Impaired Glucagon and Insulin Responses in Mice Lacking the Cl-Loader Nkcc2a

Abstract

In pancreatic β-cells, glucose metabolism and plasma membrane depolarization are accountable for the early secretory response, a mechanism regulated primarily, though not exclusively, by ATP-sensitive K+ channels. In most cells, including β-cells, the net functional balance between Cl- loaders e.g., Nkcc1 and Nkcc2a and extruders like Kcc2 keeps the intracellular Cl- concentration above equilibrium creating an outwardly directed anionic gradient, which is electrogenically dissipated by Cl- channel opening. This makes possible the additional driving force for depolarization, which in β-cells prolongs the action potentials necessary for sustained insulin secretion.β-cells express low levels of the ’kidney-specific’ Na+K+2Cl- transporter Nkcc2a. This protein and Nkcc1 are targeted by bumetanide, which by blocking Cl- loading into β-cells, impairs insulin secretion provoking intermittent hyperglycemia, as seen in patients treated with this diuretic. Since the chronic functional implications of Nkcc2a in fuel homeostasis in vivo remain unexplored, we subjected Nkcc2aKO mice to dynamic tests of glucose homeostasis and determined their secretory responses in vivo and in vitro using islets. We found that Nkcc2aKO mice exhibit: i) increased steady-state blood glucose, plasma insulin, glucagon and de novo hepatic glucose production, ii) absent glucagon responses to physiological (6h) or prolonged (16h) fasting, iii) glucose intolerance and delayed in vivo insulin responses to glucose, iv) hyperplastic and hypertrophic α- and β-cells, v) conserved islet insulin responses to glucose, and vi) normal and reduced expression levels of Nkcc1 and Kcc2, the main Cl- loader and extruder in β-cells, respectively. Together, our results suggest for the first time that mice lacking Nkcc2a exhibit a complex metabolic phenotype related to unrestrained plasma glucagon, impaired insulin and glucagon responses in vivo in spite of compensatory mechanisms related to the regulation of Cl- transport in the islet. Disclosure L.E. Kelly: None. M. DiFulvio: None.