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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. Ceramides are produced in the endoplasmic reticulum (ER) and transported to the Golgi apparatus through a transporter called CERT, where they are converted into various sphingolipid species. We show that CERT protein expression is reduced in all IR models studied because of a caspase-dependent cleavage. Inhibiting CERT activity in vitro potentiates the deleterious action of lipotoxicity on insulin signaling, whereas overexpression of CERT in vitro or in vivo decreases muscle ceramide content and improves insulin signaling. In addition, inhibition of caspase activity prevents ceramide-induced insulin signaling defects in C2C12 muscle cells. Altogether, these results demonstrate the importance of physiological ER-to-Golgi ceramide traffic to preserve muscle cell insulin signaling and identify CERT as a major actor in this process., (© 2018 by the American Diabetes Association.)

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. Global alterations in protein turnover, and accelerated degradation of long-lived proteins were found in caveolin-deficient adipocytes. Lipidation of endogenous LC3 autophagy marker and distribution of GFP-LC3 into aggregates demonstrated activated autophagy in the absence of caveolin-1 in adipocytes. Furthermore, electron microscopy revealed autophagic vacuoles in caveolin-1 deficient but not control adipocytes. Surprisingly, significant levels of lipidated LC3-II were found around lipid droplets of normal adipocytes, maintained in nutrient-rich conditions or isolated from fed mice, which do not display autophagy. Altogether, these data indicate that caveolin deficiency induce autophagy in adipocytes, a feature that is not a physiological response to fasting in normal fat cells. This likely resulted from defective insulin and lipolytic responses that converge in chronic nutrient shortage in adipocytes lacking caveolin-1. This is the first report of a pathological situation with autophagy as an adaptative response to adipocyte failure.

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. We identified several missing proteins on the lipid droplet surface of caveolin-deficient adipocytes and showed that the caveolin-1 lipid droplet pool is organized as multi-protein complexes containing cavin-1, with similar dynamics as those found in caveolae. On the lipid side, caveolin deficiency did not qualitatively alter neutral lipids in lipid droplet, but significantly reduced the relative abundance of surface phospholipid species: phosphatidylserine and lysophospholipids. Caveolin-deficient adipocytes can form only small lipid droplets, suggesting that the caveolin-lipid droplet pool might be involved in lipid droplet size regulation. Accordingly, we show that caveolin-1 concentration on adipocyte lipid droplets positively correlated with lipid droplet size in obese rodent models and human adipocytes. Moreover, rescue experiments by caveolin- green fluorescent protein in caveolin-deficient cells exposed to fatty acid overload demonstrated that caveolin-coated lipid droplets were able to grow larger than caveolin-devoid lipid droplets. Altogether, these data demonstrate that the lipid droplet-caveolin pool impacts on phospholipid and protein surface composition of lipid droplets and suggest a functional role on lipid droplet expandability.

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. Ceramide also recruits PTEN (phosphatase and tensin homologue detected on chromosome 10), a 3'-phosphoinositide phosphatase, thereby creating a repressive membrane microenvironment from which PKB cannot signal. Disrupting the structural integrity of caveolae by cholesterol depletion prevented caveolar targeting of PKCzeta and PKB and suppressed kinase-caveolin association, but, importantly, also ameliorated ceramide-induced inhibition of PKB. Consistent with this, adipocytes from caveolin-1-/- mice, which lack functional caveolae, exhibit greater resistance to ceramide compared with caveolin-1+/+ adipocytes. We conclude that the recruitment and retention of PKB within CEM contribute significantly to ceramide-induced inhibition of PKB-directed signalling.

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. To determine the functional significance of this regulation, DnaJA4 was overexpressed in COS cells, which induced a specific increase in the synthesis of cholesterol from acetate. We also observed that DnaJA4 overexpression increased the activity and the protein content of HMG-CoA reductase, the rate limiting enzyme in this pathway. At the molecular level, DnaJA4 overexpression did not alter HMG-CoA reductase stability or mRNA levels, suggesting a co-translational effect of the chaperone. In the DnaJ/Hsp40 family, DnaJA4 uniquely exhibited SREBP-regulated expression, and also responded to heat shock. Through its responsiveness to SREBP, and its stimulatory effect on cholesterol synthesis, the DnaJA4 chaperone can be viewed as a new player in cholesterol synthesis. These data suggest a link between molecular chaperones, heat stress and cholesterol synthesis.

