Insulin stimulates the activity of Na⁺/N⁺-ATPase in human peritoneal mesothelial cells

Objective: To assess the effect of insulin on the Na⁺/ K⁺-ATPase expression and activity in human peritoneal mesothelial cells (HPMC). Methods: HPMC were isolated from the omental tissue of non-uremic patients, grown to confluence and rendered quiescent by serum deprivation for 24 hours. The activity of Na⁺/K⁺-ATPase was determined by measuring the ouabain-sensitive ⁸⁶Rb uptake. To assess whether the effect of insulin was related to changes in [Na⁺](i) the sodium influx was measured with ²²Na and the activity of Na⁺/K⁺-ATPase was assessed in the presence of amiloride. Expression of Na⁺/K⁺-ATPaseα₁, α₂ and β₁-subunit mRNAs was determined by RT/PCR. Results: Exposure of HPMC to insulin resulted in a time- and dose-dependent increase in the Na⁺/K⁺-ATPase activity. After 60 minutes the ouabain-sensitive ⁸⁶Rb uptake (cpm/10⁴ cells) was increased from 6650 ± 796 in control cells to 9763 ± 1212 in HPMC exposed to 100 mU/mL insulin (1.5-fold increase; n = 4, P < 0.05). In addition, incubation of HPMC with 100 mU/mL insulin resulted in a time-dependent increase in the ²²Na influx. pre-exposure of HPMC to 1 mM amiloride reduced the activity of Na⁺/K⁺-ATPase but did not block the stimulatory effect of insulin. RT/PCR analysis revealed that HPMC constitutively expressed α₁- and β₁-subunit mRNAs while the α₂-subunit mRNA was barely detectable. Exposure of HPMC to insulin for up to 24 hours was not associated with any changes in the expression of either α₁, α₂ or β₁-subunit. Conclusion: Insulin stimulates the Na⁺/K⁺-ATPase activity in HPMC in a time- and dose-dependent manner. This effect appears to mediated by an increase in [Na⁺](i) and is not related to alterations in Na⁺/K⁺-ATPase subunit mRNAs expression.