Implication of phosphatidylethanolamine N-methyltransferase in adipocyte differentiation

Phosphatidylethanolamine N-methyltransferase (PEMT) is a small integral membrane protein that converts phosphatidylethanolamine (PE) into phosphatidylcholine (PC). It has been previously reported that, unexpectedly, PEMT deficiency protected from high-fat diet (HFD)-induced obesity and insulin resistance, pointing to a possible role of this enzyme in the regulation of adipose cell metabolism. Using mouse 3T3-L1 preadipocytes as a biological system, we demonstrate that PEMT expression is strongly increased during the differentiation of preadipocytes into mature adipose cells. Knockdown of PEMT reduced the expression of early and late adipogenic markers, inhibited lipid droplet formation, reduced triacylglycerol content and decreased the levels of leptin release from the adipocytes, suggesting that PEMT is a novel and relevant regulator of adipogenesis. Investigation into the mechanisms whereby PEMT regulates adipocyte differentiation revealed that extracellularly regulated kinases (ERK1/2) and AKT are essential factors in this process. Specifically, the activities of ERK1/2 and AKT, which are decreased during adipocyte differentiation, were elevated upon Pemt knockdown. Moreover, treatment of cells with exogenous ceramide 1-phosphate (C1P), which we reported to be a negative regulator of adipogenesis, decreased PEMT expression, suggesting that PEMT is also a relevant factor in the anti-adipogenic action of C1P. Altogether, the data presented here identify PEMT as a novel regulator of adipogenesis and a mediator of the anti-adipogenic action of C1P.     

Deletion of phospholipase D1 decreases bone mass and increases fat mass via modulation of Runx2, β-catenin-osteoprotegerin,PPAR-γ and C/EBPα signaling axis

In osteoporosis, mesenchymal stem cells (MSCs) prefer to differentiate into adipocytes at the expense of osteoblasts. Although the balance between adipogenesis and osteogenesis has been closely examined, the mechanism of commitment determination switch is unknown. Here we demonstrate that phospholipase D1 (PLD1) plays a key switch in determining the balance between bone and fat mass. Ablation of Pld1 reduced bone mass but increased fat in mice. Mechanistically, Pld1^/? MSCs inhibited osteoblast differentiaion with diminished Runx2 expression, while osteoclast differentiation was accelerated in Pld1^?/? bone marrow-derived macrophages. Pld1^?/? osteoblasts showed decreased expression of osteogenic makers. Increased number and resorption activity of osteoclasts in Pld1^?/? mice were corroborated with upregulation of osteoclastogenic markers. Moreover, Pld1^?/? osteoblasts reduced β-catenin mediated-osteoprotegerin (OPG) with increased RANKL/OPG ratio which resulted in accelerated osteoclast differentiation. Thus, low bone mass with upregulated osteoclasts could be due to the contribution of both osteoblasts and osteoclasts during bone remodeling. Moreover, ablation of Pld1 further increased bone loss in ovariectomized mice, suggesting that PLD1 is a negative regulator of osteoclastogenesis. Furthermore, loss of PLD1 increased adipogenesis, body fat mass, and hepatic steatosis along with upregulation of PPAR-γ and C/EBPα. Interestingly, adipocyte-specific Pld1 transgenic mice rescued the compromised phenotypes of fat mass and adipogenesis in Pld1 knockout mice. Collectively, PLD1 regulated the bifurcating pathways of mesenchymal cell lineage into increased osteogenesis and decreased adipogenesis, which uncovered a previously unrecognized role of PLD1 in homeostasis between bone and fat mass.     

Phospholipids of Entamoeba invadens.

