The production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) are known to be modulated by a variety of factors. Recent study showed that endotoxin-induced NO synthesis and iNOS expression were greatly enhanced by elevation of extracellular glucose concentration in murine macrophages. Although this was suggested to be due to the activation of protein kinase C (PKC) via sorbitol pathway, there was lack of evidence for this speculation. This study was performed to delineate the underlying intracellular mechanisms of glucose-enhancing effect on endotoxin-induced NO production in Raw264.7 macrophages. The levels of NO release induced by lipopolysaccharide (LPS) significantly increased by the treatment of glucose in a concentration dependent manner and also, this effect was observed in LPS-preprimed cells. Concurrent incubation of cells with PKC inhibitors, H-7 or chelerythrine, and LPS resulted in the diminution of NO production regardless of glucose conc entration but this was not in the case of LPS-prepriming, that is, chelerythrine showed a minimal effect on the glucose- enhancing effect PMA, a PKC activator, did not show any significant effect on glucose-associated NO production. Modulation of sorbitol pathway with zopolrestat, an aldose reductase inhibitor, did not affect LPS-induced NO production and iNOS expression under high glucose condition. And also, sodium pyruvate, which is expected to normalize cytosolic NADH/NAD+ ratio, did not show any significant effect at concentrations of up to 10 mM. Glucosamine marginally increased the endotoxin-induced nitrite release in both control and high glucose treated group 6-diazo-5-oxonorleucine (L-DON) and azaserine, glutamine: fructose-6-phosphate amidotransferase (GFAT) inhibitors, significantly diminished the augmentation effect of high glucose on endotoxin-induced NO production. On the other hand, negative modulation of GFAT inhibitors was not reversed by the treatment of glucosamine , suggesting the minimal involvement, if any, of glucosamine pathway in glucose-enhancing effect. In summary, these results strongly suggest that the hexosamine biosynthesis pathway and the activation of PKC via sorbitol pathway do not contribute to the augmenting effect of high glucose on endotoxin induced NO production in macrophage-like Raw264.7 cells.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; Aldehyde Reductase ; Azaserine ; Cytosol ; Glucosamine ; Glucose* ; Glutamine ; Hand ; Macrophages* ; Nitric Oxide Synthase Type II ; Nitric Oxide* ; Protein Kinase C ; Pyruvic Acid ; Sodium ; Sorbitol

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