AIMTo set up an IR-HIRc cell model for screening the inhibitor of GFAT (glutamine: fructose-6-phosphate amidotransferase) , the key enzyme in the hexosamine biosynthesis pathway (HBP).

METHODSFor GFAT activity assay, the GDH method was improved by adjusting the value of pH in the reaction system and the concentrations of the reactants. The sensitivity to insulin in the cells was estimated by the measurement of insulin-induced glucose-uptake. The IR-HIRc model was set up by the stimulation of long-action insulin for 36 h. The IR-HIRc model and GDH method was used for screening GFAT inhibitor.

RESULTSWith the administration of 25 nmol x L(-1) long-action insulin in HIRe cells for 36 hours, the GFAT activity increased by 47% and the insulin-induced glucose-uptake decreased by 21%. Azaserine, a GFAT inhibitor, inhibited GFAT activity significantly in a dose-dependent manner in IR-HIRc model.

CONCLUSIONWith the stimulation of 25 nmol x L(-1) long-action insulin for 36 h, excess hexosamine flux and insulin resistant in IR-HIRc cell model was set up, which can be used for screening

Animals ; Azaserine ; administration & dosage ; pharmacology ; Cell Line ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; metabolism ; Glucose ; metabolism ; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) ; antagonists & inhibitors ; metabolism ; Hexosamines ; biosynthesis ; Insulin ; pharmacology ; Insulin Resistance ; Models, Biological ; Rats ; Recombinant Proteins ; metabolism