The major phosphoglycerides present in Entamoeba invadens are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. Furthermore, three different sphingolipids could be isolated from the amoeba. In addition to sphingomyelin and a phosphonolipid, ceramide phosphonylethanolamine, a previously unknown sphingolipid was present. This sphingolipid contained a long chain base, inositol, and phosphorus in the ratio of 0.97:0.97: 1.0 and could be identified as ceramide phosphorylinositol. The various individual phospholipids showed different rates of turnover. Phosphatidic acid and phosphatidylinositol had, relative to the other phospholipids, a short half-time of about 12 h. Phosphatidylethanolamine and ceramide phosphorylinositol had a half-time of about 24 and 30 h, respectively. The major phospholipid, phosphatidylcholine and also sphingomyelin and phosphatidylserine showed no turnover. In contrast to the phosphoglycerides, the sphingolipid composition of the amoeba cultivated in different media was rather variable, while the total sphingolipid content remained at 21% of the total amount of phospholipids. The amount of ceramide phosphorylinositol was almost doubled in the cells cultivated on the serum-free medium (T), whereas the amount of sphingomyelin and ceramide phosphonylethanolamine decreased. Evidence is presented that these alterations in the sphingolipid composition of E. invadens are related to the amount of unsaturated fatty acids which were present in the culture medium.     

Study of lactoferrin gene expression in human and mouse adipose tissue,human preadipocytes and mouse 3T3-L1 fibroblasts. Association with adipogenic and inflammatory markers

Lactoferrin is considered an epithelial protein present in different gland secretions. Administration of exogenous lactoferrin is also known to modulate adipogenesis and insulin action in human adipocytes. Here, we aimed to investigate lactoferrin gene expression (real-time polymerase chain reaction) and protein (enzyme-linked immunosorbent assay) levels in human (n=143) and mice adipose tissue samples, in adipose tissue fractions and during human preadipocyte and 3T3-L1 cell line differentiation, evaluating the effects of inducers (rosiglitazone) and disruptors (inflammatory factors) of adipocyte differentiation. Lactoferrin (LTF) gene and protein were detectable at relatively high levels in whole adipose tissue and isolated adipocytes in direct association with low-density lipoprotein-related protein 1 (LRP1, its putative receptor). Obese subjects with type 2 diabetes and increased triglycerides had the lowest levels of LTF gene expression in subcutaneous adipose tissue. Specifically, LTF gene expression was significantly increased in adipocytes, mainly from lean subjects, increasing during differentiation in parallel to adipogenic genes and gene markers of lipid droplets. The induction or disruption of adipogenesis led to concomitant changes (increase and decrease, respectively) of lactoferrin levels during adipocyte differentiation also in parallel to gene markers of adipogenesis and lipid droplet development. The administration of lactoferrin led to autopotentiated increased expression of the LTF gene. The decreased lactoferrin mRNA levels in association with obesity and diabetes were replicated in mice adipose tissue. In conclusion, this is the first observation, to our knowledge, of lactoferrin gene expression in whole adipose tissue and isolated adipocytes, increasing during adipogenesis and suggesting a possible contribution in adipose tissue physiology through LRP1.     

CD38 deficiency suppresses adipogenesis and lipogenesis in adipose tissues through activating Sirt1/PPARγ signaling pathway

It has been recently reported that CD38 was highly expressed in adipose tissues from obese people and CD38‐deficient mice were resistant to high‐fat diet (HFD)‐induced obesity. However, the role of CD38 in the regulation of adipogenesis and lipogenesis is unknown. In this study, to explore the roles of CD38 in adipogenesis and lipogenesis in vivo and in vitro, obesity models were generated with male CD38^?/? and WT mice fed with HFD. The adipocyte differentiations were induced with MEFs from WT and CD38^?/? mice, 3T3‐L1 and C3H10T1/2 cells in vitro. The lipid accumulations and the alternations of CD38 and the genes involved in adipogenesis and lipogenesis were determined with the adipose tissues from the HFD‐fed mice or the MEFs, 3T3‐L1 and C3H10T1/2 cells during induction of adipocyte differentiation. The results showed that CD38^?/? male mice were significantly resistant to HFD‐induced obesity. CD38 expressions in adipocytes were significantly increased in WT mice fed with HFD, and the similar results were obtained from WT MEFs, 3T3‐L1 and C3H10T1/2 during induction of adipocyte differentiation. The expressions of PPARγ, AP2 and C/EBPα were markedly attenuated in adipocytes from HFD‐fed CD38^?/? mice and CD38^?/? MEFs at late stage of adipocyte differentiation. Moreover, the expressions of SREBP1 and FASN were also significantly decreased in CD38^?/? MEFs. Finally, the CD38 deficiency‐mediated activations of Sirt1 signalling were up‐regulated or down‐regulated by resveratrol and nicotinamide, respectively. These results suggest that CD38 deficiency impairs adipogenesis and lipogenesis through activating Sirt1/PPARγ‐FASN signalling pathway during the development of obesity.     

