Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes: differential effects on glucosamine 6-phosphate, UDP-N-acetylglucosamine, and ATP levels

Glucose and glucosamine (GlcN) cause insulin resistance over several hours by increasing metabolite flux through the hexosamine biosynthesis pathway (HBP). To elucidate the early events underlying glucose-induced desensitization, we treated isolated adipocytes with either glucose or GlcN and then measured intracellular levels of glucose-6-P (G-6-P), GlcN-6-P, UDP-Glc-NAc, and ATP. Glucose treatment rapidly increased G-6-P levels (t((1/2)) < 1 min), which plateaued by 15 min and remained elevated for up to 4 h (glucose ED(50) = 4mm). In glucose-treated cells, GlcN-6-P was undetectable; however, GlcN treatment (2 mm) caused a rapid and massive accumulation of GlcN-6-P. Levels increased by 5 min ( approximately 400 nmol/g) and continued to rise over 2 h (t((1/2)) approximately 20 min) before reaching a plateau at >1,400 nmol/g (ED(50) = 900 microm). Thus, at high GlcN concentrations, unrestricted flux into the HBP greatly exceeds the biosynthetic capacity of the pathway leading to a rapid buildup of GlcN-6-P. The GlcN-induced rise in GlcN-6-P levels was correlated with ATP depletion, suggesting that ATP loss is caused by phosphate sequestration (with the formation of GlcN-6-P) or the energy demands of phosphorylation. As expected, GlcN and glucose increased UDP-GlcNAc levels (t((1/2)) approximately 14-18 min), but greater levels were obtained with GlcN (4-5-fold for GlcN, 2-fold for glucose). Importantly, we found that low doses of GlcN (<250 microm, ED(50) = 80 microm) could markedly elevate UDP-GlcNAc levels without increasing GlcN-6-P levels or depleting ATP levels. These studies on the dynamic actions of glucose and GlcN on hexosamine levels should be useful in exploring the functional role of the HBP and in avoiding the potential pitfalls in the pharmacological use of GlcN.     

Glucosamine-induced activation of glycogen biosynthesis in isolated adipocytes. Evidence for a rapid allosteric control mechanism within the hexosamine biosynthesis pathway

Enhanced flux through the hexosamine biosynthesis pathway (HBP) induces insulin resistance and facilitates lipid storage through the up-regulation of enzyme mRNA levels. Both actions occur over several hours and require gene expression. We now identify a regulatory arm of the HBP that involves rapid allosteric activation of glycogen synthase (GS) and stimulation of glycogen biosynthesis (GBS). When insulin-pretreated adipocytes were exposed to 2 mM GlcN, incorporation of [14C]glucose into glycogen doubled by 10 min (t(1/2) of <5 min), whereas UDP-glucose levels were concomitantly decreased during this time (t(1/2) of 1.4 min; >90% depletion). Stimulation of GBS and depletion of UDP-glucose both correlated with an early and rapid rise in the levels of glucosamine-6-phosphate (GlcN-6-P), a known activator of GS. The lowering of GlcN-6-P levels by removing extracellular GlcN (>80% reduction by 45 min) was accompanied by the restoration of UDP-glucose levels. Prolonged GlcN treatment (20 min to 2 h) inhibited GBS, which corresponded to a massive intracellular accumulation of GlcN-6-P (t(1/2) of approximately 32 min; >1,400 nmol/g). From these data, we conclude the following. 1) GlcN treatment elevated intracellular GlcN-6-P levels within minutes, resulting in allosteric activation of GS, stimulation of GBS, and a reduction in steady-state levels of UDP-glucose due to increased precursor utilization. 2) Prolonged treatment with high concentrations of GlcN caused massive accumulation of GlcN-6-P that adversely affected cellular metabolism and reduced GBS. 3) The biphasic actions of GlcN on GBS may explain many of the discrepant reports on the role of the HBP in glycogen metabolism.     

