Glutamine:fructose-6-phosphate Amidotransferase Activity Research Articles

Excess Protein O-GlcNAcylation Links Metabolic Derangements to Right Ventricular Dysfunction in Pulmonary Arterial Hypertension.

The hexosamine biosynthetic pathway (HBP) converts glucose to uridine-diphosphate-N-acetylglucosamine, which, when added to serines or threonines, modulates protein function through protein O-GlcNAcylation. Glutamine-fructose-6-phosphate amidotransferase (GFAT) regulates HBP flux, and AMP-kinase phosphorylation of GFAT blunts GFAT activity and O-GlcNAcylation. While numerous studies demonstrate increased right ventricle (RV) glucose uptake in pulmonary arterial hypertension (PAH), the relationship between O-GlcNAcylation and RV function in PAH is unexplored. Therefore, we examined how colchicine-mediated AMP-kinase activation altered HBP intermediates, O-GlcNAcylation, mitochondrial function, and RV function in pulmonary artery-banded (PAB) and monocrotaline (MCT) rats. AMPK activation induced GFAT phosphorylation and reduced HBP intermediates and O-GlcNAcylation in MCT but not PAB rats. Reduced O-GlcNAcylation partially restored the RV metabolic signature and improved RV function in MCT rats. Proteomics revealed elevated expression of O-GlcNAcylated mitochondrial proteins in MCT RVs, which fractionation studies corroborated. Seahorse micropolarimetry analysis of H9c2 cardiomyocytes demonstrated colchicine improved mitochondrial function and reduced O-GlcNAcylation. Presence of diabetes in PAH, a condition of excess O-GlcNAcylation, reduced RV contractility when compared to nondiabetics. Furthermore, there was an inverse relationship between RV contractility and HgbA1C. Finally, RV biopsy specimens from PAH patients displayed increased O-GlcNAcylation. Thus, excess O-GlcNAcylation may contribute to metabolic derangements and RV dysfunction in PAH.

Inhibition of the hexosamine biosynthesis pathway potentiates cisplatin cytotoxicity by decreasing BiP expression in non-small-cell lung cancer cells.

Platinum anticancer agents are essential components in chemotherapeutic regimens for non-small-cell lung cancer (NSCLC) patients ineligible for targeted therapy. However, platinum-based regimens have reached a plateau of therapeutic efficacy; therefore, it is critical to implement novel approaches for improvement. The hexosamine biosynthesis pathway (HBP), which produces amino-sugar N-acetyl-glucosamine for protein glycosylation, is important for protein function and cell survival. Here we show a beneficial effect by the combination of cisplatin with HBP inhibition. Expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of HBP, was increased in NSCLC cell lines and tissues. Pharmacological inhibition of GFAT activity or knockdown of GFATimpaired cell proliferation and exerted synergistic or additive cytotoxicity to the cells treated with cisplatin. Mechanistically, GFAT positively regulated the expression of binding immunoglobulin protein (BiP; also known as glucose-regulated protein 78, GRP78), an endoplasmic reticulum chaperone involved in unfolded protein response (UPR). Suppressing GFAT activity resulted in downregulation of BiP that activated inositol-requiring enzyme 1α, a sensor protein of UPR, and exacerbated cisplatin-induced cell apoptosis. These data identify GFAT-mediated HBP as a target for improving platinum-based chemotherapy for NSCLC.

O-GlcNAc Protein Modification in Cancer Cells Increases in Response to Glucose Deprivation through Glycogen Degradation

When cellular glucose concentrations fall below normal levels, in general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases. However, recent reports demonstrated increased O-GlcNAcylation by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was unchanged, the enzyme contained less O-GlcNAc, and its activity was increased. Moreover, O-GlcNAcase activity was reduced. The studied cells contain glycogen, and we show that its degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and that this may be useful for cancer cells to survive.