Proadipogenic effects of lactoferrin in human subcutaneous and visceral preadipocytes

Lactoferrin has been associated with insulin sensitivity in vivo and in vitro studies. We aimed to test the effects of lactoferrin on human subcutaneous and visceral preadipocytes. Human subcutaneous and visceral preadipocytes were cultured with increasing lactoferrin (hLf, 0.1, 1, 10 μM) under differentiation conditions. The effects of lactoferrin on adipogenesis were studied through the expression of different adipogenic and inflammatory markers, AMPK activation and Retinoblastoma 1 (RB1) activity. The response to insulin was evaluated through ^Ser473AKT phosphorylation. In both subcutaneous and visceral preadipocytes, lactoferrin (1 and 10 μM) increased adipogenic gene expressions and protein levels (fatty acid synthase, PPARγ, FABP4, ADIPOQ, ACC and STAMP2) and decreased inflammatory markers (IL8, IL6 and MCP1) dose-dependently in parallel to increased insulin-induced ^Ser473AKT phosphorylation. In addition to these adipogenic effects, lactoferrin decreased significantly AMPK activity (reducing ^pThr172AMPK and ^pSer79ACC) and RB1 activity (increasing the ^pser807/811RB1/RB1 ratio). In conclusion, these results suggest that lactoferrin promotes adipogenesis in human adipocytes by enhancing insulin signaling and inhibiting RB1 and AMPK activities.     

Sphingolipids Are Required for Efficient Triacylglycerol Loss in Conjugated Linoleic Acid Treated Adipocytes

Conjugated linoleic acid (CLA) reduces adiposity in human and mouse adipocytes. This outcome is achieved through a variety of biological responses including increased energy expenditure and fatty acid oxidation, increased inflammation, repression of fatty acid biosynthesis, attenuated glucose transport, and apoptosis. In the current study, profiling of 261 metabolites was conducted to gain new insights into the biological pathways responding to CLA in 3T3-L1 adipocytes. Sphinganine and sphingosine levels were observed to be highly elevated in CLA treated adipocytes. Exogenous chemicals that increased endogenous ceramide levels decreased lipid levels in adipocytes, and activated AMP-activated protein kinase (AMPK) as well as NF-κB, both of which are typically activated in CLA treated adipocytes. Concurrent inhibition of ceramide de novo biosynthesis and recycling from existing sphingolipid pools attenuated the lipid lowering effect normally associated with responses to CLA, implicating ceramides as an important component of the lipid lowering response in CLA treated adipocytes.     

Lipodystrophic gene Agpat2 deficiency aggravates hyperlipidemia and atherosclerosis in Ldlr−/− mice

AimsDysfunction of adipose tissue increases the risk of cardiovascular disease. It was well established that obesity aggravates atherosclerosis, but the effect of adipose tissue loss on atherosclerosis has been less studied. AGPAT2 is the first causative gene of congenital generalized lipodystrophy (CGL), but the role of AGPAT2 on atherosclerosis has not been reported. Hypertriglyceridemia is one of the clinical manifestations of CGL patients, but it is usually absent in CGL mouse model on a normal diet. This study will investigate the effect of Agpat2 on hyperlipidemia and atherosclerosis.Methods and resultsIn this study, Agpat2 knockout (Agpat2^−/−) mice were generated using CRISPR/Cas system, which showed severe loss of adipose tissue and fatty liver, consistent with previous reports. Agpat2^−/− mice were then crossed with hypercholesterolemic and atherosclerotic prone LDL receptor knockout (Ldlr^−/−) mice to obtain double knockout mouse model (Agpat2^−/−Ldlr^−/−). Plasma lipid profile, insulin resistance, fatty liver, and atherosclerotic lesions were observed after 12 weeks of the atherogenic high-fat diet (HFD) feeding. We found that compared with Ldlr^−/− mice, Agpat2^−/−Ldlr^−/− mice showed significantly higher plasma total cholesterol and triglycerides after HFD feeding. Agpat2^−/−Ldlr^−/− mice also developed hyperglycemia and hyperinsulinemia, with increased pancreatic islet area. The liver weight of Agpat2^−/−Ldlr^−/− mice was about 4 times higher than that of Ldlr^−/− mice. The liver lipid deposition was severe and Sirius red staining showed liver fibrosis. In addition, in Agpat2^−/−Ldlr^−/− mice, the area of atherosclerotic lesions in aortic arch and aortic root was significantly increased.ConclusionsOur results show that Agpat2 deficiency led to more severe hyperlipidemia, liver fibrosis and aggravation of atherosclerosis in Ldlr^−/− mice. This study provided additional insights into the role of adipose tissue in hyperlipidemia and atherosclerosis.     