Expression of GFAT1 and OGT in podocytes: Transport of glucosamine and the implications for glucose uptake into these cells

Glutamine:fructose‐6‐phosphate amidotransferase (GFAT) and N‐acetylglucosaminyltransferase (OGT) participate in glucosamine (GlcN) production and its utilization in O‐glycosylation, one of key post‐translational modifications of nuclear and cytoplasmic proteins. For this purpose, cells require a high rate of intracellular production of GlcN and/or significant GlcN delivery. We studied the expression of GFAT1 and OGT and measured uptake of glucose and GlcN in cultured rat podocytes, the main cellular component of glomerular filtration barrier. RT‐PCR revealed the presence of both GFAT1 and OGT mRNA. Immunofluorescence of GFAT1 has shown staining signal diffused within the cytoplasm of the cell body and processes. However, OGT was distinctly visible around the nucleus and, in diffuse form, within the cytoplasm of cell bodies and processes. Glucose was transported (1.3 ± 0.2 nmol/min/mg protein) mainly by facilitative transporter systems whilst GlcN uptake (1.1 ± 0.2 nmol/min/mg protein) in a significant part, involved a sodium‐dependent transporter. There was interplay between glucose and GlcN uptake. In the presence of GlcN (50 µM), the rate of glucose uptake decreased by about 50%. The rate of GlcN uptake decreased by 28% in the presence of 5.6 mM glucose. Our results suggest that cultured podocytes possess limited ability to synthesize GlcN internally and therefore may need to receive GlcN from the extracellular environment. J. Cell. Physiol. 225: 577–584, 2010. © 2010 Wiley‐Liss, Inc.     

Isolation of a glucosamine-specific kinase, a unique enzyme of Vibrio cholerae

We showed previously that chitin catabolism by the marine bacterium Vibrio furnissii involves at least three signal transduction systems and many genes, several of which were molecularly cloned, and the corresponding proteins were characterized. The predicted amino acid sequences of these proteins showed a high degree of identity to the corresponding proteins from Vibrio cholerae, whose complete genomic sequence has recently been determined. We have therefore initiated studies with V. cholerae. We report here a novel ATP-dependent glucosamine kinase of V. cholerae encoded by a gene designated gspK. The protein, GspK (31.6 kDa), was purified to apparent homogeneity from recombinant Escherichia coli. The product of the reaction was shown to be GlcN-6-P by matrix-assisted laser desorption/ionization-time of flight (MALDI mass spectrometry) and NMR. The K(m) values for GlcN, ATP, and MgCl(2) were 0.45, 2.4, and 2.2 mm, respectively, and the V(max) values were in the range 180-200 nmol/microg/min (approximately 6 nmol/pmol/min). Kinase activity was not observed with any other sugar, including: galactosamine, mannosamine, Glc, GlcNAc, GalNAc, mannose, 2-deoxyglucose, and oligosaccharides of chitosan. The enzyme is also ATP-specific. The kinase can be used to specifically determine micro quantities of GlcN in acid hydrolysates of glycoconjugates. The physiological function of this enzyme remains to be determined.     

Coordinated regulation of glutamine:fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine. Role of hexosamine biosynthesis in enzyme regulation

We reported previously that glutamine:F-6-P amidotransferase (GFAT) plays an integral role in the development of insulin resistance by directing the flow of incoming glucose into the hexosamine biosynthesis pathway. To determine whether the enzymatic activity of GFAT is altered during desensitization of the glucose transport system, we treated isolated rat adipocytes with various combinations of insulin, glucose, and glutamine. Treatment with insulin or glucose alone (or in combination) failed to reduce cytosolic GFAT activity after 4 h, whereas combined treatment with all three components elicited a progressive loss of GFAT activity that was rapid (t1/2 of 2 h) and extensive (70% loss). A pronounced loss of GFAT activity was also seen in cells exposed to glucosamine, an agent known to directly enter the hexosamine pathway (55% loss at 4 h, ED50 of 360 microM). Moreover, a close correlation was observed between the induction of desensitization and the loss of GFAT activity as a function of glucose, insulin, glutamine, and glucosamine concentrations. When total intracellular hexosamine products were measured, we found that hexosamine formation was unaltered by insulin or glucose (or a combination) but was elevated by greater than 4-fold in the presence of insulin, glucose, and glutamine (t1/2 of 22 min), a condition known to cause both desensitization and loss of GFAT activity. Additional studies indicated that the loss of GFAT activity under desensitizing conditions is not due to allosteric regulation since removal of potential allosteric factors from the cytosol of desensitized cells by G-25 column chromatography failed to restore enzyme activity. Overall, these studies indicate that 1) GFAT is an insulin-regulated enzyme; however, control of enzyme activity is not due to a direct action of insulin, but rather is mediated by insulin-induced enhancement of glucose uptake; 2) the routing of incoming glucose through the hexosamine pathway and the formation of hexosamine products appears to regulate GFAT activity; and 3) the progressive loss of GFAT activity over several hours is probably not due to allosteric regulation.     