Glutamine fructose-6-phosphate amidotransferase (GFAT) gene expression and activity in patients with type 2 diabetes: Inter-relationships with hyperglycaemia and oxidative stress

Objective: Cell culture and animal model studies have strongly suggested a role for the rate-limiting enzyme for hexosamine biosynthesis, glutamine:fructose-6-phosphate amidotransferase (GFAT) in insulin resistance. However, there are very few clinical studies and none on Asian Indians, a high-risk group for type 2 diabetes (T2DM), which examined the role of GFAT in insulin resistance and T2DM. Design and method: The study group comprised of T2DM subjects without any complications ( n = 25) and control non-diabetic subjects ( n = 23). GFAT mRNA expression and activity were measured by semi-quantitative RT-PCR and fluorimetry, respectively. Oxidative damage was assessed in plasma by the extent of lipid peroxidation [thiobarbituric acid reactive substances (TBARS)] and protein carbonyl content (PCO) using standard methods. Result: The mean (± SE) GFAT activity was significantly higher in diabetic (30.22 ± 2.40 pM/mg protein/min) compared to control subjects (20.10 ± 1.12 pM/mg protein/min) ( p < 0.001). Plasma levels of diabetic patients also exhibited increased lipid peroxidation and protein carbonylation. GFAT activity was positively correlated ( p < 0.005) with GFAT mRNA, HbA 1c, insulin resistance (HOMA-IR), postprandial plasma glucose and levels of TBARS and PCO. In multiple logistic regression analysis, the association between GFAT activity and T2DM persisted even after adjusting for age, gender, BMI and HOMA-IR (OR = 1.202, p = 0.026). Conclusion: Increased GFAT activity appears to be associated with insulin resistance, postprandial hyperglycaemia and oxidative stress in T2DM and may point towards a potential pathway amenable for therapeutic intervention.

Glutamine protects isolated rat heart from ischemia/reperfusion injury through the hexosamine biosynthesis pathway

A number of reports have demonstrated that glutamine protects the heart from ischemia/reperfusion (I/R) injury; however, the mechanisms underlying this protection have yet to be fully elucidated. Glutamine: fructose-6-phosphate amidotransferase (GFAT) regulates the entry of glucose into the hexosamine biosynthesis pathway (HBP) and recent studies have shown that activation of this pathway is cardioprotective. Since glutamine is essential for GFAT activity the goal of this study was to determine whether the cardioprotective effects of glutamine was due to increased flux through the HBP and elevated levels of O-linked N-acetyl-glucosamine (O-GlcNAc) on proteins. Hearts from male rats were isolated and perfused with Krebs-Henseleit buffer containing 5mM glucose and global, no-flow ischemia was induced for 20min followed by 60 min reperfusion.30 min pre-treatment with 5 mM glutamine significantly improved functional recovery (RPP = 15.6±5.7% Vs. 59.4±6.1%; p<0.05) and decreased cardiac Troponin I release (25.4±3.0 Vs. 4.7±1.9; p<0.05) during reperfusion. This protection was associated with a significant increase in the levels of UDP-GlcNAc, protein O-GlcNAc and ATP. Pre-treatment with 80μM azaserine, an inhibitor of GFAT, completely reversed the protection seen with glutamine and prevented the increase in UDP-GlcNAc and protein O-GlcNAc.O-GlcNAc transferase (OGT) catalyzes the formation of O-GlcNAc and inhibition of OGT with 5mM alloxan, also reversed the protection associated with glutamine. These data support the hypothesis that glutamine cardioprotection is due, at least in part, to enhanced flux through the HBP and increased protein O-GlcNAc levels. This study was supported by NIH grant HL076165.

Hexosamines, insulin resistance, and the complications of diabetes: current status

The hexosamine biosynthesis pathway (HBP) is a relatively minor branch of glycolysis. Fructose 6-phosphate is converted to glucosamine 6-phosphate, catalyzed by the first and rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). The major end product is UDP-N-acetylglucosamine (UDP-GlcNAc). Along with other amino sugars generated by HBP, it provides essential building blocks for glycosyl side chains, of proteins and lipids. UDP-GlcNAc regulates flux through HBP by regulating GFAT activity and is the obligatory substrate of O-GlcNAc transferase. The latter is a cytosolic and nuclear enzyme that catalyzes a reversible, posttranslational protein modification, transferring GlcNAc in O-linkage (O-GlcNAc) to specific serine/threonine residues of proteins. The metabolic effects of increased flux through HBP are thought to be mediated by increasing O-GlcNAcylation. Several investigators proposed that HBP functions as a cellular nutrient sensor and plays a role in the development of insulin resistance and the vascular complications of diabetes. Increased flux through HBP is required and sufficient for some of the metabolic effects of sustained, increased glucose flux, which promotes the complications of diabetes, e.g., diminished expression of sarcoplasmic reticulum Ca(2+)-ATPase in cardiomyocytes and induction of TGF-beta and plasminogen activator inhibitor-1 in vascular smooth muscle cells, mesangial cells, and aortic endothelial cells. The mechanism was consistent with enhanced O-GlcNAcylation of certain transcription factors. The role of HBP in the development of insulin resistance has been controversial. There are numerous papers showing a correlation between increased flux through HBP and insulin resistance; however, the causal relationship has not been established. More recent experiments in mice overexpressing GFAT in muscle and adipose tissue or exclusively in fat cells suggest that the latter develop in vivo insulin resistance via cross talk between fat cells and muscle. Although the relationship between HBP and insulin resistance may be quite complex, it clearly deserves further study in concert with its role in the complications of diabetes.

