Trevor J. Biden

Ceramide Generation Is Sufficient to Account for the Inhibition of the Insulin-stimulated PKB Pathway in C2C12 Skeletal Muscle Cells Pretreated with Palmitate

We have employed C2C12 myotubes to investigate lipid inhibition of insulin-stimulated signal transduction and glucose metabolism. Cells were preincubated for 18 h in the absence or presence of free fatty acids (FFAs) and stimulated with insulin, and the effects on glycogen synthesis and signaling intermediates were determined. While the unsaturated FFAs oleate and linoleate inhibited both basal and insulin-stimulated glycogen synthesis, the saturated FFA palmitate reduced only insulin-stimulated glycogen synthesis, and was found to inhibit insulin-stimulated phosphorylation of glycogen synthase kinase-3 and protein kinase B (PKB). However, no effect of palmitate was observed on tyrosine phosphorylation, p85 association, or phosphatidylinositol 3-kinase activity in IRS-1 immunoprecipitates. In contrast, palmitate promoted phosphorylation of mitogen-activated protein MAP) kinases. Ceramide, a derivative of palmitate, has recently been associated with similar inhibition of PKB, and here, ceramide levels were found to be elevated 2-fold in palmitate-treated C2C12 cells. Incubation of C2C12 cells with ceramide closely reproduced the effects of palmitate, leading to inhibition of glycogen synthesis and PKB and to stimulation of MAP kinase. We conclude that palmitate-induced insulin resistance occurs by a mechanism distinct from that of unsaturated FFAs, and involves elevation of ceramide byde novo synthesis, leading to PKB inhibition without affecting IRS-1 function.

Alterations in the Expression and Cellular Localization of Protein Kinase C Isozymes ε and θ Are Associated With Insulin Resistance in Skeletal Muscle of the High-Fat–Fed Rat

We have tested the hypothesis that changes in the levels and cellular location of protein kinase C (PKC) isozymes might be associated with the development of insulin resistance in skeletal muscles from the highfat–fed rat. Lipid measurements showed that triglyceride and diacylglycerol, an activator of PKC, were elevated four- and twofold, respectively. PKC activity assays indicated that the proportion of membraneassociated calcium-independent PKC was also increased. As determined by immunoblotting, total (particulate plus cytosolic) PKC α, ε, and ζ levels were not different between control and fat-fed rats. However, the ratio of particulate to cytosolic PKC ε in red muscles from fat-fed rats was increased nearly sixfold, suggesting chronic activation. In contrast, the amount of cytosolic PKC θ was downregulated to 45% of control, while the ratio of particulate to cytosolic levels increased, suggesting a combination of chronic activation and downregulation. Interestingly, while insulin infusion in glucose-clamped rats increased the proportion of PKC θ in the particulate fraction of red muscle, this was potentiated by fat-feeding, suggesting that the translocation is a consequence of altered lipid flux rather than a proximal event in insulin signaling. PKC ε and θ measurements from individual rats correlated with triglyceride content of red gastrocnemius muscle; they did not correlate with plasma glucose, which was not elevated in fat-fed rats, suggesting that they were not simply a consequence of hyperglycemia. Our results suggest that these specific alterations in PKC ε and PKC θ might contribute to the link between increased lipid availability and muscle insulin resistance previously described using high-fat–fed rats.

Improved glucose homeostasis and enhanced insulin signalling in Grb14-deficient mice

Gene targeting was used to characterize the physiological role of growth factor receptor‐bound (Grb)14, an adapter‐type signalling protein that associates with the insulin receptor (IR). Adult male Grb14−/− mice displayed improved glucose tolerance, lower circulating insulin levels, and increased incorporation of glucose into glycogen in the liver and skeletal muscle. In ex vivo studies, insulin‐induced 2‐deoxyglucose uptake was enhanced in soleus muscle, but not in epididymal adipose tissue. These metabolic effects correlated with tissue‐specific alterations in insulin signalling. In the liver, despite lower IR autophosphorylation, enhanced insulin‐induced tyrosine phosphorylation of insulin receptor substrate (IRS)‐1 and activation of protein kinase B (PKB) was observed. In skeletal muscle, IR tyrosine phosphorylation was normal, but signalling via IRS‐1 and PKB was increased. Finally, no effect of Grb14 ablation was observed on insulin signalling in white adipose tissue. These findings demonstrate that Grb14 functions in vivo as a tissue‐specific modulator of insulin action, most likely via repression of IR‐mediated IRS‐1 tyrosine phosphorylation, and highlight this protein as a potential target for therapeutic intervention.

