Glucose transport by osmotic shock and vanadate is impaired by glucosamine

In 3T3-L1 adipocytes, we previously reported that glucosamine impairs insulin stimulation of glucose transport, which is accompanied by impaired insulin stimulation of serine/threonine kinase Akt. To examine the role of Akt in glucosamine-induced insulin resistance, we investigated time course for insulin stimulation of Akt activity and glucose transport during recovery from glucosamine-induced insulin resistance. After induction of insulin resistance by glucosamine, we washed cells to remove glucosamine and incubated them for various times. After one hour, insulin stimulated-glucose transport was significantly increased and continued to increase up to 6-24 h. Insulin stimulation of Akt, however, did not increase after 1-3 h and began to slightly increase after 6 h. Next, we investigated effects of osmotic shock and vanadate on glucose transport in glucosamine-treated cells and found that glucosamine completely inhibited their actions in these cells. These data suggest that an Akt-independent mechanism is operative in glucosamine-induced insulin resistance and glucosamine impairs glucose transport stimulated by various stimuli involving and not involving Akt activation.     

The inhibition of semicarbazide-sensitive amine oxidase by aminohexoses

Semicarbazide-sensitive amine oxidase (EC 1.4.3.6; amine:oxygen oxidoreductase (deaminating) (copper-containing); SSAO) is a multifunctional protein. It acts under inflammatory conditions as a vascular-adhesion protein (VAP-1), mediating the adhesion of lymphocytes to vascular endothelial cells. The relationships, if any, between this adhesion function and the enzymatic functions (amine-substrate specificity and catalysis) of SSAO have not yet been defined. Since cell surface amino sugars and their derivatives are known to be involved in cell-to-cell recognition, we have investigated their possible effects on the enzyme activity of SSAO. The aminohexoses galactosamine, glucosamine and mannosamine were not oxidatively deaminated by SSAO. However, their presence during the assay of benzylamine oxidation resulted in a time-dependent inhibition. This inhibition was shown to follow saturation kinetics with respect to hexosamine concentration. Although time-dependent, the inhibition of SSAO activity was found to be reversible by dilution. In contrast, there is no such inhibition when the N-acetylamino sugar derivatives or the parent sugars (galactose, glucose and mannose) replaced the amino sugars in the reaction mixture. These results suggest that the interactions between SSAO and aminohexoses are specific and, therefore, that the cell-adhesion functions and amine-recognition functions of VAP-1/SSAO may be interlinked.     

O-GlcNAc modification of FoxO1 increases its transcriptional activity: A role in the glucotoxicity phenomenon?

O-GlcNAc glycosylations on serines or threonines are reversible post-translational modifications that control the localisation, the activity or the stability of cytosolic and nuclear proteins. These dynamic modifications are tightly dependent on the availability of glucose and on its flux through the hexosamine biosynthetic pathway. We recently showed that treatments that increase protein O-GlcNAc glycosylation (high-glucose concentrations, glucosamine) or inhibit their deglycosylation (PUGNAc), induced O-GlcNAc modification of FoxO1 in HEK293 cells. O-GlcNAc glycosylation of FoxO1 resulted in an increased of its activity towards a glucose 6-phosphatase promoter-luciferase reporter gene (G6Pase-luc). This effect appeared to be independent of FoxO1 sub-cellular re-localisation, since it was also observed with the constitutively nuclear FoxO1-AAA mutant. In liver-derived HepG2 cells, glucosamine and PUGNAc increased the expression of G6Pase mRNA, and synergistic effects were observed when both agents were present together. In addition, the expression of PGC1 alpha gene, which is known to be under the control of FoxO1, was also increased by glucosamine and PUGNAc. In HepG2 cells stably expressing the G6Pase-luc reporter gene, glucosamine and PUGNAc also increased the activity of the G6Pase promoter. The stimulation of the G6Pase reporter gene by these agents was abolished by two different FoxO1 siRNAs, thereby demonstrating the involvement of endogenous FoxO1 in the observed effects. Since G6Pase plays a key role in glucose production by the liver, increased in its expression through FoxO1 O-GlcNAc modification may be of considerable importance in the context of glucotoxicity associated with chronic hyperglycaemia. Moreover, since FoxO1 also plays important roles in several aspects of cell biology, including cell proliferation, survival and apoptosis, the regulation of FoxO1 activity by O-GlcNAc modification may have implications for other crucial biological processes.     