Phosphorylation of Mouse Glutamine-Fructose-6-phosphate Amidotransferase 2 (GFAT2) by cAMP-dependent Protein Kinase Increases the Enzyme Activity

A protein encoded by a new gene with approximately 75% homology to glutamine-fructose-6-phosphate amidotransferase (GFAT) was termed GFAT2 on the basis of this similarity. The mouse GFAT2 cDNA was cloned, and the protein was expressed with either an N-terminal glutathione S-transferase or His tag. The purified protein expressed in mammalian cells had GFAT activity. The Km values for the two substrates of reaction, fructose 6-phosphate and glutamine, were determined to be 0.8 mm for fructose 6-phosphate and 1.2 mm for glutamine, which are within the ranges determined for GFAT1. The protein sequence around the serine 202 of GFAT2 was conserved to the serine 205 of GFAT1, whereas the serine at 235 in GFAT1 was not present in GFAT2. Previously we showed that phosphorylation of serine 205 in GFAT1 by the catalytic subunit of cAMP-dependent protein kinase (PKA) inhibits its activity. Like GFAT1, GFAT2 was phosphorylated by PKA, but GFAT2 activity increased approximately 2.2-fold by this modification. When serine 202 of GFAT2 was mutated to an alanine, the enzyme not only became resistant to phosphorylation, but also the increase in activity in response to PKA also was blocked. These results indicated that the phosphorylation of serine 202 was necessary and sufficient for these alterations by PKA. GFAT2 was modestly inhibited (15%) by UDP-GlcNAc but not through detectable O-glycosylation. GFAT2 is, therefore, an isoenzyme of GFAT1, but its regulation by cAMP is the opposite, allowing differential regulation of the hexosamine pathway in specialized tissues.

A simple and sensitive method for glutamine:fructose-6-phosphate amidotransferase assay

For measuring glutamine:fructose-6-phosphate amidotransferase (GFAT) activity in cultured cells, an enzyme method—GDH method—was set up with high-efficiency, high-sensitivity and simple operation by determining the formed glutamate. During the process of making samples, reduced glutathione (GSH, 5 mM) and glucose-6-phophate Na 2 (5 mM) were added to the buffer for scraping the cells. The range of protein content in the samples was 80–150 μg. In the GFAT activity assay, the end product reduced acetylpyridine adenine dinucleotide (APADH) was determined at 370 nm directly. The suitable concentrations of the reactants fructose-6-phosphate (F-6-P), glutamine, acetylpyridine adenine dinucleotide (APAD) and glutamate dehydrogenase (GDH) were 0.8, 6 and 0.3 mM and 6 U, respectively. However, the excess of APAD may interfere with the APADH measurement. The reaction time course was 90 min. The GFAT activity in 3T3-L1, L6, HepG2 and HIRc cells were 1.84–8.51 nmol glutamate/mg protein·min.

Overexpression of GFAT activates PAI-1 promoter in mesangial cells.

