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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.
AuthorsMarkus Meier, Harald H Klein, Jan Kramer, Maren Drenckhan, Morten Schütt
JournalThe Journal of endocrinology (J Endocrinol) Vol. 193 Issue 1 Pg. 45-51 (Apr 2007) ISSN: 0022-0795 [Print] England
PMID17400802 (Publication Type: Journal Article)
Chemical References
  • Acrylates
  • Androstadienes
  • IRS1 protein, human
  • Insulin
  • Insulin Receptor Substrate Proteins
  • PD 151746
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphoproteins
  • Glycogen
  • Receptor, Insulin
  • Protein Serine-Threonine Kinases
  • insulin receptor serine kinase
  • PTPRJ protein, human
  • Protein Tyrosine Phosphatases
  • Receptor-Like Protein Tyrosine Phosphatases, Class 3
  • Calpain
  • Wortmannin
Topics
  • Acrylates (pharmacology)
  • Androstadienes (pharmacology)
  • Calpain (antagonists & inhibitors)
  • Cell Line, Tumor
  • Glycogen (biosynthesis)
  • Humans
  • Insulin (metabolism, pharmacology)
  • Insulin Receptor Substrate Proteins
  • Liver (drug effects, metabolism)
  • Phosphatidylinositol 3-Kinases (metabolism)
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphoproteins (metabolism)
  • Protein Binding (drug effects)
  • Protein Serine-Threonine Kinases (metabolism)
  • Protein Tyrosine Phosphatases (metabolism)
  • Receptor, Insulin (metabolism)
  • Receptor-Like Protein Tyrosine Phosphatases, Class 3
  • Signal Transduction
  • Wortmannin

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