Administration of D-glucosamine into the third cerebroventricle induced feeding accompanied by hyperglycemia in rats

D-glucosamine, 2-amino-2-deoxy-D-glucose, is known to be an endogenous glucose analogue and to antagonize glucose uptake and metabolism. The present experiments were aimed to clarify effects of glucosamine and related chemical substances on ingestive behavior, as well as its direct effects on hypothalamic neurons. Infusion of 24 mumole glucosamine into the third cerebroventricle induced feeding within 30 min in 5 rats out of 7 tested, accompanied by increased ambulatory activity. No periprandial drinking was observed. Plasma glucose level increased, peaking at 30 min after the injection. Plasma insulin level tended to increase, but not significantly. Electrophoretic application of glucosamine activated glucose-sensitive neurons in the lateral hypothalamus and suppressed glucoreceptors in the ventromedial hypothalamus. These facts, together with other reported results, suggest that glucosamine can modulate physiological feeding and that carbon 2 of the glucose molecule is important in feeding modulation by glucose analogues.     

Control of amino sugar metabolism in Escherichia coli and isolation of mutants unable to degrade amino sugars

1. Growth of Escherichia coli on glucosamine results in an induction of glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] and a repression of glucosamine 6-phosphate synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16); glucose abolishes these control effects. 2. Growth of E. coli on N-acetylglucosamine results in an induction of N-acetylglucosamine 6-phosphate deacetylase and glucosamine 6-phosphate deaminase, and in a repression of glucosamine 6-phosphate synthetase; glucose diminishes these control effects. 3. The synthesis of amino sugar kinases (EC 2.7.1.8 and 2.7.1.9) is unaffected by growth on amino sugars. 4. Glucosamine 6-phosphate synthetase is inhibited by glucosamine 6-phosphate. 5. Mutants of E. coli that are unable to grow on N-acetylglucosamine have been isolated, and lack either N-acetylglucosamine 6-phosphate deacetylase (deacetylaseless) or glucosamine 6-phosphate deaminase (deaminaseless). Deacetylaseless mutants can grow on glucosamine but deaminaseless mutants cannot. 6. After growth on glucose, deacetylaseless mutants have a repressed glucosamine 6-phosphate synthetase and a super-induced glucosamine 6-phosphate deaminase; this may be related to an intracellular accumulation of acetylamino sugar that also occurs under these conditions. In one mutant the acetylamino sugar was shown to be partly as N-acetylglucosamine 6-phosphate. Deaminaseless mutants have no abnormal control effects after growth on glucose. 7. Addition of N-acetylglucosamine or glucosamine to cultures of a deaminaseless mutant caused inhibition of growth. Addition of N-acetylglucosamine to cultures of a deacetylaseless mutant caused lysis, and secondary mutants were isolated that did not lyse; most of these secondary mutants had lost glucosamine 6-phosphate deaminase and an uptake mechanism for N-acetylglucosamine. 8. Similar amounts of (14)C were incorporated from [1-(14)C]-glucosamine by cells of mutants and wild-type growing on broth. Cells of wild-type and a deaminaseless mutant incorporated (14)C from N-acetyl[1-(14)C]glucosamine more efficiently than from N[1-(14)C]-acetylglucosamine, incorporation from the latter being further decreased by acetate; cells of a deacetylaseless mutant showed a poor incorporation of both types of labelled N-acetylglucosamine.     