Effects of hyperglycemia on glomerular cells may be mediated by glucose entry into the hexosamine pathway, and mesangial cell (MC) expression of the hexosamine pathway rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) is increased in diabetic glomerulosclerosis. We hypothesized that GFAT activity would be an important determinant of gene expression in glomerular MC. When overexpressed in primary MC, GFAT produced a two- to threefold increase in the activity of plasminogen activator inhibitor-1 (PAI-1) promoter. There was a 1.4-fold increase in PAI-1 promoter activity in cells exposed to high glucose (20 mM), whereas in MC overexpressing GFAT, exposure to high glucose caused a 3.5- to 4-fold increase in promoter activity. PAI-1 promoter activation was dependent on GFAT enzyme activity because o-diazoacetyly-L-serine and 6-diazo-5-oxonorleucine, inhibitors of GFAT enzyme activity, abrogated the activation of PAI-1 promoter in MC overexpressing GFAT. Glucosamine, which is downstream of GFAT in the hexosamine pathway, produced a 2.5-fold increase in the PAI-1 promoter activity. In addition to increasing the mRNA levels for transforming growth factor-beta1 (TGF-beta1), GFAT overexpression also increased mRNA levels for the TGF-beta type I and type II receptors. TGF-beta-neutralizing antibody did not normalize PAI-1 promoter activity in MC exposed to glucosamine or those overexpressing GFAT. We conclude that GFAT expression and activity are important determinants of gene expression in MC and that flux through the hexosamine pathway activates expression of genes implicated in vascular injury pathways.

Role of the hexosamine biosynthetic pathway in diabetic nephropathy

The hexosamine biosynthetic pathway has been hypothesized to be involved in the development of insulin resistance and diabetic vascular complications. In particular, it was demonstrated that hyperglycemia-induced production of transforming growth factor-beta (TGF-beta1), a prosclerotic cytokine causally involved in the development of diabetic nephropathy. Several lines of evidence indicate that TGF-beta1 induction is mediated by the hexosamine pathway. In cultured mesangial cells, high glucose levels induce TGF-beta1 production. This effect is eliminated by inhibition of glutamine: fructose-6-phosphate-amidotransferase (GFAT), the rate-limiting enzyme of this pathway. Furthermore, stable overexpression of GFAT increased levels of TGF-beta1 protein, mRNA, and promoter activity. Inasmuch as stimulation or inhibition of GFAT increased or decreased high glucose-stimulated activity of protein kinase C (PKC), respectively, the observed effects appear to be transduced by PKC. In similar experiments, involvement of the hexosamine pathway in hyperglycemia-induced production of cytokines (TGF-alpha and basic fibroblast growth factor [bFGF]) was demonstrated in vascular smooth muscle cells. These studies also revealed a rapid increase in GFAT activity by treatment with agents that elevated levels of cyclic adenosine 3',5' monophosphate (cAMP), thus indicating that GFAT activity is tightly regulated by cAMP-dependent phosphorylation. Using immunohistochemistry and in situ hybridization, high expression of GFAT was found in human adipocytes, skeletal muscle, vascular smooth muscle cells, and renal tubular epithelial cells. whereas glomerular cells remained essentially unstained. However, significant staining occurred in glomerular cells of patients with diabetic nephropathy. Current data indicate that the flux through the hexosamine pathway, regulated by GFAT, may be causally involved in the development of diabetic vascular disease, particularly diabetic nephropathy.

Transgenic mice with increased hexosamine flux specifically targeted to beta-cells exhibit hyperinsulinemia and peripheral insulin resistance.

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 beta-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 beta-cells by glucose. Increased hexosamine flux in the beta-cell results in hyperinsulinemia, insulin resistance, and (in males) mild type 2 diabetes.

Glutamine: Fructose-6-phosphate amidotransferase activity and gene expression are regulated in a tissue-specific fashion in pregnant rats

We examined whether regulation of glutamine: fructose-6-phosphate amidotransferase (GFA), the rate-limiting enzyme of the hexosamine pathway, is tissue specific and if so whether such regulation occurs at the level of gene expression. We compared GFA activity and expression and levels of UDP-hexosamines and UDP-hexoses between insulin-sensitive (liver and muscle) tissues and a glucose-sensitive (placenta) tissue from 19 day pregnant streptozotocin diabetic and non-diabetic rats. In pregnant non-diabetic rats GFA activities averaged (1521 ± 75 pmol/mg protein min) in the placenta, 895±74 in the liver and 81 ± 11 in muscle (p < 0.001 between each tissue). In the diabetic rats, GFA activities were ~50 % decreased both in the liver (340 ± 42 pmol/mg protein-min, p < 0.05 vs control rats) and in skeletal muscle (46 ± 3, p < 0.05) compared to control rats. In the placenta, GFA activities were identical between diabetic (1519 ± 112 pmol/mg protein-min) and non-diabetic (1521 ± 75) animals. In the liver, the reduction in GFA activity could be attributed to a significant decrease in GFA mRNA concentrations, while GFA mRNA concentrations were similar in the placenta between diabetic and non-diabetic animals. UDP-N-acetyl-glucosamine (UDP-GlcNAc), the end product of the hexosamine pathway, was significantly reduced in the liver and in skeletal muscle but similar in the placenta between diabetic and non-diabetic rats. In summary, GFA activity and expression and the concentration of UDP-GlcNAc are decreased in the liver but unaltered in the placenta, although GFA activity is almost 2-fold higher in this tissue than in the liver. These data provide the first evidence for tissue specific regulation of GFA and for its regulation at the level of gene expression.

