The epithelial Na+ channel (ENaC) in pancreatic islet β-cells regulates insulin secretion and glucose metabolism

Abstract

Insulin is exclusively produced and secreted by pancreatic islet β-cells, which plays a central role in regulating glucose metabolism. In response to blood glucose elevation or other physiological stimuli, β-cells are excited to trigger intracellular events such as Ca2+ mobilization and cAMP elevation leading to exocytosis of insulin granules, although the underlying molecular mechanisms are not fully understood. Previously, we identified a chloride channel CFTR in contributing to β-cell excitability and insulin secretion. In epithelial cells, it is well noted that CFTR closely interacts with the epithelial Na+ channel (ENaC), which is best known for its role in epithelial Na+ absorption. The present study aimed to elucidate whether and how ENaC is expressed in pancreatic islet β-cells to regulate insulin secretion and glucose metabolism. In the first part of study, we analyzed human databases, primary rat/mouse pancreatic tissues as well as RINm5F, a rat β-cell line, which confirmed the expression of Scnn1a, Scnn1b and Scnn1g genes (encoding ENaC subunits, α, β and γ, respectively) in human and rodent β-cells. To our surprise, inhibiting this Na+ channel by selective blockers, amiloride or benzamil, did not retard insulin secretion, but instead triggered a slow membrane depolarization with electrical bursts, elicited substantial Ca2+ oscillations and promoted insulin secretion in RINm5F or isolated mouse β-cells. siRNA-based knockdown of ENaCα, the rate-limiting subunit of ENaC, in RINm5F cells confirmed that deficiency of ENaC induced a significant increase in insulin secretion. These findings suggested that ENaC in pancreatic islet β-cells is inversely correlated with insulin secretion. The second part of the study was carried out to investigate the mechanism underlying the ENaC-mediated suppression of insulin secretion. Proteomic analysis of RINm5F cells through mass spectrometry showed ENaC knockdown altered glucose metabolism and cAMP-related insulin secretion signaling pathways, consistently suggesting a role of ENaC deficiency in exciting β-cells to release insulin. Further study confirmed that ENaC deficiency indeed caused an increase in intracellular cAMP levels. Pharmaceutical inhibition of the cAMP-synthesized enzyme adenyl cyclase (AC) by DDA or knockdown AC6/AC8 abolished the effects of ENaC deficiency including membrane depolarization, Ca2+ increases and insulin secretion. Manipulation of intracellular sodium levels together with recording cAMP signals revealed that high concentrations of sodium inhibit the cAMP response to its agonist forskolin. It therefore suggested that ENaC-mediated Na+ entry exerts an inhibitory effect on ACs activity and thus cAMP production which modulates multiple downstream events/factors to suppress modulating insulin secretion in β-cells. Next, to evaluate the effect of ENaC on insulin secretion and glucose metabolism in vivo, a conditional knockout (cKO) mouse model with β-cell-specific knockout of ENaCα (Scnn1afl/fl, Ins2-Cre+) was built, which exhibited disturbed responses in glucose tolerance in comparison with the loxp-negative Cre control mice (Scnn1awt/wt, Ins2-Cre+). The cKO mice, at young ages before puberty, exhibited insulin hypersecretion, hypoglycemia, enhanced PKA activation in islets as compared to the Cre control mice, although these changes were found diminished when the mice grew to mature ages (> 8-week-old). However, isolated islets from young or old cKO mice consistently showed hypersecretion of insulin in vitro. Taken together, the present study has revealed a previously undefined role of ENaC in regulating β-cell function and insulin secretion: ENaC serves as a brake to restrain insulin secretion by inhibiting of cAMP production. Therefore, it provides new insights into the understanding the mechanism of insulin secretion in β-cells. Malfunction of ENaC could be a possible etiology of related disorder such as diabetes. The revealed novel role of ENaC in regulating cAMP production may have other implications beyond β-cell physiology.