Glucokinase Is a Critical Regulator of Ventromedial Hypothalamic Neuronal Glucosensing

Glucokinase Is a Critical Regulator of Ventromedial Hypothalamic Neuronal Glucosensing Ling Kang 1 , Ambrose A. Dunn-Meynell 1 2 , Vanessa H. Routh 1 3 , Larry D. Gaspers 3 , Yasufumi Nagata 4 , Teruyuki Nishimura 4 , Junichi Eiki 4 , Bei B. Zhang 5 and Barry E. Levin 1 2 3 1 Department of Neurology and Neuroscience, New Jersey Medical School, Newark, New Jersey 2 Neurology Service, Veterans Affairs Medical Center, East Orange, New Jersey 3 Department of Physiology and Pharmacology, New Jersey Medical School, Newark, New Jersey 4 Tsukuba Research Institute, Banyu Pharmaceutical, Ibaraki, Japan 5 Merck Research Laboratories, Rahway, New Jersey Address correspondence and reprint requests to Barry E. Levin, MD, Neurology Service (127C), VA Medical Center, 385 Tremont Ave., East Orange, NJ 07018-1095. E-mail: levin{at}umdnj.edu 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 glucose-induced (0.5–2.5 mmol/l) changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) oscillations, using fura-2 and changes in membrane potential (using a membrane potential–sensitive dye), were highly correlated in both glucose-excited and -inhibited neurons. Also, glucose-excited neurons increased (half-maximal effective concentration [EC 50 ] = 0.54 mmol/l) and glucose-inhibited neurons decreased (half-maximal inhibitory concentration [IC 50 ] = 1.12 mmol/l) [Ca 2+ ] i oscillations to incremental changes in glucose from 0.3 to 5 mmol/l. In untreated primary VMH neuronal cultures, the expression of glucokinase mRNA and the number of demonstrable glucosensing neurons fell spontaneously by half over 12–96 h without loss of viable neurons. Transfection of neurons with small interfering glucokinase RNA did not affect survival but did reduce glucokinase mRNA by 90% in association with loss of all demonstrable glucose-excited neurons and a 99% reduction in glucose-inhibited neurons. A pharmacological glucokinase activator produced a dose-related increase in [Ca 2+ ] i oscillations in glucose-excited neurons (EC 50 = 0.98 mmol/l) and a decrease in glucose-inhibited neurons (IC 50 = 0.025 μmol/l