Acute insulin infusion effects on ENaC subunits in mice

Abstract

Perfused tubule and cell culture studies have revealed that insulin positively regulates the activity of the epithelial sodium channel (ENaC). We investigated the effect of acute insulin injection to mice on the trafficking of ENaC subunits by immunobased approaches. Six-month-old male C57BL/CBA mice were randomly assigned one of three intraperitoneal treatments (n = 5/group): 1) control, 0.3 ml saline; 2) glucose (50% glucose in 0.3 ml saline); or 3) insulin (0.0078 units/kg·bw of insulin in 0.3 ml 50% glucose). Blood glucose levels were measured every 20 minutes by glucometer on tail blood. Blood glucose was significantly reduced by insulin (group 3) after 40 and 60 minutes, but mice were not markedly hypoglycemic (mg/dl)at 60 minutes: 126 ± 6, 256 ± 33, 87 ± 22, for groups 1–3, respectively. After 60 minutes, mice were anesthetized with pentobarbital, right kidneys were clamped and removed, and left kidneys were perfusion fixed. Immunoperoxidase labeling showed increased apical localization of γ-ENaC in the collecting duct principal cells of group 3, relative to groups 1 and 2. In agreement, western blotting of a plasma-membrane enriched fraction from whole kidney revealed an increase in the density of bands for γ-ENaC (both 85 and 70-kDa) in group 3, relative to 1 and 2, suggesting increased apical membrane residency and activation of this protein. No clear differences were found between groups for α- or β-ENaC, although α-ENaC appeared more tightly apical than did β-ENaC. These studies provide in vivo evidence of trafficking of γ-ENaC in response to acute insulin stimulation in mouse kidney, and may be part of the mechanism underlying the anti-natriuretic actions of this hormone. Funded by NIH.