Glucokinase is a critical regulator of ventromedial hypothalamic neuronal glucosensing.
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 micromol/l) held at 0.5 mmol/l glucose. Together, these data support a critical role for glucokinase in neuronal glucosensing.
Kang
Ling
L
Department of Neurology and Neuroscience, New Jersey Medical School, Newark, USA.
Dunn-Meynell
Ambrose A
AA
Routh
Vanessa H
VH
Gaspers
Larry D
LD
Nagata
Yasufumi
Y
Nishimura
Teruyuki
T
Eiki
Junichi
J
Zhang
Bei B
BB
Levin
Barry E
BE
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000494:pharmacology; D008297:Male; D009474:Neurons / Q000201:enzymology* / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014044:Tolbutamide / Q000494:pharmacology; D014697:Ventromedial Hypothalamic Nucleus / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology*
D007004:Hypoglycemic Agents; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014044:Tolbutamide; D005941:Glucokinase; D005947:Glucose; D002118:Calcium