Cytokine-induced β-cell death is independent of endoplasmic reticulum stress signaling

OBJECTIVE—Cytokines contribute to β-cell destruction in type 1 diabetes. Endoplasmic reticulum (ER) stress–mediated apoptosis has been proposed as a mechanism for β-cell death. We tested whether ER stress was necessary for cytokine-induced β-cell death and also whether ER stress gene activation was present in β-cells of the NOD mouse model of type 1 diabetes. RESEARCH DESIGN AND METHODS—INS-1 β-cells or rat islets were treated with the chemical chaperone phenyl butyric acid (PBA) and exposed or not to interleukin (IL)-1β and γ-interferon (IFN-γ). Small interfering RNA (siRNA) was used to silence C/EBP homologous protein (CHOP) expression in INS-1 β-cells. Additionally, the role of ER stress in lipid-induced cell death was assessed. RESULTS—Cytokines and palmitate triggered ER stress in β-cells as evidenced by increased phosphorylation of PKR-like ER kinase (PERK), eukaryotic initiation factor (EIF)2α, and Jun NH2-terminal kinase (JNK) and increased expression of activating transcription factor (ATF)4 and CHOP. PBA treatment attenuated ER stress, but JNK phosphorylation was reduced only in response to palmitate, not in response to cytokines. PBA had no effect on cytokine-induced cell death but was associated with protection against palmitate-induced cell death. Similarly, siRNA-mediated reduction in CHOP expression protected against palmitate- but not against cytokine-induced cell death. In NOD islets, mRNA levels of several ER stress genes were reduced (ATF4, BiP [binding protein], GRP94 [glucose regulated protein 94], p58, and XBP-1 [X-box binding protein 1] splicing) or unchanged (CHOP and Edem1 [ER degradation enhancer, mannosidase α–like 1]). CONCLUSIONS—While both cytokines and palmitate can induce ER stress, our results suggest that, in contrast to lipoapoptosis, the PERK-ATF4-CHOP ER stress–signaling pathway is not necessary for cytokine-induced β-cell death.

Protein Kinase Cδ Activation by Interleukin-1β Stabilizes Inducible Nitric-oxide Synthase mRNA in Pancreatic β-Cells

Exposure of pancreatic islets to cytokines such as interleukin (IL)-1β induces a variety of proinflammatory genes including type II nitric-oxide synthase (iNOS) which produces nitric oxide (NO). NO is thought to be a major cause of islet β-cell dysfunction and apoptotic β-cell death, which results in type I diabetes. Since protein kinase C (PKC) mediates some of the actions of cytokines in other cell types, our aim was to assess the role of PKC in IL-1β-induced iNOS expression in pancreatic β-cells. PKCδ, but not PKCα, was specifically activated in the rat INS-1 β-cell line by IL-1β as assessed by membrane translocation. Moreover, iNOS expression and NO production were significantly attenuated by the PKCδ specific inhibitor rottlerin and overexpression of a PKCδ kinase-dead mutant protein. Conversely, overexpression of PKCδ wild type protein significantly potentiated this response. These results were confirmed at the mRNA level by reverse transcriptase-polymerase chain reaction. However, a role at the level of transcriptional regulation appeared unlikely, since PKCδ was not required for the activation of NF-κB, activating protein 1, and activating transcription factor 2 signaling pathways in response to IL-1β. There was, however, a significant increase in iNOS mRNA stability mediated by PKCδ wild type, while PKCδ kinase-dead acted reciprocally, reducing iNOS mRNA stability. The results indicate that, in addition to transcriptional activation, mRNA stabilization is a key component of the mechanism by which IL-1β stimulates iNOS expression in β-cells and that PKCδ plays an essential role in this process. PKCδ activation may therefore have significant consequences with regard to cellular function and viability when β-cells are exposed to IL-1β and potentially other cytokines.