Structure of inhibited fructose-1,6-bisphosphatase from Escherichia coli: distinct allosteric inhibition sites for AMP and glucose 6-phosphate and the characterization of a gluconeogenic switch

Allosteric activation of fructose-1,6-bisphosphatase (FBPase) from Escherichia coli by phosphoenolpyruvate implies rapid feed-forward activation of gluconeogenesis in heterotrophic bacteria. But how do such bacteria rapidly down-regulate an activated FBPase in order to avoid futile cycling? Demonstrated here is the allosteric inhibition of E. coli FBPase by glucose 6-phosphate (Glc-6-P), the first metabolite produced upon glucose transport into the cell. FBPase undergoes a quaternary transition from the canonical R-state to a T-like state in response to Glc-6-P and AMP ligation. By displacing Phe(15), AMP binds to an allosteric site comparable with that of mammalian FBPase. Relative movements in helices H1 and H2 perturb allosteric activator sites for phosphoenolpyruvate. Glc-6-P binds to allosteric sites heretofore not observed in previous structures, perturbing subunits that in pairs form complete active sites of FBPase. Glc-6-P and AMP are synergistic inhibitors of E. coli FBPase, placing AMP/Glc-6-P inhibition in bacteria as a possible evolutionary predecessor to AMP/fructose 2,6-bisphosphate inhibition in mammalian FBPases. With no exceptions, signature residues of allosteric activation appear in bacterial sequences along with key residues of the Glc-6-P site. FBPases in such organisms may be components of metabolic switches that allow rapid changeover between gluconeogenesis and glycolysis in response to nutrient availability.     

Optimization of glucose feeding approaches for enhanced glucosamine and N-acetylglucosamine production by an engineered Escherichia coli

In this work, a recombinant Escherichia coli was constructed by overexpressing glucosamine (GlcN) synthase and GlcN-6-P N-acetyltransferase for highly efficient production of GlcN and N-acetylglucosamine (GlcNAc). For further enhancement of GlcN and GlcNAc production, the effects of different glucose feeding strategies including constant-rate feeding, interval feeding, and exponential feeding on GlcN and GlcNAc production were investigated. The results indicated that exponential feeding resulted in relatively high cell growth rate and low acetate formation rate, while constant feeding contributed to the highest specific GlcN and GlcNAc production rate. Based on this, a multistage glucose supply approach was proposed to enhance GlcN and GlcNAc production. In the first stage (0–2 h), batch culture with initial glucose concentration of 27 g/l was conducted, whereas the second culture stage (2–10 h) was performed with exponential feeding at μ _set = 0.20 h⁻¹, followed by feeding concentrated glucose (300 g/l) at constant rate of 32 ml/h in the third stage (10–16 h). With this time-variant glucose feeding strategy, the total GlcN and GlcNAc yield reached 69.66 g/l, which was enhanced by 1.59-fold in comparison with that of batch culture with the same total glucose concentration. The time-dependent glucose feeding approach developed here may be useful for production of other fine chemicals by recombinant E. coli.     

