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PMID:16443775

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Citation

Kang, L, Dunn-Meynell, AA, Routh, VH, Gaspers, LD, Nagata, Y, Nishimura, T, Eiki, J, Zhang, BB and Levin, BE (2006) Glucokinase is a critical regulator of ventromedial hypothalamic neuronal glucosensing. Diabetes 55:412-20

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 micromol/l) held at 0.5 mmol/l glucose. Together, these data support a critical role for glucokinase in neuronal glucosensing.

Links

PubMed

Keywords

Animals; Calcium/metabolism; Cells, Cultured; Gene Expression Regulation, Enzymologic; Glucokinase/genetics; Glucokinase/metabolism; Glucose/metabolism; Hypoglycemic Agents/pharmacology; Male; Neurons/enzymology; Neurons/physiology; RNA, Messenger/genetics; RNA, Messenger/metabolism; RNA, Small Interfering/genetics; RNA, Small Interfering/metabolism; Rats; Rats, Sprague-Dawley; Tolbutamide/pharmacology; Ventromedial Hypothalamic Nucleus/cytology; Ventromedial Hypothalamic Nucleus/drug effects; Ventromedial Hypothalamic Nucleus/enzymology; Ventromedial Hypothalamic Nucleus/physiology

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

RAT:HXK4

involved_in

GO:0001678: cellular glucose homeostasis

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

RAT:HXK4

involved_in

GO:0051594: detection of glucose

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

See also

References

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