title: 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 TNFalpha 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 CbetaII alternative splicing through phosphorylation of serine/arginine-rich protein 40, a required mechanism for insulin-stimulated glucose uptake. We hypothesized that TNFalpha 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 3beta in response to TNFalpha and the short chain C6 ceramide analog. TNFalpha 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 TNFalpha and C6 on insulin-stimulated phosphorylation of glycogen synthase kinase 3beta were prevented by myriocin and tautomycin, a PP1 inhibitor, further implicating a de novo ceramide-PP1 pathway. Alternative splicing assays demonstrated that TNFalpha abolished insulin-mediated inclusion of the protein kinase CbetaII exon. Collectively, our work demonstrates a role for PP1-like ceramide-activated protein phosphatase in mediating TNFalpha effects blocking insulin phosphorylation cascades involved in glycogen metabolism and alternative splicing.
authors.affiliation: Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Nilanjan
authors.initials: N
authors.lastname: Ghosh
authors.forename: Niketa
authors.initials: N
authors.lastname: Patel
authors.forename: Kun
authors.initials: K
authors.lastname: Jiang
authors.forename: James E
authors.initials: JE
authors.lastname: Watson
authors.forename: Jin
authors.initials: J
authors.lastname: Cheng
authors.forename: Charles E
authors.initials: CE
authors.lastname: Chalfant
authors.forename: Denise R
authors.initials: DR
authors.lastname: Cooper
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects; D000818:Animals; D002478:Cells, Cultured; D002518:Ceramides / Q000494:pharmacology; D007333:Insulin Resistance; D032342:Muscle Cells / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D064546:Protein Kinase C beta; D054645:Protein Phosphatase 1; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology*; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000502:physiology*; D051381:Rats; D000068103:Serine-Arginine Splicing Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D002518:Ceramides; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D014409:Tumor Necrosis Factor-alpha; C518407:serine-arginine-rich protein 40, rat; D000068103:Serine-Arginine Splicing Factors; D012313:RNA; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1