azaserine and Insulin-Resistance

To 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).. For 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.. With 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.. With 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

Topics: Animals; Azaserine; Cell Line; Dose-Response Relationship, Drug; Fibroblasts; Glucose; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Hexosamines; Insulin; Insulin Resistance; Models, Biological; Rats; Recombinant Proteins

Chronic hyperglycemia causes insulin resistance, termed glucose toxicity. Herein we studied chronic glucose-dependent regulation of the glucose transport system in adipocytes. 3T3-L1 adipocytes were incubated for up to 24 h with low (1 mM) or high (25 mM) glucose, and glucose transport was subsequently analyzed. 100 nM insulin was present throughout the experiments. 24 h incubation with 1 mM glucose caused a 2.3+/-0.4 fold increase in glucose transport activity, compared to the values obtained with 25 mM glucose. This difference was not observed when 24 h incubation was carried out without insulin. Glucose transport activity was not increased at 3 or 6 h incubation with 1 mM glucose, but was increased at 12 h, which closely paralleled increased expression of GLUT1. In addition to increased GLUT1 expression, more efficient translocation of GLUT1 to the plasma membrane was observed when incubated with 1 mM glucose compared to 25 mM glucose. The addition of azaserin or deprivation of glutamine at 25 mM glucose did not increase the glucose transport activity to the level obtained with 1 mM glucose. PD98059 did not affect glucose transport activity when incubated with 1 mM or 25 mM glucose. In conclusion, the present study is the first to show that, in 3T3-L1 adipocytes, chronic exposure to low (1 mM) and high (25 mM) glucose leads to different insulin-stimulated glucose transport activities. These differences result from the difference in the expression and plasma membrane distribution of GLUT1, but not of GLUT4, and the hexosamine biosynthesis pathway or extracellular signal-regulated protein kinase is not involved.

Topics: 3T3 Cells; Animals; Azaserine; Biological Transport; Glucose; Glucose Transporter Type 1; Glucose Transporter Type 4; Hexosamines; Insulin; Insulin Resistance; Kinetics; Mice; Mitogen-Activated Protein Kinases; Monosaccharide Transport Proteins; Muscle Proteins

Based on our previous finding that desensitization of the insulin-responsive glucose transport system (GTS) requires three components, glucose, insulin, and glutamine, we postulated that the routing of incoming glucose through the hexosamine biosynthesis pathway plays a key role in the development of insulin resistance in primary cultured adipocytes. Two approaches were used to test this hypothesis. First, we assessed whether glucose-induced desensitization of the GTS could be prevented by glutamine analogs that irreversibly inactivate glutamine-requiring enzymes, such as glutamine:fructose-6-phosphate amidotransferase (GFAT) the first and the rate-limiting enzyme in hexosamine biosynthesis. Both O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxonorleucine inhibited desensitization in 18-h treated cells without affecting maximal insulin responsiveness in control cells. Moreover, close agreement was seen between the ability of azaserine to prevent desensitization of the GTS in intact adipocytes (70% inhibition, ED50 = 1.1 microM), its ability to inactivate GFAT in intact adipocytes (64% inhibition, ED50 = 1.0 microM) and its ability to inactivate GFAT activity in a cytosolic adipocyte preparation (ED50 = 1.3 microM). From these results we concluded that a glutamine amidotransferase is involved in the induction of insulin resistance. As a second approach, we determined whether glucosamine, an agent known to preferentially enter the hexosamine pathway at a point distal to enzymatic amidation by GFAT, could induce cellular insulin resistance. When adipocytes were exposed to various concentrations of glucosamine for 5 h, progressive desensitization of the GTS was observed (ED50 = 0.36 mM) that culminated in a 40-50% loss of insulin responsiveness. Moreover, we estimated that glucosamine is at least 40 times more potent than glucose in mediating desensitization, since glucosamine entered adipocytes at only one-quarter of the glucose uptake rate, yet induced desensitization at an extra-cellular dose 10 times lower than glucose. In addition, we found that glucosamine-induced desensitization did not require glutamine and was unaffected by azaserine treatment. Thus, we conclude that glucosamine enters the hexosamine-desensitization pathway at a point distal to GFAT amidation. Overall, these studies indicate that a unique metabolic pathway exists in adipocytes that mediates desensitization of the insulin-responsive GTS, and reveal that an early step in this pathwa

Topics: Animals; Azaserine; Biological Transport; Glucosamine; Glucose; Glutamine; Hexosamines; Insulin Resistance; Male; Rats; Rats, Inbred Strains