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. SR-BI was mainly distributed in the cytoplasm as determined by laser-scanning confocal analysis of the immunofluorescence labeling of SR-BI or the study of an enhanced green fluorescent protein-tagged SR-BI fusion protein. Exposure of cells to either insulin or Ang II (1-2 h) induced the mobilization of SR-BI from intracellular pools to the plasma membrane. This was further confirmed by Western blotting on purified plasma membrane and by fluorescence-activated cell sorter analysis of the SR-BI receptor. Similar results were also observed in primary adipocytes. We also demonstrated that, in the presence of either insulin or Ang II, SR-BI translocation to the cell membrane is functional, because insulin and Ang II induced a significant increase in the high density lipoprotein-delivered 22-(N-7-nitrobenz-2-oxa-1,3-diazo-4-yl)-amino-23,24-bisnor-5-cholen-3-ol uptake and in total cholesterol content. These data demonstrate that SR-BI can be acutely mobilized from intracellular stores to the cell surface by insulin or Ang II, two hormones that exert lipogenic effects in adipocytes. This suggests that SR-BI might participate in the storage of lipids in the adipose tissue.

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. Lipid efflux onto apolipoprotein A-I (apoA-I), which depends on ABCA1, was comparable in adipocytes and preadipocytes, demonstrating a differential regulation of ABCA1 mRNA and cholesterol efflux. We also found that total cell cholesterol remained stable during differentiation of 3T3-L1 cells, but membrane cholesterol was lower in adipocytes than in preadipocytes, suggesting redistribution of cholesterol to the lipid droplet. Finally, we show that under standard lipolytic stimulation, 3T3-L1 adipocytes do not release cholesterol onto apoA-I, a process that required long exposures to lipolytic agents (24 h). In conclusion, despite large induction of ABCA1 mRNA during differentiation, cholesterol efflux through the ABCA1 pathway remains limited in adipocytes and requires prolonged lipolysis. This is consistent with the view of the adipocyte behaving as a cholesterol sink, with plasma cholesterol-buffering properties.

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. We show that treatment with methyl-beta-cyclodextrin, which mimics the membrane cholesterol reduction of hypertrophied adipocytes, induces insulin resistance. We also produced cholesterol depletion by mevastatin treatment, which activates SREBP-2 and its target genes. The analysis of 40 adipocyte genes showed that the response to cholesterol depletion implicated genes involved in cholesterol traffic (caveolin 2, scavenger receptor BI, and ATP binding cassette 1 genes) but also adipocyte-derived secretion products (tumor necrosis factor alpha, angiotensinogen, and interleukin-6) and proteins involved in energy metabolism (fatty acid synthase, GLUT 4, and UCP3). These data demonstrate that altering cholesterol balance profoundly modifies adipocyte metabolism in a way resembling that seen in hypertrophied fat cells from obese rodents or humans. This is the first evidence that intracellular cholesterol might serve as a link between fat cell size and adipocyte metabolic activity.

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. In MCF7, progesterone induced ADD1/SREBP1c and Metallothionein II (a well known progesterone-regulated gene) mRNAs, with comparable potency. In preadipocytes, progesterone increased ADD1/SREBP1c mRNA dose-dependently, but not SREBP1a or SREBP2. Run-on experiments demonstrated that progesterone action on ADD1/SREBP1c was primarily at the transcriptional level. The membrane-bound and mature nuclear forms of ADD1/SREBP1 protein accumulated in preadipocytes cultured with progesterone, and FAS induction could be abolished by adenovirus-mediated overexpression of a dominant negative form of ADD1/SREBP1 in these cells. Finally, in the presence of insulin, progesterone was unable to up-regulate ADD1/SREBP1c mRNA in preadipocytes, whereas its effect was restored after 24 h of insulin deprivation. Together these results demonstrate that ADD1/SREBP1c is controlled by progesterone, which, like insulin, acts by increasing ADD1/SREBP1c gene transcription. This provides a potential mechanism for the lipogenic actions of progesterone on adipose tissue.

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.e., when obesity is fully expressed. Nevertheless at 16 days of age, although overdevelopment of adipose tissue could be detected in preobese rats, C/EBP alpha mRNA levels were similar whatever the genotype. In conclusion C/EBP alpha mRNA is overexpressed in adipose tissue of obese rats, suggesting a possible role for this factor in the activation of lipid storage-related genes in adipose tissue of obese rats. However, C/EBP alpha overexpression is not temporally related to the onset of obesity.