Vitamin E alleviates non-alcoholic fatty liver disease in phosphatidylethanolamine N-methyltransferase deficient mice

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC), mainly in the liver. Pemt^?/? mice are protected from high-fat diet (HFD)-induced obesity and insulin resistance, but develop severe non-alcoholic fatty liver disease (NAFLD) when fed a HFD, mostly due to impaired VLDL secretion. Oxidative stress is thought to be an essential factor in the progression from simple steatosis to steatohepatitis. Vitamin E is an antioxidant that has been clinically used to improve NAFLD pathology. Our aim was to determine whether supplementation of the diet with vitamin E could attenuate HFD-induced hepatic steatosis and its progression to NASH in Pemt^?/? mice. Treatment with vitamin E (0.5?g/kg) for 3?weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis. We also observed abnormal ceramide metabolism in Pemt^?/? mice fed a HFD, with elevation of ceramides and other sphingolipids and higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (Cerk). Interestingly, vitamin E supplementation restored Asah1 and Cerk mRNA and sphingolipid levels. Together this study shows that vitamin E treatment efficiently prevented the progression from simple steatosis to steatohepatitis in mice lacking PEMT.     

Caudatin suppresses adipogenesis in 3T3-L1 adipocytes and reduces body weight gain in high-fat diet-fed mice through activation of hedgehog signaling

BackgroundThe regulative effects of caudatin, a C-21 steroid that is identified from Cynanchum bungee roots, on adipogenesis and obesity have not been studied. Many studies have demonstrated that the activation of hedgehog (Hh) signaling can help prevent obesity. Therefore, we hypothesized that caudatin can inhibit adipogenesis and obesity via activating the Hh signaling pathway.MethodsTo investigate the effects of caudatin on adipogenesis in 3T3-L1 preadipocytes and high-fat diet induced obesity in C57BL/6 mice, in vitro and in vivo experiments were performed. For in vitro evaluation, Oil red O staining were used to represent lipid accumulation in differentiated 3T3-L1 adipocytes. For in vivo assessment, male 5 week-old C57BL/6 mice were fed with standard chow diet, high-fat diet (HFD), HFD with 25 mg/kg caudatin, HFD with 1mg/kg purmorpharmine for 10 weeks, respectively. Hh signaling and key adipogenic marker involved in adipogenesis were evaluated by real-time PCR and western blot. The adipocyte size of white adopose tissue and lipid storage of liver were visualized by hematoxylin and eosin staining. In addition, the expression of Gli1 and peroxisome proliferator-activated receptor γ (PPARγ) in white adipose tissue were investigated by immunohistochemistry staining.ResultsCaudatin suppressed the accumulation of lipid droplets and downregulated the expression of key adipogenic factors, i.e., peroxisome proliferator-activated receptor γ PPARγ and CCAAT-enhancer binding protein α (C/EBPα), through activating Hh signaling in differentiated 3T3-L1 cells. Furthermore, caudatin and the Hh activator purmorpharmine significantly decreased body weight gain and white adipose tissue (WAT) weight in HFD-induced mice and affected adipogenic markers and Hh signaling mediators in WAT, which were in line with the in vitro experimental results.ConclusionTo our best knowledge, it is the first report to demonstrate that caudatin downregulated adipocyte differentiation and suppressed HFD-induced body weight gain through activating the Hh signaling pathway, suggesting that caudatin can potentially counteract obesity.     

