Ceramide-Activated Protein Phosphatase Involvement in Insulin Resistance via Akt, Serine/Arginine-Rich Protein 40, and Ribonucleic Acid Splicing in L6 Skeletal Muscle Cells

Abstract

Elevated TNFα levels are associated with insulin resistance, but the molecular mechanisms linking cytokine signaling to impaired insulin function remain elusive. We previously demonstrated a role for Akt in insulin regulation of protein kinase CβII alternative splicing through phosphorylation of serine/arginine-rich protein 40, a required mechanism for insulin-stimulated glucose uptake. We hypothesized that TNFα attenuated insulin signaling by dephosphorylating Akt and its targets via ceramide-activated protein phosphatase. Western blot analysis of L6 cell lysates demonstrated impaired insulin-stimulated phosphorylation of Akt, serine/arginine-rich protein 40, and glycogen synthase kinase 3β in response to TNFα and the short chain C6 ceramide analog. TNFα increased serine/threonine phosphatase activity of protein phosphatase 1 (PP1) in response to C6, but not insulin, suggesting a ceramide-specific effect. Myriocin, an inhibitor of de novo ceramide synthesis, blocked stimulation of the PP1 activity. Ceramide species measurement by liquid chromatography-mass spectrometry showed consistent increases in C24:1 and C16 ceramides. Effects of TNFα and C6 on insulin-stimulated phosphorylation of glycogen synthase kinase 3β were prevented by myriocin and tautomycin, a PP1 inhibitor, further implicating a de novo ceramide-PP1 pathway. Alternative splicing assays demonstrated that TNFα abolished insulin-mediated inclusion of the protein kinase CβII exon. Collectively, our work demonstrates a role for PP1-like ceramide-activated protein phosphatase in mediating TNFα effects blocking insulin phosphorylation cascades involved in glycogen metabolism and alternative splicing.