In isolated hepatocytes of fat-fed rats, as compared to control fed animals, the cellular uptake of [1-14C] oleate and its oxidation to CO2 were similar but the incorporation of the label into water-soluble products (mainly ketone bodies) was increased by 36.6% whereas its esterification to triacylglycerols and phospholipids decreased by 36%. While endogenous ketogenesis was slightly but not significantly increased, ketone body synthesis from both 2 mM octanoate and 0.7 mM oleate was stimulated two fold. Thus, in the fatfed rats the oxidative pathway is clearly activated whereas long chain fatty acids are preferentially channelled into the oxidation pathway at the expense of esterification. Yet, hepatocyte triacylglycerol content was 3-fold higher after fat-feeding. In this regard, lysosomal triacylglycerol lipase (EC 3.1.1.3) activity, in homogenates of hepatocytes was decreased by 32% (p less than 0.01). This findings suggest a lower breakdown of endogenous triacylglycerols, which, taken together with decreased secretion of VLDL lipoprotein triacylglycerol (Kalopissis et al. Biochem. J. 198: 373, 1981) and an in vivo increased fatty acid influx to the liver may contribute to the accumulation of lipids in the livers of fat-fed rats.

Hepatic VLDL production appears to be correlated with de novo fatty acid synthesis (lipogenesis). In this study lipogenesis was inhibited in vitro in order to establish an eventual direct effect on VLDL secretion. Hepatocytes from rats fed a standard diet were incubated with three triglyceride-rich lipoproteins: chylomicrons and VLDL obtained from rats fed a high-fat diet and VLDL obtained from rats fed a standard diet. The inhibition of lipogenesis (10 to 55%) was proportional to the concentration of the lipoproteins added. Chylomicrons and VLDL originating mainly from the intestine (prepared from fat-fed rats) inhibited lipogenesis more effectively than VLDL produced essentially by the liver (prepared from rats fed a standard diet). However the secretion of newly synthesized fatty acids in the medium did not decrease. When hepatocyte lipogenesis was inhibited by the addition of 1 mM oleic acid, total VLDL secretion (expressed as nmol of VLDL triglyceride/10(6) cells) was unchanged compared to control cells incubated without oleic acid. Our results suggest that hepatic VLDL secretion is not directly related to de novo fatty acid synthesis.

1. The effect of a high-fat diet (30% fat by wt.) on intestinal very low density lipoprotein (VLDL) secretion was studied in male rats after specific inhibition of hepatic VLDL secretion by dietary orotic acid. Total VLDL secretion (from liver and intestine) was measured in animals not receiving orotic acid. 2. Fat-feeding resulted in a 32% decreased post-Triton secretion of total serum VLDL triacylglycerols as compared to a control (low fat) diet. Concomitantly, a large stimulation of post-Triton intestinal VLDL triacylglycerols secretion was measured in fat-fed rats. Thus, the major part (64%) of circulating triacylglycerols transported as VLDL originated from the intestine in these animals, leading presumably to an increased secretion of intestinal apolipoproteins. 3. Intestinal VLDL and chylomicron secretion rates increased with the amount of fat in the diet (7, 13, 20 or 30% fat by wt.). Whereas the chylomicron secretion was linearly related to the dietary fat content, the relationship between intestinal VLDL secretion and fat content of the diet was sigmoidal. The highest stimulation of intestinal VLDL formation was observed within a narrow range of dietary fat content (between 10 and 20%).

The relationship between lipogenesis and ketogenesis and the concentration of malonyl coenzyme A (CoA) was investigated in hepatocytes from adult obese Zucker rats and their lean littermates fed either a control low-fat diet or a high-fat diet (30% lard in weight). With the control diet, lipogenesis--although strongly inhibited in the presence of either 1 mmol/L oleate, 10(-6) mol/L glucagon or 0.1 mmol/L TOFA (a hypolipidemic drug)--remained about fifteen-fold higher in the obese rats than in the lean rats. In contrast, ketogenesis under some conditions (oleate + TOFA) was not significantly lower (30%) as compared with the lean rats. After adaptation to the high-fat diet, lipogenesis was depressed fourfold in the lean rats and ninefold in the obese ones; however its magnitude remained significantly higher in the latter, namely at a value close to that measured in control-fed lean rats. Ketogenesis was comparable in lean and obese rats and much higher in the presence of 1 mmol/L oleate than of 0.3 mmol/L oleate, whereas lipogenesis did not vary with increasing oleate concentration in the medium. Acetyl-CoA carboxylase activity measured in liver homogenates was higher in the obese group, but was stepwise inhibited by increasing concentrations of oleyl-CoA regardless of the diet for both lean and obese rats, thus showing no abnormality of in vitro responsiveness to this inhibitor. With the control diet, hepatocyte malonyl-CoA levels were significantly higher in the obese rats, both in the basal state and after inhibition of lipogenesis by oleate and TOFA.(ABSTRACT TRUNCATED AT 250 WORDS)