Localization of low density lipoprotein receptor-related protein 1 to caveolae in 3T3-L1 adipocytes in response to insulin treatment

The insulin-induced translocation of low density lipoprotein receptor-related protein 1 (LRP1) from intracellular membranes to the cell surface in 3T3-L1 adipocytes was differentiation-dependent and did not occur in 3T3-L1 fibroblasts. Prompted by findings that the plasma membrane of 3T3-L1 adipocytes was rich in caveolae, we determined whether LRP1 became caveolae-associated upon insulin stimulation. The caveolae domain was isolated by the well characterized detergent solubilization and sucrose density ultracentrifugation methodology. Under basal conditions, only a trace amount of LRP1 was caveolae-associated despite the markedly elevated caveolin-1 and caveolae after adipocytic cell differentiation. Upon insulin treatment, the amount of LRP1 associated with caveolae was increased by 4-fold within 10 min, which was blocked completely by pretreatment with wortmannin prior to insulin. The caveolar localization of LRP1 in adipocytes was specific to insulin; treatment with platelet-derived growth factor-bb isoform did not promote but rather decreased caveolar localization of LRP1 below basal levels. The insulin-induced caveolar localization of LRP1 was also observed in 3T3-L1 fibroblasts where translocation of LRP1 from intracellular membranes to the cell surface was absent, suggesting that association of LRP1 with caveolae was achieved, at least in part, through lateral transmigration along the plane of plasma membranes. Immunocytochemistry studies revealed partial co-localization of LRP1 (either endogenous LRP1 or an epitope-tagged minireceptor) with caveolin-1 in cells treated with insulin, which was confirmed by co-immunoprecipitation of LRP1 with caveolin-1 in cells treated with insulin but not platelet-derived growth factor-bb. These results suggest that the localization of LRP1 to caveolae responds selectively to extracellular signals.     

3,5-dicaffeoyl‑epi-quinic acid from Atriplex gmelinii enhances the osteoblast differentiation of bone marrow-derived human mesenchymal stromal cells via WnT/BMP signaling and suppresses adipocyte differentiation via AMPK activation

BackgroundImpaired bone formation is one of the reasons behind osteoporosis. Alterations in the patterns of mesenchymal stromal cell differentiation towards adipocytes instead of osteoblasts contribute to osteoporosis progression. Natural anti-osteoporotic agents are effective and safe alternatives for osteoporosis treatment.PurposeIn this context, 3,5-dicaffeoyl‑epi-quinic acid (DCEQA) which is a derivative of chlorogenic acid with reported bioactivities was studied for its osteogenic differentiation enhancing potential in vitro.MethodsAnti-osteoporotic effects of DCEQA were investigated in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) which were induced to differentiate into osteoblasts or adipocytes with or without DCEQA treatment. Changes in the osteogenic and adipogenic markers such as ALP activity and lipid accumulation, respectively, were observed along with differentiation-specific activation of mitogen activated protein kinase (MAPK) pathways.ResultsAt 10 μM concentration, DCEQA increased the proliferation of bone marrow-derived human mesenchymal stromal cells (hBM-MSCs) during osteoblast differentiation. The expression of osteogenic markers ALP, osteocalcin, Runx2, BMP2 and Wnt 10a was upregulated by DCEQA treatment. The ALP activity and extracellular mineralization were also increased. DCEQA elevated the phosphorylation levels of p38 and JNK MAPKs as well as the activation of β-catenin and Smad1/5. DCEQA suppressed the lipid accumulation and downregulated expression of adipogenic markers PPARγ, C/EBPα and SREBP1c in adipo-induced hBM-MSCs. DCEQA also decreased the phosphorylation of p38 and ERK MAPKs and stimulated the activation of AMPK in hBM-MSC adipocytes.ConclusionDCEQA was suggested to enhance osteoblast differentiation via stimulating Wnt/BMP signaling. The adipocyte differentiation inhibitory effect of DCEQA was suggested to arise from its ability to increase AMPK phosphorylation. Overall, DCEQA was shown to possess osteogenesis enhancing and adipogenesis inhibitory properties which might facilitate its use against osteoporotic conditions.     

