Meier Markus, Klein Harald H, Kramer Jan, Drenckhan Maren, Schütt Morten
Department of Internal Medicine I, University of Lübeck, Ratzeburger Allee 160, D- 23538 Lübeck, Germany.
J Endocrinol. 2007 Apr;193(1):45-51. doi: 10.1677/joe.1.07087.
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.
钙蛋白酶是一类非溶酶体细胞质半胱氨酸蛋白酶。由于已发现蛋白酶钙蛋白酶家族成员钙蛋白酶10（CAPN10）是一个假定的糖尿病易感基因，因此有人认为钙蛋白酶可能参与2型糖尿病的发生发展。然而，钙蛋白酶在胰岛素信号传导和/或胰岛素作用中的功能作用尚不清楚。我们研究了钙蛋白酶1和2抑制剂PD151746对人肝癌G2细胞（HepG2）中胰岛素信号传导和胰岛素作用的影响。将HepG2细胞在无（-PD）或含（+PD）5.33微摩尔/升PD151746的条件下孵育不同时间，然后用100纳摩尔/升胰岛素刺激0（t(0)）、5（t(5)）、15（t(15)）、30（t(30)）、45（t(45)）和60（t(60)）分钟。细胞溶解后，测定胰岛素受体激酶活性、胰岛素受体底物-1（IRS-1）的酪氨酸磷酸化、与IRS-1相关的磷脂酰肌醇-3激酶（PI3激酶）、PI3激酶活性、Akt的苏氨酸（Thr(308)）磷酸化、蛋白酪氨酸磷酸酶ε（PTPε）的量以及糖原合酶活性。与未用钙蛋白酶抑制剂预孵育的细胞相比（-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），用PD151746孵育导致胰岛素刺激的糖原合成显著减少。用PD151746孵育12小时后，细胞中PTPε的蛋白量显著增加（-PD：t(1)，0.85±0.18相对单位（RU）；t(8)，0.87±0.18 RU；t(12)，0.
