title: Calpain inhibition impairs glycogen syntheses in HepG2 hepatoma cells without altering insulin signaling.
abstract: Calpains are a family of non-lysosomal cytoplasmatic cysteine proteases. Since calpain 10 (CAPN10), a member of the calpain family of proteases, has been found to represent a putative diabetes susceptibility gene, it was argued that calpains may be involved in the development of type 2 diabetes. The functional role of calpains in insulin signaling and/or insulin action is, however, not clear. We investigated the effects of the calpains 1 and 2 inhibitor PD151746 on insulin signaling and insulin action in human hepatoma G2 cells (HepG2). HepG2 cells were incubated without (-PD) or with (+PD) 5.33 micromol/l PD151746 for different times and then stimulated with 100 nmol/l insulin for 0 (t(0)), 5 (t(5)), 15 (t(15)), 30 (t(30)), 45 (t(45)), and 60 (t(60)) min. After solubilization of the cells, insulin receptor kinase activity, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1-associated phosphatidylinositol-3 kinase (PI3-kinase), PI3-kinase activity, Thr(308) phosphorlyation of Akt, amount of protein tyrosine phosphatase-epsilon (PTPepsilon), and glycogen synthase activity were determined. Incubation with PD151746 resulted in a significant reduction of insulin-stimulated glycogen synthesis compared with cells not pre-incubated with the calpain inhibitor (-PD: t(0), 4.90 +/- 1.20%; t(5), 5.90 +/- 1.02%; t(15), 5.29 +/- 0.95%; t(30), 5.60 +/- 1.10%; t(45), 5.52 +/- 0.90%; t(60), 5.67 +/- 0.97%;+PD: t(0), 4.56 +/- 1.10%; t(5), 6.16 +/- 1.05%; t(15), 7.52 +/- 1.09%; t(30), 7.68 +/- 1.10%; t(45), 8.28 +/- 0.89%; t(60), 7.69 +/- 0.98%; P < 0.05). Incubation with PD151746 significantly increased the protein amount of PTPepsilon in the cells after 12 h (-PD: t(1), 0.85 +/- 0.18 RU (Relative unit); t(8), 0.87 +/- 0.18 RU; t(12), 0.9 +/- 0.13 RU; +PD: t(1), 0.92 +/- 0.21 RU; t(8), 1.1 +/- 0.15 RU; t(12), 1.34 +/- 0.16 RU; P < 0.05). Calpain inhibition with PD151746 had no effect on the insulin stimulation of the investigated insulin signaling parameters. These results in HepG2 cells suggest that calpains play a role in the hepatic regulation of insulin-stimulated glycogen synthesis independent of the PI3-kinase/Akt signaling pathway.
authors.affiliation: Department of Internal Medicine I, University of Lübeck, Ratzeburger Allee 160, D- 23538 Lübeck, Germany. markus.meier@uni-luebeck.de
authors.forename: Markus
authors.initials: M
authors.lastname: Meier
authors.forename: Harald H
authors.initials: HH
authors.lastname: Klein
authors.forename: Jan
authors.initials: J
authors.lastname: Kramer
authors.forename: Maren
authors.initials: M
authors.lastname: Drenckhan
authors.forename: Morten
authors.initials: M
authors.lastname: Schütt
mesh_terms: D000179:Acrylates / Q000494:pharmacology*; D000730:Androstadienes / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors*; D045744:Cell Line, Tumor; D006003:Glycogen / Q000096:biosynthesis*; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D054631:Receptor-Like Protein Tyrosine Phosphatases, Class 3; D015398:Signal Transduction; D000077191:Wortmannin
chemical_list: D000179:Acrylates; D000730:Androstadienes; D002154:Calpain; D006003:Glycogen; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D017027:Protein Tyrosine Phosphatases; D011972:Receptor, Insulin; D054631:Receptor-Like Protein Tyrosine Phosphatases, Class 3; D000077191:Wortmannin; C526292:IRS1 protein, human; C117345:PD 151746; C081827:insulin receptor serine kinase; C486288:PTPRJ protein, human