Abstract
Ceramides are known to promote insulin resistance in a number of metabolically important tissues including skeletal muscle, the predominant site of insulin-stimulated glucose disposal. Depending on cell type, these lipid intermediates have been shown to inhibit protein kinase B (PKB/Akt), a key mediator of the metabolic actions of insulin, via two distinct pathways: one involving the action of atypical protein kinase C (aPKC) isoforms, and the second dependent on protein phosphatase-2A (PP2A). The main aim of this study was to explore the mechanisms by which ceramide inhibits PKB/Akt in three different skeletal muscle-derived cell culture models; rat L6 myotubes, mouse C2C12 myotubes and primary human skeletal muscle cells. Our findings indicate that the mechanism by which ceramide acts to repress PKB/Akt is related to the myocellular abundance of caveolin-enriched domains (CEM) present at the plasma membrane. Here, we show that ceramide-enriched-CEMs are markedly more abundant in L6 myotubes compared to C2C12 myotubes, consistent with their previously reported role in coordinating aPKC-directed repression of PKB/Akt in L6 muscle cells. In contrast, a PP2A-dependent pathway predominantly mediates ceramide-induced inhibition of PKB/Akt in C2C12 myotubes. In addition, we demonstrate for the first time that ceramide engages an aPKC-dependent pathway to suppress insulin-induced PKB/Akt activation in palmitate-treated cultured human muscle cells as well as in muscle cells from diabetic patients. Collectively, this work identifies key mechanistic differences, which may be linked to variations in plasma membrane composition, underlying the insulin-desensitising effects of ceramide in different skeletal muscle cell models that are extensively used in signal transduction and metabolic studies.
Highlights
Once bound to its receptor, insulin stimulates a signalling network that functions to regulate whole-body glucose homeostasis by coordinating numerous physiological processes
In an initial attempt to identify factors underpinning the distinct mechanisms involved in mediating the repressive effects of ceramide upon PKB/Akt in L6 and C2C12 myotubes through PKCf and phosphatase 2A (PP2A) respectively, we hypothesised that differences in caveolin-enriched microdomains (CEM) abundance and/or composition may account for these differential modes of action
Because ceramide is known to accumulate within CEMs [21], we assessed whether ceramide concentration would correlate with CEM caveolin-3 content in detergent-resistant membranes (DRM) fractions isolated from these two muscle cell lines
Summary
Once bound to its receptor, insulin stimulates a signalling network that functions to regulate whole-body glucose homeostasis by coordinating numerous physiological processes. Various studies have suggested that ectopic accumulation of ceramide in response to oversupply of saturated fatty acids including palmitate may underlie the development of insulin resistance in this tissue [4,5,6]. We and others have demonstrated that ceramide can impair insulin action through inhibition of protein kinase B (PKB/ Akt), a key signal transduction intermediate that plays a pivotal role in coordinating the insulin-dependent uptake and utilization of glucose [7,8]. In differentiated rat L6 myotubes, treatment with palmitate or exogenous ceramide leads to the activation of the atypical protein kinase C isoform PKCf which in turn directly interacts with and phosphorylates the pleckstrin homolog (PH) domain of PKB/Akt at Thr. Whilst exposure of C2C12 myotubes to palmitate has been shown to result in the activation of aPKCf, PKB/Akt becomes repressed primarily through its dephosphorylation by protein phosphatase 2A (PP2A) [12]
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