Immunoregulatory properties of fractions from human pregnancy urine: evidence that human chorionic gonadotropin is not responsible.

The immunologically privileged position of the histoin-compatible fetus and placenta is a striking example of a physiologic immunoregulatory mechanism. This study was designed to examine the effects of human chorionic gonadotropin (HCG) on the recognitive proliferative phase and the cytotoxic effector phase of in vitro cell-mediated immune responsiveness, since HCG has previously been reported to be immunosuppressive in vitro and in vivo. Commercial preparations of HCG were found to be potent inhibitors of lymphocyte proliferative responses to nonspecific mitogens like phytohemagglutinin (PHA), specific antigens such as streptolysin-O (SLO), and allogeneic cells as measured in the one-way mixed leukocyte response. Cytotoxic effector function of lymphocytes as measured by antibody-dependent cellular cytotoxicity (ADCC) and mitogen-induced cellular cytotoxicity were also markedly inhibited by these preparations. However, the 50% inhibitory concentration varied widely from lot to lot of these commercial materials. After dialysis, a portion of the inhibitory activity was lost from some but not all HCG lots. The dialysate from those lots with diminished activity was found to be immunosuppressive in vitro but contained no HCG detectable by radioimmunoassay. Following dialysis, the immunosuppressive activity of the various HCG lots remained variable and correlated poorly with values for HCG obtained by a double antibody radioimmunoassay. HCG preparations purified to a homogeneity sufficient for amino acid sequence were found to be only minimally immunosuppressive to the in vitro PHA response and had almost no effect on proliferative responses to antigens and allogeneic cells. These data do not support the concept of a primary immunoregulatory role for HCG, but they suggest that other uncharacterized compounds partially co-purified from pregnant urine along with HCG may have such immunoregulatory activity. Further characterization and identification of this immunoregulatory material(s) is essential, since it appears to have many of the properties of an ideal immunosuppressive compound: a) nontoxicity, b) ready reversibility, c) activity at very low concentration, and d) activity on a broad range of cellular immune functions.     

Immunosuppressive effect on T cell activation by interleukin- 16-cDNA-transfected human squamous cell line

It is well known that it is difficult to induce an immunotolerance with allogeneic skin transplantation. We attempted to find the immunosuppressive protocol for prolonging skin allograft rejection by using interleukin-16 because IL-16 is considered one of the natural ligands to CD4 molecules. First we examined whether synergistic immunosuppressive effects of recombinant IL-16 plus anti-CD4 mAbs are induced in mixed lymphocyte reaction (MLR). Next we used IL-16-cDNA-transfected OSC-20 (human oral squamous cell carcinoma cell line) as an in vitro model of the epidermal keratinocyte equivalent and examined whether this transfectant could inhibit the activation of allogeneic T cells. Our data indicated that IL-16 clearly inhibited human MLR and that IL-16 increased synergistically the immunosuppressive effect of anti-CD4 mAb. We also used IL-16 transfectant and this produced more than 50 ng/ml of IL-16 in the supernatant by which human MLR was significantly inhibited. Furthermore, this transfectant also inhibited the activation of allogeneic lymphocytes stimulated directly with transfectant cells. These results indicated that the IL-16-producing allogeneic skin graft might have a local immunosuppressive action that would prolong graft survival.     

Development and comparison of two 3T3-L1 adipocyte models of insulin resistance: increased glucose flux vs glucosamine treatment