Mechanism of hexosamine-induced insulin resistance in transgenic mice overexpressing glutamine:fructose-6-phosphate amidotransferase: decreased glucose transporter GLUT4 translocation and reversal by treatment with thiazolidinedione.

Hexosamines have been hypothesized to mediate aspects of glucose sensing and toxic effects of hyperglycemia. For example, insulin resistance results when the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), is overexpressed in muscle and adipose tissue of transgenic mice. The glucose infusion rates required to maintain euglycemia at insulin infusion rates of 0.5, 2, 15, and 20 mU/kg x min were 39-90% lower in such transgenic mice, compared with their control littermates (P < or = 0.01). No differences were observed in hepatic glucose output, serum insulin levels, or muscle ATP levels. Uptake of 2-deoxyglucose, measured under conditions of hyperinsulinemia, was significantly lower in transgenic hindlimb muscle, compared with controls (85.9 +/- 17.8 vs. 166.8 +/- 15.1 pmol deoxyglucose/g x min). The decrease in glucose uptake by transgenic muscle was associated with a disruption in the translocation of the insulin-stimulated glucose transporter GLUT4. Fractionation of muscle membranes on a discontinuous sucrose gradient revealed that insulin stimulation of control muscle led to a 28.8% increase in GLUT4 content in the 25% fraction and a 61.2% decrease in the 35% fraction. In transgenic muscle, the insulin-stimulated shifts in GLUT4 distribution were inhibited by over 70%. Treatment of the transgenic animals with the thiazolidinedione troglitazone completely reversed the defect in glucose disposal without changing GFA activity or the levels of uridine 5'-diphosphate-N-acetylglucosamine. Overexpression of GFA in skeletal muscle thus leads to defects in glucose transport similar to those seen in type 2 diabetes. These data support the hypothesis that excess glucose metabolism through the hexosamine pathway may be responsible for the diminished insulin sensitivity and defective glucose uptake that are seen with hyperglycemia.

Mechanism of Hexosamine-Induced Insulin Resistance in Transgenic Mice Overexpressing Glutamine:Fructose-6-Phosphate Amidotransferase: Decreased Glucose Transporter GLUT4 Translocation and Reversal by Treatment with Thiazolidinedione

Hexosamines have been hypothesized to mediate aspects of glucose sensing and toxic effects of hyperglycemia. For example, insulin resistance results when the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), is overexpressed in muscle and adipose tissue of transgenic mice. The glucose infusion rates required to maintain euglycemia at insulin infusion rates of 0.5, 2, 15, and 20 mU/kg·min were 39–90% lower in such transgenic mice, compared with their control littermates (P ≤ 0.01). No differences were observed in hepatic glucose output, serum insulin levels, or muscle ATP levels. Uptake of 2-deoxyglucose, measured under conditions of hyperinsulinemia, was significantly lower in transgenic hindlimb muscle, compared with controls (85.9 ± 17.8 vs. 166.8 ± 15.1 pmol deoxyglucose/g·min). The decrease in glucose uptake by transgenic muscle was associated with a disruption in the translocation of the insulin-stimulated glucose transporter GLUT4. Fractionation of muscle membranes on a discontinuous sucrose gradient revealed that insulin stimulation of control muscle led to a 28.8% increase in GLUT4 content in the 25% fraction and a 61.2% decrease in the 35% fraction. In transgenic muscle, the insulin-stimulated shifts in GLUT4 distribution were inhibited by over 70%. Treatment of the transgenic animals with the thiazolidinedione troglitazone completely reversed the defect in glucose disposal without changing GFA activity or the levels of uridine 5′-diphosphate-N-acetylglucosamine. Overexpression of GFA in skeletal muscle thus leads to defects in glucose transport similar to those seen in type 2 diabetes. These data support the hypothesis that excess glucose metabolism through the hexosamine pathway may be responsible for the diminished insulin sensitivity and defective glucose uptake that are seen with hyperglycemia.