ENZYMIC AND CEREBRAL METABOLIC EFFECTS OF 2-DEOXY-d-GLUCOSE

—The time course of effects of 2-deoxy-d -glucose on cerebral glucose metabolism has been studied in vivo and the inhibitory actions of 2-deoxy-d -glucose and 2-deoxy-d -glucose-6-phosphate on cerebral glycolytic enzymes in vitro. Mice were given 2-deoxy-d -glucose 3 g/kg intraperitoneally. Blood 2-deoxy-d -glucose/glucose ratio was 2–3 from 5 to 30 min after injection, the hyperglycaemic response to 2-deoxy-d -glucose having been suppressed with propranolol. Maximal cerebral 2-deoxy-d -glucose uptake observed was 1μ11 μmol/g/min between 5 and 10 min after injection. At 10 min brain concentrations of 2-deoxy-d -glucose and 2-deoxy-d -glucose-6-phosphate were 5·82 and 3·12 μmol/g. Analysis of the fate of d -[U-¹⁴C] glucose given subcutaneously 5 min before death showed that glucose uptake was reduced to 40–60 per cent of control from 5 to 30 min after 2-deoxy-d -glucose. However brain glucose concentration rose three to five-fold 20–30 min after 2-deoxy-d -glucose. The majority of glucose entering the brain after 10 min of 2-deoxy-d -glucose treatment was recovered as glucose. Conversion of brain glucose to other acid soluble components was reduced to 1/3 at 10 min and 1/5 at 20–30 min. Glucose-6-phosphate concentration rose from 5 min onwards and was maintained at twice control concentration from 10–30 min. However, because of the rapid entry of 2-deoxy-d -glucose and its conversion to 2-deoxy-d -glucose-6-phosphate, the 2-deoxy-d -glucose 6-P/glucose 6-P ratio was between 19 and 32. Brain adenosine triphosphate concentration did not change, creatine phosphate concentration fell after 25 min. Measurement of enzyme activities in cerebral homogenates (using 1 mivs substrate concentration) showed that hexokinase (EC 2.7.1.1) was 40 per cent inhibited by 5 mm -deoxy-d -glucose (but not by 2-deoxy-d -glucose 6-P). Glucose 6-P dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.43) and phosphoglucomutase (EC 2.7.5.1) were not affected by either 2-deoxy-d -glucose (5 mm ) or 2-deoxy-d -glucose 6-P (5 or 20 mm ). Hexose-phosphate isomerase (EC 5.3.1.9) was 70 per cent inhibited by 20 mm -d -deoxy-d -glucose 6-P. Phosphofructokinase (EC 2.7.1.11) was inhibited by 17 per cent by 2-deoxy-d -glucose 6-P (20 mm ). During the initial impairment of cerebral function by 2-deoxy-d -glucose there is competitive inhibition of glucose transport into the brain; later, glycolysis is more powerfully depressed by the inhibition of isomerase produced by the high intracerebral concentration of 2-deoxyglucose-6-phosphate.     

Why does Escherichia coli grow more slowly on glucosamine than on N-acetylglucosamine? Effects of enzyme levels and allosteric activation of GlcN6P deaminase (NagB) on growth rates

Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.     

A Saccharomyces cerevisiae mutant unable to convert glucose to glucose-6-phosphate accumulates excessive glucose in the endoplasmic reticulum due to core oligosaccharide trimming

D-Glucose is the preferred carbon and energy source for most eukaryotic cells. Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P). The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P. In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose. However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal. Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu(3)Man(9)GlcNac(2) effectively reduced the accumulation of glucose. Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5Delta or alg6Delta, also diminished glucose accumulation. These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER). Consistent with this conclusion, both [(14)C]glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P. The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway. Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca(2+) uptake. This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation.     

Kinetics of insulin action on protein synthesis in isolated adipocytes. Ability of glucose to selectively desensitize the glucose transport system without altering insulin stimulation of protein synthesis

When adipocytes were exposed to [3H]leucine for times ranging from 5 to 180 s, leucine was found to enter cells rapidly and equilibrate with the cell interior within 5 s. After an additional 15-30 s [3H]leucine was incorporated into nascent protein, and the rate of incorporation was linear for up to 6 h in both control and insulin-treated cells. Since treatment of adipocytes with 10 ng/ml insulin enhanced the rate of leucine incorporation 2-3-fold with minimal or no effect on the rate of protein degradation or leucine uptake, we conclude that the predominant effect of insulin is on enhancement of protein synthesis. To assess the time required for insulin to stimulate protein synthesis, we preincubated cells with 10 ng/ml of insulin for various times from 2 to 30 min and then measured [3H]leucine incorporation into protein during a 4-min assay. These results revealed that the insulin stimulation of protein synthesis is rapid (t 1/2 of 4.4 min), but 9-fold slower than insulin activation of glucose transport (t 1/2 less than 0.5 min under identical conditions). In contrast to the rapidity of insulin activation, we found that deactivation proceeded at much slower rates (t 1/2 of 32 and 21 min for protein synthesis and glucose transport, respectively). Desensitization of the glucose transport system has previously been shown to occur after adipocytes are exposed to high glucose and insulin. To examine the specificity of desensitization, we treated cells for 6 h with 20 mM glucose and 25 ng/ml insulin and then examined insulin sensitivity and maximal insulin responsiveness of both the glucose transport and protein synthesis systems. After treatment, the glucose transport was markedly insulin-resistant (60% loss in maximal insulin responsiveness and a marked loss in insulin sensitivity), whereas the protein synthesis system exhibited neither diminished insulin responsiveness nor loss of insulin sensitivity. In fact, insulin sensitivity actually increased, as indicated by the finding that less insulin was required to stimulate protein synthesis (insulin ED50 values of 0.25 and 18 ng/ml at 0 and 6 h of treatment). From these studies we conclude that desensitization of the glucose transport system by glucose and insulin treatment appears to be specific for this particular effector system and does not reflect a state of generalized cellular insulin resistance.     

