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

Department of Molecular Medicine (N.G., N.P., K.J., D.R.C.), College of Medicine, University of South Florida, Tampa, Florida 33612; The Research Service (J.E.W., D.R.C.), James A. Haley Veterans Hospital, Tampa, Florida 33612; Moffitt Cancer Center (J.C.), Tampa, Florida 33612; and Department of Biochemistry (C.E.C.), Virginia Commonwealth University and Hunter Holmes McGuire Veterans Medical Center, Richmond, Virginia 23298

Address all correspondence and requests for reprints to: Denise R. Cooper, Ph.D., J. A. Haley Veterans Hospital VAR 151, 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612. E-mail: dcooper{at}health.usf.edu.

Elevated TNF 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 CßII alternative splicing through phosphorylation of serine/arginine-rich protein 40, a required mechanism for insulin-stimulated glucose uptake. We hypothesized that TNF 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 3ß in response to TNF and the short chain C6 ceramide analog. TNF 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 TNF and C6 on insulin-stimulated phosphorylation of glycogen synthase kinase 3ß were prevented by myriocin and tautomycin, a PP1 inhibitor, further implicating a de novo ceramide-PP1 pathway. Alternative splicing assays demonstrated that TNF abolished insulin-mediated inclusion of the protein kinase CßII exon. Collectively, our work demonstrates a role for PP1-like ceramide-activated protein phosphatase in mediating TNF effects blocking insulin phosphorylation cascades involved in glycogen metabolism and alternative splicing.

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