Regulation of glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase.

Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme in hexosamine biosynthesis, an important pathway for cellular glucose sensing. Human GFA has two potential sites for phosphorylation by cAMP-dependent protein kinase A (PKA). To test whether GFA activity is regulated by cAMP-dependent phosphorylation, rat aortic smooth muscle cells were treated in vivo with cAMP-elevating agents, 10 micromol/l forskolin, 1 mmol/l 8-Br-cAMP, or 3-isobutyl-1-methylxanthine. All treatments resulted in rapid and significant increases (2- to 2.4-fold) in GFA activity assayed in cytosolic extracts. Maximal effects of forskolin were observed at 10 micromol/l and 60 min. Preincubation of cells with cycloheximide did not abolish the effect of forskolin. Incubation of cytosolic extracts at 37 degrees C for 10 min in a buffer without phosphatase inhibitors led to a 79% decrease of GFA activity. This loss of activity was inhibited by the addition of phosphatase inhibitors (5 mmol/l sodium orthovanadate, 50 mmol/l sodium fluoride, or 5 mmol/l EDTA, but not 100 nmol/l okadaic acid), suggesting that GFA undergoes rapid dephosphorylation by endogenous phosphatases. Purified GFA is phosphorylated in vitro by purified PKA, resulting in a 1.7-fold increase in GFA activity. Treatment of GFA with purified protein kinase C had no effect. We conclude that GFA activity may be modulated by cAMP-dependent phosphorylation.

Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance in multiple insulin sensitive tissues.

We determined the effect of infusion of glucosamine (GlcN), which bypasses the rate limiting reaction in the hexosamine pathway, on insulin-stimulated rates of glucose uptake and glycogen synthesis in vivo in rat tissues varying with respect to their glutamine:fructose-6-phosphate amidotransferase (GFA) activity. Three groups of conscious fasted rats received 6-h infusions of either saline (BAS), insulin (18 mU/kg x min) and saline (INS), or insulin and GlcN (30 micromol/ kg x min, GLCN). [3-(3)H]glucose was infused to trace whole body glucose kinetics and glycogen synthesis, and rates of tissue glucose uptake were determined using a bolus injection of [1-(14)C]2-deoxyglucose at 315 min. GlcN decreased insulin-stimulated glucose uptake (315-360 min) by 49% (P < 0.001) at the level of the whole body, and by 31-53% (P < 0.05 or less) in the heart, epididymal fat, submandibular gland and in soleus, abdominis and gastrocnemius muscles. GlcN completely abolished glycogen synthesis in the liver. GlcN decreased insulin-stimulated glucose uptake similarly in the submandibular gland (1.3 +/- 0.2 vs. 2.0 +/- 0.3 nmol/mg protein x min, GLCN vs. INS, P < 0.05) and gastrocnemius muscle (1.4 +/- 0.3 vs. 3.1 +/- 0.5 nmol/mg protein x min), although the activity of the hexosamine pathway, as judged from basal GFA activity, was 10-fold higher in the submandibular gland (286 +/- 35 pmol/mg protein x min) than in gastrocnemius muscle (27 +/- 3 pmol/mg protein x min, P < 0.001). These data raise the possibility that overactivity of the hexosamine pathway may contribute to glucose toxicity not only in skeletal muscle but also in other insulin sensitive tissues. They also imply that the magnitude of insulin resistance induced between tissues is determined by factors other than GFA.

Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance.

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 +/- 3.5 compared with 129.4 +/- 9.4 mg/kg per min (P < 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.