Glucosamine regulates hepatic lipid accumulation by sensing glucose levels or feeding states of normal and excess

Dose-dependent lipid accumulation was induced by glucose in HepG2 cells. GlcN also exerted a promotory effect on lipid accumulation in HepG2 cells under normal glucose conditions (NG, 5 mM) and liver of normal fed zebrafish larvae. High glucose (HG, 25 mM)-induced lipid accumulation was suppressed by l-glutamine-d-fructose 6-phosphate amidotransferase inhibitors. ER stress inhibitors did not suppress HG or GlcN-mediated lipid accumulation. HG and GlcN stimulated protein expression, DNA binding and O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP). Furthermore, both HG and GlcN increased nuclear sterol regulatory element-binding protein-1 (SREBP-1) levels in HepG2 cells. In contrast to its stimulatory effect under NG, GlcN suppressed lipid accumulation in HepG2 cells under HG conditions. Similarly, GlcN suppressed lipid accumulation in livers of overfed zebrafish. In addition, GlcN activity on DNA binding and O-GlcNAcylation of ChREBP was stimulatory under NG and inhibitory under HG conditions. Moreover, GlcN enhanced ChREBP, SREBP-1c, ACC, FAS, L-PK and SCD-1 mRNA expression under NG but inhibited HG-induced upregulation in HepG2 cells. The O-GlcNAc transferase inhibitor, alloxan, reduced lipid accumulation by HG or GlcN while the O-GlcNAcase inhibitor, PUGNAc, enhanced lipid accumulation in HepG2 cells and liver of zebrafish larvae. GlcN-induced lipid accumulation was inhibited by the AMPK activator, AICAR. Phosphorylation of AMPK (p-AMPK) was suppressed by GlcN under NG while increased by GlcN under HG. PUGNAc downregulated p-AMPK while alloxan restored GlcN- or HG-induced p-AMPK inhibition. Our results collectively suggest that GlcN regulates lipogenesis by sensing the glucose or energy states of normal and excess fuel through AMPK modulation.     

Design and synthesis of novel cell wall inhibitors of Mycobacterium tuberculosis GlmM and GlmU

GlmM and GlmU are key enzymes in the biosynthesis of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc), an essential precursor of peptidoglycan and the rhamnose–GlcNAc linker region in the mycobacterial cell wall. These enzymes are involved in the conversion of two important precursors of UDP-GlcNAc, glucosamine-6-phosphate (GlcN-6-P) and glucosamine-1-phosphate (GlcN-1-P). GlmM converts GlcN-6-P to GlcN-1-P, GlmU is a bifunctional enzyme, whereby GlmU converts GlcN-1-P to GlcNAc-1-P and then catalyzes the formation of UDP-GlcNAc from GlcNAc-1-P and uridine triphosphate. In the present study, methyl 2-amino-2-deoxyl-α-d-glucopyranoside 6-phosphate (1α), methyl 2-amino-2-deoxyl-β-d-glucopyranoside 6-phosphate (1β), two analogs of GlcN-6-P, were synthesized as GlmM inhibitors; 2-azido-2-deoxy-α-d-glucopyranosyl phosphate (2) and 2-amino-2,3-dideoxy-3-fluoro-α-d-glucopyranosyl phosphate (3), analogs of GlcN-1-P, were synthesized firstly as GlmU inhibitors. Compounds 1α, 1β, 2, and 3 as possible inhibitors of mycobacterial GlmM and GlmU are reported herein. Compound 3 showed promising inhibitory activities against GlmU, whereas 1α, 1β and 2 were inactive against GlmM and GlmU even at high concentrations.     