Muscle lipid accumulation and protein kinase C activation in the insulin-resistant chronically glucose-infused rat

Chronic glucose infusion results in hyperinsulinemia and causes lipid accumulation and insulin resistance in rat muscle. To examine possible mechanisms for the insulin resistance, alterations in malonyl-CoA and long-chain acyl-CoA (LCA-CoA) concentration and the distribution of protein kinase C (PKC) isozymes, putative links between muscle lipids and insulin resistance, were determined. Cannulated rats were infused with glucose (40 mg ⋅ kg-1 ⋅ min-1) for 1 or 4 days. This increased red quadriceps muscle LCA-CoA content (sum of 6 species) by 1.3-fold at 1 day and 1.4-fold at 4 days vs. saline-infused controls (both P < 0.001 vs. control). The concentration of malonyl-CoA was also increased (1.7-fold at 1 day, P < 0.01, and 2.2-fold at 4 days, P < 0.001 vs. control), suggesting an even greater increase in cytosolic LCA-CoA. The ratio of membrane to cytosolic PKC-ε was increased twofold in the red gastrocnemius after both 1 and 4 days, suggesting chronic activation. No changes were observed for PKC-α, -δ, and -θ. We conclude that LCA-CoAs accumulate in muscle during chronic glucose infusion, consistent with a malonyl-CoA-induced inhibition of fatty acid oxidation (reverse glucose-fatty acid cycle). Accumulation of LCA-CoAs could play a role in the generation of muscle insulin resistance by glucose oversupply, either directly or via chronic activation of PKC-ε.

A lipidomic screen of palmitate-treated MIN6 β-cells links sphingolipid metabolites with endoplasmic reticulum (ER) stress and impaired protein trafficking

Saturated fatty acids promote lipotoxic ER (endoplasmic reticulum) stress in pancreatic β-cells in association with Type 2 diabetes. To address the underlying mechanisms we employed MS in a comprehensive lipidomic screen of MIN6 β-cells treated for 48 h with palmitate. Both the overall mass and the degree of saturation of major neutral lipids and phospholipids were only modestly increased by palmitate. The mass of GlcCer (glucosylceramide) was augmented by 70% under these conditions, without any significant alteration in the amounts of either ceramide or sphingomyelin. However, flux into ceramide (measured by [3H]serine incorporation) was augmented by chronic palmitate, and inhibition of ceramide synthesis decreased both ER stress and apoptosis. ER-to-Golgi protein trafficking was also reduced by palmitate pre-treatment, but was overcome by overexpression of GlcCer synthase. This was accompanied by increased conversion of ceramide into GlcCer, and reduced ER stress and apoptosis, but no change in phospholipid desaturation. Sphingolipid alterations due to palmitate were not secondary to ER stress since they were neither reproduced by pharmacological ER stressors nor overcome using the chemical chaperone phenylbutyric acid. In conclusion, alterations in sphingolipid, rather than phospholipid, metabolism are more likely to be implicated in the defective protein trafficking and enhanced ER stress and apoptosis of lipotoxic β-cells.

Protein Kinase C Function in Muscle, Liver, and β-Cells and Its Therapeutic Implications for Type 2 Diabetes

Increased lipid availability is strongly associated with both β-cell dysfunction and insulin resistance, two key facets of type 2 diabetes. Isoforms of the protein kinase C (PKC) family have been viewed as candidates for mediating the effects of fat oversupply because they are lipid-dependent kinases with wide-ranging roles in signal transduction, including the positive and negative modulation of insulin action. Until recently, their involvement was based on correlative studies, but now causative roles for distinct PKC isoforms have also been addressed, in both pancreatic β-cells and insulin-sensitive tissues. Our goal here, therefore, is to review the hitherto disparate fields of PKC function in insulin signaling/resistance on the one hand and in regulating β-cell biology on the other hand. By integrating these two areas, we provide a reappraisal of the current paradigm regarding PKC and type 2 diabetes. In particular, we propose that PKCe warrants further investigation, not merely as a treatment for insulin resistance as previously supposed, but also as a positive regulator of insulin availability. The protein kinase C (PKC) family comprises 10 isoforms that have been subdivided into three groups (Fig. 1) based on sequence homology and mechanisms of activation (rev. in 1). While differentiated by their sensitivity to Ca2+, both the conventional PKCs (cPKCα, -β, and -γ) and novel PKCs (nPKCδ, -e, -η, and -θ) are dependent on diacylglycerol (DAG) for full activation. These isoforms are therefore responsive to the stimulation of G protein–coupled receptors or receptor tyrosine kinases, which activate phospholipase C, inducing the hydrolysis of phosphatidylinositol 4,5-bisphosphate at the plasma membrane and the resultant generation of DAG and Ca2+. Evidence for the acute elevation of DAG in this fashion by insulin was reported in early studies (2), although the identities of the putative phospholipase(s) and phospholipid substrates involved were never clarified. …