Glucose Metabolism to Glucosamine Is Necessary for Glucose Stimulation of Transforming Growth Factor-α Gene Transcription

Transforming growth factor-alpha (TGFalpha) gene transcription can be increased when arterial smooth muscle cells are exposed to supraphysiological concentrations of glucose, and this effect of glucose can be mimicked by glucosamine. To determine whether the metabolism of glucose to glucosamine is required for this glucose effect, the rate-limiting step in glucose metabolism to glucosamine through the enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) was blocked using pharmacological and antisense strategies. We found that blockage of GFAT activity or expression significantly blunted the glucose-induced increase of TGFalpha expression. Blockage of GFAT also resulted in a decreased RL2 signal on intracellular proteins as detected by Western blotting and indirect immunofluorescence. The RL2 monoclonal antibody recognizes an epitope on proteins that contain N-acetylglucosamine and thus is a measure of protein glycosylation. Conversely, treatment of the cells with glucose and glucosamine resulted in an increase in the RL2 epitope on intracellular proteins. These results indicate that the metabolism of glucose to glucosamine is necessary for the transcriptional stimulation of TGFalpha expression in vascular smooth muscle cells by glucose. Furthermore, the level of glycosylation of some intracellular proteins can be modulated in response to physiological changes in the extracellular glucose concentration and the net activity of GFAT.

Glutamine:fructose-6-phosphate amidotransferase activity in cultured human skeletal muscle cells: relationship to glucose disposal rate in control and non-insulin-dependent diabetes mellitus subjects and regulation by glucose and insulin.

We examined the activity of the rate-limiting enzyme for hexosamine biosynthesis, glutamine:fructose-6-phosphate amidotransferase (GFA) in human skeletal muscle cultures (HSMC), from 17 nondiabetic control and 13 subjects with non-insulin-dependent diabetes. GFA activity was assayed from HSMC treated with low (5 mM) or high (20 mM) glucose and low (22 pM) or high (30 microM) concentrations of insulin. In control subjects GFA activity decreased with increasing glucose disposal rate (r = -0.68, P < 0.025). In contrast, a positive correlation existed between GFA and glucose disposal in the diabetics (r = 0.86, P < 0.005). Increased GFA activity was also correlated with body mass index in controls but not diabetics. GFA activity was significantly stimulated by high glucose (22%), high insulin (43%), and their combination (61%). GFA activity and its regulation by glucose and insulin were not significantly different in diabetic HSMC. We conclude that glucose and insulin regulate GFA activity in skeletal muscle. More importantly, our results are consistent with a regulatory role for the hexosamine pathway in human glucose homeostasis. This relationship between hexosamine biosynthesis and the regulation of glucose metabolism is altered in non-insulin-dependent diabetes.

Increased glutamine:fructose-6-phosphate amidotransferase activity in skeletal muscle of patients with NIDDM.

Overactivity of the hexosamine pathway mediates glucose-induced insulin resistance in rat adipocytes. Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme of this pathway. We determined GFA activity in human skeletal muscle biopsies and rates of insulin-stimulated whole-body, oxidative, and nonoxidative glucose disposal using the euglycemic insulin clamp technique combined with indirect calorimetry (insulin infusion rate (1.5 mU x kg-1 x min-1)) in 12 male patients with NIDDM (age 54 +/- 2 years, BMI 27.5 +/- 0.9 kg/m2, fasting plasma glucose 8.5 +/- 0.6 mmol/l) and 9 matched normal men. GFA activity was detectable in human skeletal muscles and completely inhibited by uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) in all subjects. GFA activity was 46% increased in the NIDDM patients compared with the normal subjects (9.5 +/- 1.3 vs. 6.5 +/- 1.2 pmol, P < 0.05). Whole-body glucose uptake was 58% decreased in patients with NIDDM (20 +/- 3 micromol x kg body wt-1 x min-1) compared with normal subjects (47 +/- 4 micromol x kg body wt-1 x min-1, P < 0.001). This decrease was attributable to decreases in both glucose oxidation (9 +/- 1 vs. 15 +/- 1 micromol x kg-1 x min-1, NIDDM patients vs. control subjects, P < 0.002) and nonoxidative glucose disposal (11 +/- 2 vs. 31 +/- 4 micromol x kg-1 x min-1, P < 0.001). In patients with NIDDM, both HbA1c (r= 0.51, P < 0.05) and BMI (r= -0.57, P < 0.05) correlated with whole-body glucose uptake. HbA1c but not BMI or insulin sensitivity was correlated with basal GFA activity (r = -0.57,P < 0.01) in NIDDM patients and control subjects. We conclude that GFA is found in human skeletal muscle and that all this activity is sensitive to feedback inhibition by UDP-GlcNAc. Chronic hyperglycemia is associated with an increase in skeletal muscle GFA activity, suggesting that increased activity of the hexosamine pathway may contribute to glucose toxicity and insulin resistance in humans.