Regulation of hexokinase activity by protein kinase C in peritoneal mesothelium cell

Abstract

To investigate the effect of protein kinase C (PKC) on the activity of hexokinase (HK) in rat peritoneal mesothelium cell (PMC) and the functional consequences in glucose uptake. omentum obtained from albino male rats of the Sprague Dawley strain, PMC were isolated and subculture by enzymatic disaggregation. The antibody to cytokeratin was used for identified by immunofluorescence technique. After treated with PKC activator--phorbol esters (PMA and PDD) and classical PKC (cPKC) inhibitor--Gö6976, total HK activity in PMC were measured by a standard G6PDH-coupled assay, glucose disappearance in the culture medium were tested as glucose net utilization by colorimetric method. The PMC were polygonal when confluent and reacted positively for cytokeratin. PMA induced HK activity in a time and dose-dependent manner and increased net glucose utilization. At concentration of 0.01, 0.1 and 1 micromol/L PMA for 24 h, the increase in HK activity were 30.7%, 59.9% and 99.1%, respectively above the level in the control (P < 0.05). After treated with 1 micromol/L PMA for 6 h, the HK activity of PMC began to up-regulation, the peak reached at 24 h (21.3% vs 100.4%, P < 0.05), and accompanied by increase in net glucose utilization (2 mmol/L, P < 0.05). The expression of HK induced by 0.1 micromol/L PDD were similar to 1 micromol/L PMA, 4 alpha-PDD whose structure resemble to PDD didn't have effect on HK. Gö6976 blocked the activity of HK induced by PMA. After incubated with Gö6976 at concentration of 10, 20, 30, 40 and 50 micromol/L for 30 min, PMC were cultured in 1 micromol/L PMA for 24 h, the decrease in HK activity were 14.6%, 26.8%, 34.2%, 44.2% and 54.9% (P < 0.05). Elevated HK activity by PKC was accompanied with corresponding increase in net glucose utilization, which might take part in the accelerated glucose uptake after long-term peritoneal dialysis. The blocking of HK activity by cPKC inhibitor might be an effective method for increasing ultrafiltration in peritoneal dialysis.