Kinetic characterization of human glutamine-fructose-6-phosphate amidotransferase I: potent feedback inhibition by glucosamine 6-phosphate

Glutamine-fructose-6-phosphate amidotransferase (GFAT) catalyzes the first committed step in the pathway for biosynthesis of hexosamines in mammals. A member of the N-terminal nucleophile class of amidotransferases, GFAT transfers the amino group from the L-glutamine amide to D-fructose 6-phosphate, producing glutamic acid and glucosamine 6-phosphate. The kinetic constants reported previously for mammalian GFAT implicate a relatively low affinity for the acceptor substrate, fructose 6-phosphate (Fru-6-P, K(m) 0.2-1 mm). Utilizing a new sensitive assay that measures the production of glucosamine 6-phosphate (GlcN-6-P), purified recombinant human GFAT1 (hGFAT1) exhibited a K(m) for Fru-6-P of 7 microm, and was highly sensitive to product inhibition by GlcN-6-P. In a second assay method that measures the stimulation of glutaminase activity, a K(d) of 2 microm was measured for Fru-6-P binding to hGFAT1. Further, we report that the product, GlcN-6-P, is a potent competitive inhibitor for the Fru-6-P site, with a K(i) measured of 6 microm. Unlike other members of the amidotransferase family, where glutamate production is loosely coupled to amide transfer, we have demonstrated that hGFAT1 production of glutamate and GlcN-6-P are strictly coupled in the absence of inhibitors. Similar to other amidotransferases, competitive inhibitors that bind at the synthase site may inhibit the synthase activity without inhibiting the glutaminase activity at the hydrolase domain. GlcN-6-P, for example, inhibited the transfer reaction while fully activating the glutaminase activity at the hydrolase domain. Inhibition of hGFAT1 by the end product of the pathway, UDP-GlcNAc, was competitive with a K(i) of 4 microm. These data suggest that hGFAT1 is fully active at physiological levels of Fru-6-P and may be regulated by its product GlcN-6-P in addition to the pathway end product, UDP-GlcNAc.     

Effects of high-fat diet and exercise training on intracellular glucose metabolism in rats

We examined the effects of high-fat diet (HFD) and exercise training on insulin-stimulated whole body glucose fluxes and several key steps of glucose metabolism in skeletal muscle. Rats were maintained for 3 wk on either low-fat (LFD) or high-fat diet with or without exercise training (swimming for 3 h per day). After the 3-wk diet/exercise treatments, animals underwent hyperinsulinemic euglycemic clamp experiments for measurements of insulin-stimulated whole body glucose fluxes. In addition, muscle samples were taken at the end of the clamps for measurements of glucose 6-phosphate (G-6-P) and GLUT-4 protein contents, hexokinase, and glycogen synthase (GS) activities. Insulin-stimulated glucose uptake was decreased by HFD and increased by exercise training (P < 0.01 for both). The opposite effects of HFD and exercise training on insulin-stimulated glucose uptake were associated with similar increases in muscle G-6-P levels (P < 0.05 for both). However, the increase in G-6-P level was accompanied by decreased GS activity without changes in GLUT-4 protein content and hexokinase activities in the HFD group. In contrast, the increase in G-6-P level in the exercise-trained group was accompanied by increased GLUT-4 protein content and hexokinase II (cytosolic) and GS activities. These results suggest that HFD and exercise training affect insulin sensitivity by acting predominantly on different steps of intracellular glucose metabolism. High-fat feeding appears to induce insulin resistance by affecting predominantly steps distal to G-6-P (e.g., glycolysis and glycogen synthesis). Exercise training affected multiple steps of glucose metabolism both proximal and distal to G-6-P. However, increased muscle G-6-P levels in the face of increased glucose metabolic fluxes suggest that the effect of exercise training is quantitatively more prominent on the steps proximal to G-6-P (i.e., glucose transport and phosphorylation).     

The crystal and solution studies of glucosamine-6-phosphate synthase from Candida albicans

Glucosamine 6-phosphate (GlcN-6-P) synthase is an ubiquitous enzyme that catalyses the first committed step in the reaction pathway that leads to formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor of macromolecules that contain amino sugars. Despite sequence similarities, the enzyme in eukaryotes is tetrameric, whereas in prokaryotes it is a dimer. The activity of eukaryotic GlcN-6-P synthase (known as Gfa1p) is regulated by feedback inhibition by UDP-GlcNAc, the end product of the reaction pathway, whereas in prokaryotes the GlcN-6-P synthase (known as GlmS) is not regulated at the post-translational level. In bacteria and fungi the enzyme is essential for cell wall synthesis. In human the enzyme is a mediator of insulin resistance. For these reasons, Gfa1p is a target in anti-fungal chemotherapy and in therapeutics for type-2 diabetes. The crystal structure of the Gfa1p isomerase domain from Candida albicans has been analysed in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy-d-mannitol 6-phosphate (ADMP). A solution structure of the native Gfa1p has been deduced using small-angle X-ray scattering (SAXS). The tetrameric Gfa1p can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains. UDP-GlcNAc binds, together with a metal cation, in a well-defined pocket on the surface of the isomerase domain. The residues responsible for tetramerisation and for binding UDP-GlcNAc are conserved only among eukaryotic sequences. Comparison with the previously studied GlmS from E. coli reveals differences as well as similarities in the isomerase active site. This study of Gfa1p focuses on the features that distinguish it from the prokaryotic homologue in terms of quaternary structure, control of the enzymatic activity and details of the isomerase active site.     

