Glucokinase Is a Critical Regulator of Ventromedial Hypothalamic Neuronal Glucosensing

Abstract

To test the hypothesis that glucokinase is a critical regulator of neuronal glucosensing, glucokinase activity was increased, using a glucokinase activator drug, or decreased, using RNA interference combined with calcium imaging in freshly dissociated ventromedial hypothalamic nucleus (VMN) neurons or primary ventromedial hypothalamus (VMH; VMN plus arcuate nucleus) cultures. To assess the validity of our approach, we first showed that glucoseinduced (0.5-2.5 mmol/l) changes in intracellular Ca U nlike most neurons in the brain that utilize glucose to fuel their metabolic needs (1), a select group of neurons use glucose as a signaling molecule to alter their firing rate as a means of glucosensing (2,3). Glucose-excited neurons increase, whereas glucose-inhibited neurons decrease, their firing rate as ambient glucose levels rise (2-7). During situations of low glucose availability, glucose-inhibited neurons are activated and glucose-excited neurons inactivated (4 -8).The ventromedial hypothalamus (VMH) area contains both the ventromedial hypothalamic nucleus (VMN) and arcuate nucleus. Both contain glucosensing neurons that respond to differing levels of glucose and are linked to pathways involved in the regulation of glucose homeostasis (3-11) and the counterregulatory responses to hypoglycemia (12-19). Our work (6,7) and that of others (8) strongly support a role for glucokinase (hexokinase IV) as a key regulator of neuronal glucosensing, which is similar to its purported role in pancreatic ␤-cell glucosensing (20,21). We previously demonstrated that inhibition of glucokinase activity reduced glucose-excited and increased glucose-inhibited neuronal activity at 2.5 mmol/l glucose, the concentration at which they are normally active and inactive, respectively (6,7).Recurrent hypoglycemia is common in patients with type 1 diabetes, especially in children (22)(23)(24)(25). This leads to hypoglycemia-associated autonomic failure, in which counterregulatory responses to subsequent bouts of hypoglycemia are severely blunted (26 -28). Our previous studies suggested that the development of hypoglycemiaassociated autonomic failure might be associated with changes in the ability of VMH neurons to sense and respond to glucose (6,28). Furthermore, its development is associated with upregulation of glucokinase mRNA in the VMH (6,28,29). This upregulation might be a compensatory response that makes glucosensing neurons more sensitive to glucose by shifting their concentration-response to the left. If so, this could underlie the development of hypoglycemia-associated autonomic failure because it would require lower levels of glucose to be reached before counterregulatory responses were initiated. This would predict that increasing glucokinase activity would produce a leftward shift in glucose sensitivity in VMH glucosensing neurons such as it does in pancreatic ␤-cells, using a drug that increases glucokinase activity (30). It would also predict that reducing glucokinase activity would inhibit the response to glucose. Here, we ...

Physiological glucose is critical for optimized neuronal viability and AMPK responsiveness in vitro

“…Our results are consistent with others in the literature, in which extracellular glucose levels are shown to be critical for eliciting relevant physiological responses from glucose sensing neurons in the hypothalamus (Wang, et al, 2004;Song, et al, 2005;Kang, et al, 2006;Canabal, et al, 2007b), as well as for determining the oxidative capacity of astroglia in vitro (Abe, et al, 2006). However, it is common practice in many studies to maintain cultures in high glucose and then transfer them into media containing lower glucose concentrations prior to experimentation (Ioudina, et al, 2004;Abe, et al, 2006;Bak, et al, 2006;Morgenthaler, et al, 2006).…”

“…For these studies, a previously established primary dissociated cortical neuronal culture (Dawson, et al, 1993;Landree, et al, 2004) was maintained in a glucose-free media supplemented with glutamine and B27 and either a physiological concentration of 3 mM glucose (NB-A 3 ), or a non-physiological high concentration of 25 mM glucose (NB-A 25 ). The present study was designed to further develop previous studies that considered the role of media glucose concentrations present during short-term culture (used by DIV4) and/or experimental medias (Wang, et al, 2004;Song, et al, 2005;Morgenthaler, et al, 2006;Abe, et al, 2006;Bak, et al, 2006;Kang, et al, 2006;Canabal, et al, 2007a;Canabal, et al, 2007b).…”

“…In these situations, brain glucose can rise to 4.5 mM or drop as low as 0.16 mM (Silver, et al, 1994). Select studies acknowledge these relationships, and emphasize the importance of in vitro culture conditions for CNS research (Wang, et al, 2004;Song, et al, 2005;Morgenthaler, et al, 2006;Abe, et al, 2006;Bak, et al, 2006;Kang, et al, 2006;Canabal, et al, 2007a;Canabal, et al, 2007b); however, most studies do not. It is not unusual for culture media to contain 25 mM glucose, a level perhaps seen in the plasma of obese ob/ob mice (Schwartz, et al, 1996), but never seen in the brain.…”

Neuroendocrine control of satiation

“…Thus, their dynamic range spans the normal range of extracellular glucose levels in the brain, which are about ∼10% of systemic blood glucose levels [135], and they are plausible normal physiological controls of eating. GE neurons require glucose metabolism for excitation [134,136,137]; however, GI neurons comprise distinct populations, which either require glucose metabolism [136,137] or sense the glucose molecule directly [138].…”

Type 2 Diabetes, Metabolic Syndrome, Lipid Metabolism

“…Conversely, glucose sensing is abolished in both neuron populations when AMPK activity is suppressed and the mechanism for this remains unknown but does not appear to be only a general protective one. Glucose levels in the hypothalamus are involved in the orexigenic process via GLUT2, K-ATP channels and glucokinase [2][3][4] with complex relations between electrophysiological activity and physiological response. For more on the subject, read the chapter on 'Neuroendocrinology' prepared by Nicolas de Roux.…”