Glucokinase Mediated Glucosensing in Hypothalamic Neurons

Abstract

Glucosensing is the ability of specialized cells to detect changes in extracellular glucose concentration and convert this information into a change in membrane potential; this allows glucose levels to influence the activity of cells that regulate glucose-altering or glucose-dependent physiological processes. Two major glucosensing cellular populations are pancreatic and hypothalamic cells; the mechanism of glucosensing in pancreatic beta cells is established, but that of hypothalamic neurons remains controversial. Beta cells glucosense by expressing a specialized type of hexokinase (the rate-limiting enzyme in glucose metabolism) called glucokinase (GCK). GCK, unlike standard hexokinase, is not saturated at physiological levels of glucose, allowing glucose metabolism in cells expressing GCK to increase when the extracellular glucose concentration is raised. This increase in metabolism is then converted into a membrane depolarization. Furthermore, S-nitrosylation can enhance the activity of GCK, and therefore enhance glucose metabolism and glucosensing. Here we use GT1-7 cells to explore whether a similar mechanism of glucosensing exists in hypothalamic neurons. The addition of a glucose concentration at which GCK is sensitive (and standard hexokinase is saturated) leads to an increase in cellular metabolism, as measured by NADH autofluorescence; but only in the presence of isoproterenol, a beta-adrenergic receptor agonist which increases intracellular calcium to activate neuronal nitric oxide synthase and facilitate S-nitrosylation. We also use a FRET-based GCK sensor to measure GCK activation under low and high glucose concentrations and in the presence of isoproterenol. These studies reveal the presence of a functional GCK and the sensitivity of GCK to post-translational/signaling cascade modulation. Further, the lack of metabolic response to glucose alone may be linked to high basal activity of GCK in this serum-grown cell line. These results point to a functional, receptor-potentiated glucose sensing mechanism in neurons that is mediated through post-translational activation of GCK.