Reduced endoplasmic reticulum (ER)-to-Golgi protein trafficking contributes to ER stress in lipotoxic mouse beta cells by promoting protein overload

Aims/hypothesisSaturated fatty acids augment endoplasmic reticulum (ER) stress in pancreatic beta cells and this is implicated in the loss of beta cell mass that accompanies type 2 diabetes. However, the mechanisms underlying the induction of ER stress are unclear. Our aim was to establish whether saturated fatty acids cause defects in ER-to-Golgi protein trafficking, which may thereby contribute to ER stress via protein overload.MethodsCells of the mouse insulinoma cell line MIN6 were transfected with temperature-sensitive vesicular stomatitis virus G protein (VSVG) tagged with green fluorescent protein to quantify the rate of ER-to-Golgi protein trafficking. I14 antibody, which detects only correctly folded VSVG, was employed to probe the folding environment of the ER. ER stress markers were monitored by western blotting.ResultsPretreatment with palmitate, but not oleate, significantly reduced the rate of ER-to-Golgi protein trafficking assessed using VSVG. This was not secondary to ER stress, since thapsigargin, which compromises chaperone function by depletion of ER calcium, markedly inhibited VSVG folding and promoted strong ER stress but only slightly reduced protein trafficking. Blockade of ER-to-Golgi protein trafficking with brefeldin A (BFA) was sufficient to trigger ER stress, but neither BFA nor palmitate compromised VSVG folding.Conclusions/interpretationReductions in ER-to-Golgi protein trafficking potentially contribute to ER stress during lipoapoptosis. In this case ER stress would be triggered by protein overload, rather than a disruption of the protein-folding capacity of the ER.

Protein kinase C delta activation by IL-1beta stabilises inducible NO synthase mRNA in pancreatic beta-cells

Exposure of pancreatic islets to cytokines such as interleukin (IL)-1β induces a variety of proinflammatory genes including type II nitric-oxide synthase (iNOS) which produces nitric oxide (NO). NO is thought to be a major cause of islet β-cell dysfunction and apoptotic β-cell death, which results in type I diabetes. Since protein kinase C (PKC) mediates some of the actions of cytokines in other cell types, our aim was to assess the role of PKC in IL-1β-induced iNOS expression in pancreatic β-cells. PKCδ, but not PKCα, was specifically activated in the rat INS-1 β-cell line by IL-1β as assessed by membrane translocation. Moreover, iNOS expression and NO production were significantly attenuated by the PKCδ specific inhibitor rottlerin and overexpression of a PKCδ kinase-dead mutant protein. Conversely, overexpression of PKCδ wild type protein significantly potentiated this response. These results were confirmed at the mRNA level by reverse transcriptase-polymerase chain reaction. However, a role at the level of transcriptional regulation appeared unlikely, since PKCδ was not required for the activation of NF-κB, activating protein 1, and activating transcription factor 2 signaling pathways in response to IL-1β. There was, however, a significant increase in iNOS mRNA stability mediated by PKCδ wild type, while PKCδ kinase-dead acted reciprocally, reducing iNOS mRNA stability. The results indicate that, in addition to transcriptional activation, mRNA stabilization is a key component of the mechanism by which IL-1β stimulates iNOS expression in β-cells and that PKCδ plays an essential role in this process. PKCδ activation may therefore have significant consequences with regard to cellular function and viability when β-cells are exposed to IL-1β and potentially other cytokines.