Hyperglycemia induces PAI-1 gene expression in adipose tissue by activation of the hexosamine biosynthetic pathway.

We examined whether acute in vivo increases in either plasma glucose or insulin concentrations stimulate PAI-1 gene expression in fat tissue. We studied chronically catheterized unstressed and awake, lean (approximately 300 g, n=12) and obese (approximately 450 g, n=12) Sprague-Dawley rats. Hyperglycemia (approximately 18mM) was induced for 3 h by glucose infusion during a pancreatic clamp (somatostatin inhibited endogenous insulin secretion). Compared with equivalent saline infusion, hyperglycemia induced a 6-7 fold increase in PAI-1 gene expression in both lean and obese rats (P<0.001). When the rate of cellular glucose uptake was matched during a euglycemic hyperinsulinemic (approximately 60 microU/ml) clamp, PAI-1 gene expression in both obese and lean rats was proportionately and significantly increased (P<0.001). We further examined whether induction of the hexosamine biosynthetic pathway would mimic the effects of hyperglycemia and hyperinsulinemia on PAI-1 gene expression. Indeed, infusion of glucosamine (GlcN, 30 micromol/kg/min), induced a approximately 3-4 fold increase (P<0.01) in PAI-1 gene expression in both lean and obese animals. While obese rats had a four times greater fat mass then the lean rats, PAI-1 gene expression remained significantly higher when expressed as per gram fat. Our results support the hypothesis that increased glucose uptake induces PAI-1 gene expression in adipose tissue, probably through the activation of the hexosamine biosynthetic pathway. These findings may account for some of the fibrinolytic alterations seen in obese type 2 diabetic humans.     

Glucosamine regulation of glucose metabolism in cultured human skeletal muscle cells: divergent effects on glucose transport/phosphorylation and glycogen synthase in non-diabetic and type 2 diabetic subjects.

Chronic exposure (48 h) to glucosamine resulted in a dose-dependent reduction of basal and insulin-stimulated glucose uptake activities in human skeletal muscle cell cultures from nondiabetic and type 2 diabetic subjects. Insulin responsiveness of uptake was also reduced. There was no change in total membrane expression of either GLUT1, GLUT3, or GLUT4 proteins. While glucosamine treatment had no significant effects on hexokinase activity measured in cell extracts, glucose phosphorylation in intact cells was impaired after treatment. Under conditions where glucose transport and phosphorylation were down regulated, the fractional velocity (FV) of glycogen synthase was increased by glucosamine treatment. Neither the total activity nor protein expression of glycogen synthase were influenced by glucosamine treatment. The stimulation of glycogen synthase by glucosamine was not due totally to soluble mediators. Reflective of the effects on transport/phosphorylation, total glycogen content and net glycogen synthesis were reduced after glucosamine treatment. These effects were similar in nondiabetic and type 2 cells. In summary: 1) Chronic treatment with glucosamine reduces glucose transport/phosphorylation and storage into glycogen in skeletal muscle cells in culture and impairs insulin responsiveness as well. 2) Down-regulation of glucose transport/phosphorylation occurs at a posttranslational level of GLUTs. 3) Glycogen synthase activity increases with glucosamine treatment. 4) Nondiabetic and type 2 muscle cells display equal sensitivity and responsiveness to glucosamine. Increased exposure of skeletal muscle to glucosamine, a substrate/precursor of the hexosamine pathway, alters intracellular glucose metabolism at multiple sites and can contribute to insulin resistance in this tissue.     

Regulation of insulin-stimulated glucose transport by chronic glucose exposure in 3T3-L1 adipocytes.

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.     

