The hexosamine biosynthetic pathway has been hypothesized to be involved in mediating some of the toxic effects of hyperglycemia. Glutamine:fructose-6-phosphate amidotransferase (GFA), the first and rate limiting enzyme of the hexosamine biosynthetic pathway, was overexpressed in skeletal muscle and adipose tissue of transgenic mice. A 2.4-fold increase of GFA activity in muscle of the transgenic mice led to weight-dependent hyperinsulinemia in random-fed mice. The hyperinsulinemic-euglycemic clamp technique confirmed that transgenic mice develop insulin resistance, with a glucose disposal rate of 68.5 Ϯ 3.5 compared with 129.4 Ϯ 9.4 mg/kg per min ( P Ͻ 0.001) for littermate controls. The decrease in the glucose disposal rate of the transgenic mice is accompanied by decreased protein but not mRNA levels of the insulin-stimulated glucose transporter (GLUT4). These data support the hypothesis that excessive flux through the hexosamine biosynthesis pathway mediates adverse regulatory and metabolic effects of hyperglycemia, specifically insulin resistance of glucose disposal. These mice can serve as a model system to study the mechanism for the regulation of glucose homeostasis by hexosamines.

Overexpression of the rate-limiting enzyme for hexosamine synthesis (glutamine:fructose-6-phosphate amidotransferase) in muscle and adipose tissue of transgenic mice was previously shown to result in insulin resistance and hyperleptinemia. Explanted muscle from transgenic mice was not insulin resistant in vitro, suggesting that muscle insulin resistance could be mediated by soluble factors from fat tissue. To dissect the relative contributions of muscle and fat to hexosamine-induced insulin resistance, we overexpressed glutamine:fructose-6-phosphate amidotransferase 2.5-fold, specifically in fat under control of the aP2 promoter. Fasting glucose, insulin, and triglycerides were unchanged in the transgenic mice; leptin and beta-hydroxybutyrate levels were 91% and 29% higher, respectively. Fasted transgenic mice have mild glucose intolerance and skeletal muscle insulin resistance in vivo. In fasting transgenic mice, glucose disposal rates with hyperinsulinemia were decreased 27% in females and 10% in males. Uptake of 2-deoxy-D-glucose into muscle was diminished by 45% in female and 21% in male transgenics. Serum adiponectin was also lower in the fasted transgenics, by 37% in females and 22% in males. TNF alpha and resistin mRNA levels in adipose tissue were not altered in the fasted transgenics; levels of mRNA for leptin were increased and peroxisome proliferator-activated receptor gamma decreased. To further explore the relationship between adiponectin and insulin sensitivity, we examined mice that have been refed for 6 h after a 24-h fast. Refeeding wild-type mice resulted in decreased serum adiponectin and increased leptin. In transgenic mice, however, the regulation of these hormones by refeeding was lost for adiponectin and diminished for leptin. Refed transgenic female and male mice no longer exhibited decreased serum adiponectin in the refed state, and they were no longer insulin resistant as by lower or unchanged insulin and glucose levels. We conclude that increased hexosamine levels in fat, mimicking excess nutrient delivery, are sufficient to cause insulin resistance in skeletal muscle. Changes in serum adiponectin correlate with the insulin resistance of the transgenic animals.

Hexosamines have been shown to mediate effects of hyperglycemia and so-called "glucose toxicity" in insulin-sensitive tissues. To determine the effects of hexosamines on insulin synthesis and secretion, transgenic mice were created to overexpress the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), specifically in ␤-cells. GFA activity in islets of heterozygous transgenic mice was elevated 76% compared with littermate controls. The increased GFA activity led to 1.4-and 2.1-fold increased pancreatic insulin content in 2-and 10-month-old transgenic mice, respectively (P < 0.005). Fasting insulin levels were 1.6-fold higher than in littermate controls (P < 0.05). Hyperinsulinemia was evident despite a 28% reduction in insulin mRNA levels. The fasting glucose levels in the transgenic mice equaled that of controls aged 2-4 months but exceeded that of the controls aged 6-10 months (means ± SE 6.9 ± 0.2 vs. 5.9 ± 0.2 mmol/l, P < 0.001). By 8 months, the males were overweight and mildly diabetic (fasting glucose 8.8 ± 0.5 mmol/l) despite persistent hyperinsulinemia. Insulin resistance was confirmed in both males and females using the euglycemic-hyperinsulinemic clamp technique; glucose disposal rates decreased by 48% in transgenic mice (P < 0.01). Triglyceride levels did not differ, and free fatty acid levels were lower in the transgenic animals. ATP levels were unchanged in the transgenic islets. We conclude that hexosamine biosynthesis is involved in the regulation of insulin content in ␤-cells by glucose. Increased hexosamine flux in the ␤-cell results in hyperinsulinemia, insulin resistance, and (in males) mild type 2 diabetes. Diabetes

The hexosamine biosynthesis pathway has been hypothesized to mediate some of the regulatory as well as the deleterious effects of glucose. We have stably overexpressed the cDNA for human glutamine:fructose-6-phosphate amidotransferase (GFA), the rate-limiting enzyme in the hexosamine biosynthesis pathway, in rat-1 fibroblasts. Two cell lines expressing the human RNA were selected by Northern analysis, and they exhibited 51-95% increases in GFA activity. Insulin-stimulated glycogen synthase (GS) activity and net glycogen synthesis were assayed, and GFA cells revealed decreased insulin sensitivity for both GS and net glycogen synthesis. The ED50 for insulin stimulation of GS was 2.45 +/- 0.4 nmol/l insulin in controls and 5.29 +/- 1.01 nmol/l in GFA cells (P < 0.005). For insulin-stimulated glycogen synthesis, the ED50 was 3.43 +/- 0.88 nmol/l in controls and 5.54 +/- 0.98 nmol/l in GFA cells (P < 0.005). There were no significant differences in maximally insulin-stimulated or total GS activities, insulin binding or receptor number, or glucose uptake between GFA and control cells. We also examined the effects of glucose on GS activity. GFA cells had a twofold increase in GS activity at low glucose (0.5 mmol/l) when compared with controls (P < 0.025). Both GFA and control cells had an approximately 75-80% decrease in GS activity as glucose concentration was increased from 0.5 to 20 mmol/l. This change in GS activity was not observed until after 12 h in culture. GFA cells were more sensitive to the effects of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)