in transiently transfected adipocytes from genetically obese rats.

Previous studies have shown that the adipose tissue of young genetically obese Zucker rats was characterized by a coordinate overtranscription of lipogenic genes, suggesting that the fa mutation triggers transcription factor(s) acting in common on the promoters of these genes. To test this hypothesis, we developed a system of transient transfection of rat adipocytes with constructs containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthetase (FAS) promoters fused to gene reporter CAT. Those transfected cells expressed high levels of promoter-driven chloramphenicol acetyltransferase (CAT) activity through correctly initiated transcription as shown by primer extension analysis.

Evidence for a sustained genetic effect on fat storage capacity in cultured adipose cells from Zucker rats.

Using mature adipocytes and preadipocytes from genetically obese Zucker rats, we investigated the cells' ability to maintain abnormal fat storage capacity when withdrawn from their in vivo environment. Long-term adipocyte cultures from obese rats displayed an increase in both glucose consumption (GC) and enzyme activities, including fatty acid synthase (4-fold), glycerol-3-phosphate dehydrogenase (4.5-fold), lipoprotein lipase (LPL; 6-fold), and malic enzyme (2.

Differential polypeptide expression in adipose tissue of lean and obese Zucker rats. Evidence of specifically repressed peptides in 7-day-old pre-obese rats.

Using two-dimensional electrophoresis on total extracts of adipose tissue from young lean (Fa/fa) and obese (fa/fa) Zucker rats, we have investigated the existence of early events at the protein level, before obvious obesity. Our results indicate that the two genotypes do not differ at 3 days of age in terms of polypeptide pattern. By 7 days of age, two polypeptides are transiently repressed in the fatty genotype, leading us to suggest their potential involvement in the onset of obesity.

Genetic regulation of fatty acid synthetase expression in adipose tissue: overtranscription of the gene in genetically obese rats.

We have investigated the molecular mechanism of the overactivity of fatty acid synthetase (FAS) in adipose tissue from the genetically obese Zucker rat. Purified FAS from lean and obese rat adipose tissues displayed kinetics constants, molecular weight, and immunological properties that were identical. Western blot analysis revealed that FAS overactivity in obese versus lean rat adipose tissue was paralleled by a proportionate increase in FAS mass, i.

Gene expression of lipid storage-related enzymes in adipose tissue of the genetically obese Zucker rat. Co-ordinated increase in transcriptional activity and potentiation by hyperinsulinaemia.

The genetically obese Zucker rat displays excessive fat storage capacity which is due to a tissue-specific increase in the activities of a number of lipid storage-related enzymes in adipose tissue. The aim of this study was to investigate the molecular mechanism responsible for this phenomenon. Lean (Fa/fa) and obese (fa/fa) Zucker rats were studied during the early stages of adipose tissue overdevelopment, both before (at 16 days of age) and after (at 30 days of age) the emergence of hyperinsulinaemia, in order to delineate the effects of the fatty genotype independently of those of hyperinsulinaemia.

Impairment of adipsin expression is secondary to the onset of obesity in db/db mice.

The nature of the primary biochemical lesions in genetically obese mice, which might prove to be useful models for human obesity, remains totally obscure. The recent finding that the expression of adipsin was virtually suppressed in both db/db and ob/ob adult mice has opened new perspectives, suggesting a potential role for this defect in the pathogenesis of obesity. To be of etiological significance, adipsin deficiency must be present very early in life when excess fat storage starts to develop.

Chylomicron-remnant uptake by freshly isolated hepatocytes. Effect of heparin and of hepatic triacylglycerol lipase.

Chylomicron remnants labelled biologically with [3H]cholesterol were efficiently taken up by freshly isolated hepatocytes during a 3 h incubation in Krebs bicarbonate medium. Their [3H]cholesteryl ester was hydrolysed (74% net hydrolysis), and 0.1 mM-chloroquine could partially inhibit this hydrolysis, provided that hepatocytes were first preincubated for 2 h 30 min at 37 degrees C.

Adipsin mRNA amounts are not decreased in the genetically obese Zucker rat.

Adipsin gene expression as assessed by mRNA amounts was examined in adipose tissue of genetically obese rats at the onset (16 days of age) or at later stages (30 and 60 days of age) of obesity. Amounts of mRNA were equivalent in obese and lean rats at 16 days of age. In adult rats, we observed a 2-fold decrease in adipsin mRNA in the obese rats compared with control lean rats, which was abolished by weaning the animals on a high-fat diet.