Cyclooxygenase-2 dependent metabolism of 20-HETE increases adiposity and adipocyte enlargement in mesenchymal stem cell-derived adipocytes

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a product of the cytochrome P450 (CYP)-catalyzed ω-hydroxylation of arachidonic acid, induces oxidative stress and, in clinical studies, is associated with increased body mass index (BMI) and the metabolic syndrome. This study was designed to examine the effects of exogenous 20-HETE on mesenchymal stem cell (MSC)-derived adipocytes. The expression levels of CYP4A11 and CYP4F2 (major 20-HETE synthases in humans) in MSCs decreased during adipocyte differentiation; however, exogenous administration of 20-HETE (0.1–1 μM) increased adipogenesis in a dose-dependent manner in these cells (P < 0.05). The inability of a 20-HETE analog to reproduce these effects suggested the involvement of a metabolic product of 20-HETE in mediating its pro-adipogenic effects. A cyclooxygenase (COX)-1 selective inhibitor enhanced, whereas a COX-2 selective or a dual COX-1/2 inhibitor attenuated adipogenesis induced by 20-HETE. The COX-derived metabolite of 20-HETE, 20-OH-PGE₂, enhanced adipogenesis and lipid accumulation in MSCs. The pro-adipogenic effects of 20-HETE and 20-OH-PGE₂ resulted in the increased expression of the adipogenic regulators PPARγ and β-catenin in MSC-derived adipocytes. Taken together we show for the first time that 20-HETE-derived COX-2-dependent 20-OH-PGE₂ enhances mature inflamed adipocyte hypertrophy in MSC undergoing adipogenic differentiation.     

Involvement of de novo ceramide synthesis in pro-inflammatory adipokine secretion and adipocyte–macrophage interaction

Interaction between adipocytes and macrophages has been suggested to play a central role in the pathogenesis of obesity. Ceramide, a sphingolipid de novo synthesized from palmitate, is known to stimulate pro-inflammatory cytokine secretion from multiple types of cells. To clarify whether de novo synthesized ceramide contributes to cytokine dysregulation in adipocytes and macrophages, we observed cytokine secretion in mature 3T3-L1 adipocytes (L1) and RAW264.7 macrophages (RAW) cultured alone or co-cultured under the suppression of de novo ceramide synthesis.Palmitate enhanced ceramide accumulation and stimulated the expression and secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in L1. The suppression of serine-palmitoyl transferase, a rate-limiting enzyme of de novo ceramide synthesis, by myriocin or siRNA attenuated those palmitate-induced alterations, and a ceramide synthase inhibitor fumonisin B1 showed similar results. In contrast, the inhibitor of sphingosine kinase or a membrane-permeable ceramide analogue augmented the cytokine secretion. Myriocin effects on the palmitate-induced changes were not abrogated by toll-like receptor-4 blockade. Although palmitate stimulated RAW to secrete tumor necrosis factor-α (TNF-α), it did not significantly increase ceramide content, and neither myriocin nor fumonisin B1 attenuated the TNF-α hypersecretion. The co-culture of L1 with RAW markedly augmented IL-6 and MCP-1 levels in media. Myriocin or fumonisin B1 significantly lowered these cytokine levels and suppressed the gene expression of TNF-α and MCP-1 in RAW and of IL-6 and MCP-1 in L1.In conclusion, de novo synthesized ceramide partially mediates the palmitate effects on pro-inflammatory adipokines and is possibly involved in the interaction with macrophages.     

Inhibition of ceramide production reverses TNF-induced insulin resistance

Ceramide has been implicated as a mediator of insulin resistance induced by tumor necrosis factor-alpha (TNF) in adipocytes. Adipocytes contain numerous caveolae, sphingolipid and cholesterol-enriched lipid microdomains, that are also enriched in insulin receptor (IR). Since caveolae may be important sites for crosstalk between tyrosine kinase and sphingolipid signaling pathways, we examined the role of increased caveolar pools of ceramide in regulating tyrosine phosphorylation of the IR and its main substrate, insulin receptor substrate-1 (IRS-1). Neither exogenous short-chain ceramide analogs nor pharmacologic increases in endogenous caveolar pools of ceramide inhibited insulin-induced tyrosine phosphorylation of the IR and IRS-1. However, inhibition of TNF-induced caveolar ceramide production reversed the decrease in IR tyrosine phosphorylation in response to TNF. These results suggest that TNF-independent increases in caveolar pools of ceramide are not sufficient to inhibit insulin signaling but that in conjunction with other TNF-dependent signals, caveolar pools of ceramide are a critical component for insulin resistance by TNF.