Hydrogen peroxide induces activation of insulin signaling pathway via AMP-dependent kinase in podocytes

Abstract

Podocytes are cells that form the glomerular filtration barrier in the kidney. Insulin signaling in podocytes is critical for normal kidney function. Insulin signaling is regulated by oxidative stress and intracellular energy levels. We cultured rat podocytes to investigate the effects of hydrogen peroxide (H2O2) on the phosphorylation of proximal and distal elements of insulin signaling. We also investigated H2O2-induced intracellular changes in the distribution of protein kinase B (Akt). Western blots showed that H2O2 (100μM) induced rapid, transient phosphorylation of the insulin receptor (IR), the IR substrate-1 (IRS1), and Akt with peak activities at 5min (Δ 183%, P<0.05), 3min (Δ 414%, P<0.05), and 10min (Δ 35%, P<0.05), respectively. Immunostaining cells with an Akt-specific antibody showed increased intensity at the plasma membrane after treatment with H2O2>. Furthermore, H2O2 inhibited phosphorylation of the phosphatase and tensin homologue (PTEN; peak activity at 10min; Δ −32%, P<0.05) and stimulated phosphorylation of the AMP-dependent kinase alpha subunit (AMPKα; 78% at 3min and 244% at 10min). The stimulation of AMPK was abolished with an AMPK inhibitor, Compound C (100μM, 2h). Moreover, Compound C significantly reduced the effect of H2O2 on IR phosphorylation by about 40% (from 2.07±0.28 to 1.28±0.12, P<0.05). In addition, H2O2 increased glucose uptake in podocytes (from 0.88±0.04 to 1.29±0.12nmol/min/mg protein, P<0.05), and this effect was attenuated by Compound C.Our results suggested that H2O2 activated the insulin signaling pathway and glucose uptake via AMPK in cultured rat podocytes. This signaling may play a potential role in the prevention of insulin resistance under conditions associated with oxidative stress.