Vanadate restores glucose 6-phosphate in diabetic rats: a mechanism to enhance glucose metabolism

Vanadate mimics the metabolic actions of insulin. In diabetic rodents, vanadate also sensitizes peripheral tissues to insulin. We have analyzed whether this latter effect is brought about by a mechanism other than the known insulinomimetic actions of vanadium in vitro. We report that the levels of glucose 6-phosphate (G-6-P) in adipose, liver, and muscle of streptozotocin-treated (STZ)-hyperglycemic rats are 77, 50, and 58% of those in healthy control rats, respectively. Normoglycemia was induced by vanadium or insulin therapy or by phlorizin. Vanadate fully restored G-6-P in all three insulin-responsive peripheral tissues. Insulin did not restore G-6-P in muscle, and phlorizin was ineffective in adipose and muscle. Incubation of diabetic adipose explants with glucose and vanadate in vitro increased lipogenic capacity three- to fourfold (half-maximally effective dose = 11 +/- 1 microM vanadate). Lipogenic capacity was elevated when a threshold level of approximately 7.5 +/- 0.3 nmol G-6-P/g tissue was reached. In summary, 1) chronic hyperglycemia largely reduces intracellular G-6-P in all three insulin-responsive tissues; 2) vanadate therapy restores this deficiency, but insulin therapy does not restore G-6-P in muscle tissue; 3) induction of normoglycemia per se (i.e., by phlorizin) restores G-6-P in liver only; and 4) glucose and vanadate together elevate G-6-P in adipose explants in vitro and significantly restore lipogenic capacity above the threshold of G-6-P level. We propose that hyperglycemia-associated decrease in peripheral G-6-P is a major factor responsible for peripheral resistance to insulin. The mechanism by which vanadate increases peripheral tissue capacity to metabolize glucose and to respond to the hormone involves elevation of this hexose phosphate metabolite and the cellular consequences of this elevated level of G-6-P.     

ArPIKfyve-PIKfyve interaction and role in insulin-regulated GLUT4 translocation and glucose transport in 3T3-L1 adipocytes

Insulin activates glucose transport by promoting translocation of the insulin-sensitive fat/muscle-specific glucose transporter GLUT4 from an intracellular storage compartment to the cell surface. Here we report that an optimal insulin effect on glucose uptake in 3T3-L1 adipocytes is dependent upon expression of both PIKfyve, the sole enzyme for PtdIns 3,5-P(2) biosynthesis, and the PIKfyve activator, ArPIKfyve. Small-interfering RNAs that selectively ablated PIKfyve or ArPIKfyve in this cell type depleted the PtdIns 3,5-P(2) pool and reduced insulin-activated glucose uptake to a comparable degree. Combined loss of PIKfyve and ArPIKfyve caused further PtdIns 3,5-P(2) ablation that correlated with greater attenuation in insulin responsiveness. Loss of PIKfyve-ArPIKfyve reduced insulin-stimulated Akt phosphorylation and the cell surface accumulation of GLUT4 or IRAP, but not GLUT1-containing vesicles without affecting overall expression of these proteins. ArPIKfyve and PIKfyve were found to physically associate in 3T3-L1 adipocytes and this was insulin independent. In vitro labeling of membranes isolated from basal or insulin-stimulated 3T3-L1 adipocytes documented substantial insulin-dependent increases of PtdIns 3,5-P(2) production on intracellular membranes. Together, the data demonstrate for the first time a physical association between functionally related PIKfyve and ArPIKfyve in 3T3-L1 adipocytes and indicate that the novel ArPIKfyve-PIKfyve-PtdIns 3,5-P(2) pathway is physiologically linked to insulin-activated GLUT4 translocation and glucose transport.