Obesity-induced CerS6-dependent C16:0 ceramide production promotes weight gain and glucose intolerance.

Ceramides increase during obesity and promote insulin resistance. Ceramides vary in acyl-chain lengths from C14:0 to C30:0 and are synthesized by six ceramide synthase enzymes (CerS1-6). It remains unresolved whether obesity-associated alterations of specific CerSs and their defined acyl-chain length ceramides contribute to the manifestation of metabolic diseases. Here we reveal that CERS6 mRNA expression and C16:0 ceramides are elevated in adipose tissue of obese humans, and increased CERS6 expression correlates with insulin resistance. Conversely, CerS6-deficient (CerS6(Δ/Δ)) mice exhibit reduced C16:0 ceramides and are protected from high-fat-diet-induced obesity and glucose intolerance. CerS6 deletion increases energy expenditure and improves glucose tolerance, not only in CerS6(Δ/Δ) mice, but also in brown adipose tissue- (CerS6(ΔBAT)) and liver-specific (CerS6(ΔLIVER)) CerS6 knockout mice. CerS6 deficiency increases lipid utilization in BAT and liver. These experiments highlight CerS6 inhibition as a specific approach for the treatment of obesity and type 2 diabetes mellitus, circumventing the side effects of global ceramide synthesis inhibition.

Adipose triglyceride lipase-null mice are resistant to high-fat diet-induced insulin resistance despite reduced energy expenditure and ectopic lipid accumulation.

Adipose triglyceride lipase (ATGL) null (-/-) mice store vast amounts of triacylglycerol in key glucoregulatory tissues yet exhibit enhanced insulin sensitivity and glucose tolerance. The mechanisms underpinning these divergent observations are unknown but may relate to the reduced availability of circulating fatty acids. The aim of this study was to determine whether the enhancements in insulin stimulated glucose metabolism in ATGL-/- mice persist when challenged with a high-fat diet. ATGL-/- mice fed a low-fat diet exhibit improved whole-body insulin sensitivity and glucose tolerance compared with wild-type mice. Wild-type mice became hyperlipidemic and insulin-resistant when challenged with a high-fat diet (HFD, 60% fat) for 4 wk. ATGL-/- mice fed a HFD had elevated circulating fatty acids but had reduced fasting glycemia compared to pre-high-fat diet levels and were refractory to glucose intolerance and insulin resistance. This protection from high-fat diet-induced metabolic perturbations was associated with a preference for fatty acid utilization but reduced energy expenditure and no change in markers of mitochondrial capacity or density. The protection from high-fat diet-induced insulin resistance in ATGL-/- mice was due to increased cardiac and liver insulin-stimulated glucose clearance despite increased lipid content in these tissues. Additionally, there was no difference in skeletal muscle insulin-stimulated glucose disposal, but there was a reduction observed in brown adipose tissue. Overall, these results show that ATGL-/- mice are protected from HFD-induced insulin resistance and reveal a tissue specific disparity between lipid accumulation and insulin sensitivity.

Pigment epithelium-derived factor contributes to insulin resistance in obesity.

Obesity is a major risk factor for insulin resistance; however, the factors linking these disorders are not well defined. Herein, we show that the noninhibitory serine protease inhibitor, pigment epithelium-derived factor (PEDF), plays a causal role in insulin resistance. Adipocyte PEDF expression and serum levels are elevated in several rodent models of obesity and reduced upon weight loss and insulin sensitization. Lean mice injected with recombinant PEDF exhibited reduced insulin sensitivity during hyperinsulinemic-euglycemic clamps. Acute PEDF administration activated the proinflammatory serine/threonine kinases c-Jun terminal kinase and extracellular regulated kinase in both muscle and liver, which corresponded with reduced insulin signal transduction. Prolonged PEDF administration stimulated adipose tissue lipolysis, resulted in ectopic lipid deposition, and reduced insulin sensitivity, while neutralizing PEDF in obese mice enhanced insulin sensitivity. Overall, these results identify a causal role for PEDF in obesity-induced insulin resistance.

Metabolic remodeling in adipocytes promotes ciliary neurotrophic factor-mediated fat loss in obesity.

Obesity is characterized by an expanded adipose tissue mass, and reversing obesity reduces the risk of insulin resistance and cardiovascular disease. Ciliary neurotrophic factor (CNTF) reverses obesity by promoting the preferential loss of white adipose tissue. We evaluated the cellular and molecular mechanisms by which CNTF regulates adiposity. Obese mice fed a high-fat diet were treated with saline or recombinant CNTF for 10 d, and adipose tissue was removed for analysis. Another group fed a high-fat diet was pair fed to CNTF mice. In separate experiments, 3T3-L1 adipocytes were treated with CNTF to examine metabolic responses and signaling. CNTF reduced adipose mass that resulted from reductions in adipocyte area and triglyceride content. CNTF treatment did not affect lipolysis but resulted in decreases in fat esterification and lipogenesis and enhanced fatty acid oxidation. The enhanced fat oxidation was associated with the expression of peroxisome proliferator-activated receptor coactivator-1alpha (PGC1alpha) and nuclear respiratory factor 1 and increases in oxidative phosphorylation subunits and mitochondrial biogenesis as determined by electron microscopy. Studies in cultured adipocytes revealed that CNTF activates p38 MAPK and AMP-activated protein kinase. Inhibiting p38 activation prevented the CNTF-induced increase in PGC1alpha but not AMP-activated protein kinase activation. Diminished food intake with pair feeding induced similar decreases in fat mass, but this was related to increased expression of uncoupling protein 1. We conclude that CNTF reprograms adipose tissue to promote mitochondrial biogenesis, enhancing oxidative capacity and reducing lipogenic capacity, thereby resulting in triglyceride loss.

Apoptosis in skeletal muscle myotubes is induced by ceramides and is positively related to insulin resistance.

Fatty acid-induced apoptosis occurs in pancreatic beta-cells and contributes to the metabolic syndrome. Skeletal muscle insulin resistance is mediated by fatty acid oversupply, which also contributes to the metabolic syndrome. Therefore, we examined whether fatty acids induce apoptosis in skeletal muscle myotubes, the proapoptotic signaling involved, and the effects on insulin sensitivity. Exposure of L6 myotubes to palmitate induced apoptosis, as demonstrated by increased caspase-3 activation, phosphatidylserine exposure on the plasma membrane, and terminal deoxynucleotide transferase dUTP nick end labeling and DNA laddering, both markers of DNA fragmentation. Ceramide content was concomitantly increased, indicating a potential role for ceramides in palmitate-induced apoptosis. Supporting this notion, reducing stearoyl-CoA desaturase-1 (SCD-1) protein content with short interfering RNA resulted in ceramide accumulation and was associated with increased apoptosis in the absence of palmitate. Furthermore, the membrane-permeable C(2)-ceramide enhanced apoptosis in myotubes, whereas the ceramide synthase inhibitor, fumonisin B(1), abrogated the proapoptotic effects of palmitate. Insulin-stimulated glucose uptake was inhibited by palmitate treatment, whereas the addition of effector caspase inhibitors [Ac-DEVD-aldehyde (DEVD-CHO), Z-DQMD-FMK] independently restored >80% of the insulin-stimulated glucose uptake. These effects were observed independently from changes in the protein content of insulin signaling proteins, suggesting that proteosomal degradation is not involved in this process. We conclude that lipoapoptosis occurs in skeletal muscle myotubes, at least partially via de novo ceramide accumulation, and that inhibiting downstream apoptotic signaling improves glucose uptake in vitro.