Glucose-induced insulin resistance of phosphatidylinositol 3'-OH kinase and AKT/PKB is mediated by the hexosamine biosynthesis pathway

Hyperglycemia is responsible for many of the vascular complications and metabolic derangements seen in diabetes. One potential regulator of the effects of glucose is the hexosamine biosynthesis pathway (HBP). Glutamine: fructose-6-phosphate amidotransferase (GFA), the first and rate-limiting enzyme in this pathway, catalyzes the transfer of an amino group from glutamine to fructose-6-phosphate to form glucosamine-6-phosphate. Overexpression of GFA in rat-1 fibroblasts results in insulin resistance for glycogen synthase (GS) activity, and renders these cells more sensitive to the effects of glucose. Using rat-1 cells, we examine further the mechanisms whereby hexosamines lead to insulin resistance. Insulin stimulated GS activity was found to occur via a PI-3 kinase (PI-3K)-dependent pathway as wortmannin, an inhibitor of PI-3K, blocked insulin's ability to stimulate GS activity. Subsequently, we examined the effects of hexosamines on PI-3K and Akt/PKB activity. Cells were cultured in 1 mM glucose (low glucose, LG), 20 mM glucose (high glucose, HG), or 1 mM glucose plus 3 mM glucosamine (GlcN) for 16--20 h. After treatment with insulin (100 nM) for 5 min, cell extracts were assayed for IRS-1 associated and total PI-3K activity. At LG, insulin increased PI-3K activity by 43%. There was no insulin stimulation of PI-3K activity in cells cultured in HG or GlcN. There was a trend for IRS-1 protein levels to decrease in HG but not GlcN. PI-3K protein levels were not altered by HG or GlcN. Finally PKB activity was assayed. At LG, insulin stimulated PKB activity. Again, both HG and GlcN significantly reduced insulin's ability to stimulate PKB activity. We conclude that the hexosamine-mediated insulin resistance of GS activity seen in rat-1 cells is mediated by hexosamine regulation of PI-3K and PKB.     

Hexosamines are unlikely to function as a nutrient-sensor in 3T3-L1 adipocytes: a comparison of UDP-hexosamine levels after increased glucose flux and glucosamine treatment

Whether the hexosamine biosynthesis pathway acts as a nutrient-sensing pathway is still unclear. Glucose is directed into this pathway by GFAT. Because the activity of GFAT is tightly regulated, we examined whether UDP-hexosamine levels can increase significantly and dose-dependently in response to elevated glucose concentrations. In glucosamine-treated 3T3-L1 adipocytes, inhibition of insulin-stimulated glucose uptake was highly correlated with UDP-hexosamine levels (r=−0.992; p<0.0001 for UDP-GlcNAc and r=−0.996; p<0.0001 for UDP-GalNAc). Incubation of 3T3-L1 adipocytes with 0.1 μM insulin for 24 h in medium containing 1 and 5 mM glucose increased the rate of glucose uptake by 365% and 175% compared to untreated cells, respectively. This increase was not observed when the cells were incubated for 24 h with insulin in medium containing 10 or 25 mM glucose. However, treatment of cells with insulin and 1, 5, 10, or 25 mM glucose resulted in similar increases in levels of UDP-GlcNAc and UDP-GalNAc that always amounted to approx 30–40% above baseline values. This led us to conclude that despite exposure of adipocytes to conditions of extreme and prolonged glucose disposal, the increases in cellular UDP-hexosamines were minimal and not dependent on the extracellular glucose concentration. Taken together, our results are in line with the hypothesis that in glucosamine-treated adipocytes UDP-hexosamines influence insulin-stimulated glucose uptake. However, our observations in glucose-treated adipocytes argue against the possibility that UDP-hexosamines function as a nutrient-sensor, and question the role of the hexosamine biosynthesis pathway in the pathogenesis of insulin resistance.     

Activation of PI 3-kinase by the hexosamine biosynthesis pathway

It has been shown that hyperglycaemia-induced defects in glucose transport and insulin action are mediated by increased flux of excess glucose through the hexosamine biosynthesis pathway (HBP). We have previously demonstrated that in rat adipocytes, increased flux through the HBP activates protein kinase C (PKC). The aim of the present study was to explore the mechanism for HBP-mediated activation of PKC. We show that activation of the HBP by either high glucose or glucosamine causes the translocation of PKC-zeta/lambda and PKC-epsilon but not other PKC isoforms tested (alpha, beta, delta). This translocation was inhibited by wortmannin, a PI 3-kinase inhibitor. Both high glucose and glucosamine caused widespread cellular activation of PI 3-kinase. We demonstrate that HBP-mediated activation of PI 3-kinase has an insulin-like effect to translocate GLUT4. We conclude that an acute increase of glucose flux through the HBP activates PI 3-kinase.