Insulin resistance can be induced in vivo by intravenous infusion of glucosamine or in cells by incubation with glucosamine. However, a publication (Hresko, R. C., et al. (1998) J. Biol. Chem. 273, 20658-20668) suggests a trivial explanation of glucosamine-induced insulin resistance whereby intracellular ATP pools are depleted presumably due to the phosphorylation of glucosamine to glucosamine 6-phosphate, a hexosamine pathway intermediate. The reduced ATP level impaired insulin receptor (IR) autophosphorylation and tyrosine kinase activity toward substrates. The present work describes the development and comparison of two methods for inducing insulin resistance, by treating 3T3-L1 adipocytes overnight using either 25 mM glucose/5 nM insulin or 2 mM glucosamine. Under these conditions basal glucose transport rates were comparable with controls. Insulin-stimulated 2-deoxyglucose uptake, however, was reduced by approximately 45% in response to both high glucose/insulin and glucosamine treatment, relative to control cells. The total relative amounts of the insulin-responsive glucose transporter, Glut4, remained constant under both treatment conditions. The relative phosphotyrosine (Tyr(P)) contents of the insulin receptor and its substrate 1 (IRS-1) were assessed in whole cell homogenates. With both methods to induce insulin resistance, IR/IRS-1 Tyr(P) levels were virtually indistinguishable from those in control cells. Insulin-stimulated phosphorylation of Akt on Ser(473) was not impaired in insulin-resistant cells. Furthermore, the relative Tyr(P) content of the PDGF receptor was comparable in high glucose/insulin- or glucosamine-treated 3T3-L1 adipocytes upon subsequent challenge with PDGF. Finally, the relative amounts of glutamine:fructose-6-phosphate amidotransferase and O-linked N-acetylglucosamine transferase, two important hexosamine pathway enzymes, were similar in both treatments when compared with controls. Thus, 3T3-L1 adipocytes can be used as a model system for studying insulin resistance induced by increased influx of glucose. Under appropriate experimental conditions, glucosamine treatment can mimic the effects of increased glucose flux without impairment of tyrosine phosphorylation-based signaling.     

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes

To study molecular mechanisms for glucosamine-induced insulin resistance, we induced complete and reversible insulin resistance in 3T3-L1 adipocytes with glucosamine in a dose- and time-dependent manner (maximal effects at 50 mM glucosamine after 6 h). In these cells, glucosamine impaired insulin-stimulated GLUT-4 translocation. Glucosamine (6 h) did not affect insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 and -2 and weakly, if at all, impaired insulin stimulation of phosphatidylinositol 3-kinase. Glucosamine, however, severely impaired insulin stimulation of Akt. Inhibition of insulin-stimulated glucose transport was correlated with that of Akt activity. In these cells, glucosamine also inhibited insulin stimulation of p70 S6 kinase. Glucosamine did not alter basal glucose transport and insulin stimulation of GLUT-1 translocation and mitogen-activated protein kinase. In summary, glucosamine induced complete and reversible insulin resistance in 3T3-L1 adipocytes. This insulin resistance was accompanied by impaired insulin stimulation of GLUT-4 translocation and Akt activity, without significant impairment of upstream molecules in insulin-signaling pathway.     

GLUT2 is a high affinity glucosamine transporter

When expressed in Xenopus oocytes, GLUT1, 2 and 4 transport glucosamine with V(max) values that are three- to four-fold lower than for glucose. The K(m)s for glucosamine and glucose of GLUT1 and GLUT4 were similar. In contrast, GLUT2 had a much higher apparent affinity for glucosamine than for glucose (K(m)=0.8+/-0.1 mM vs. approximately 17-20 mM). Glucosamine transport by GLUT2 was confirmed in mammalian cells and, using hepatocytes from control or GLUT2-null mice, HgCl(2)-inhibitable glucosamine uptake by liver was shown to be exclusively through GLUT2. These data have implications for glucosamine effects on impaired glucose metabolism and for structure-function studies of transporter sugar binding sites.     

Distinct effects of glucose and glucosamine on vascular endothelial and smooth muscle cells: Evidence for a protective role for glucosamine in atherosclerosis

Accelerated atherosclerosis is one of the major vascular complications of diabetes. Factors including hyperglycemia and hyperinsulinemia may contribute to accelerated vascular disease. Among the several mechanisms proposed to explain the link between hyperglycemia and vascular dysfunction is the hexosamine pathway, where glucose is converted to glucosamine. Although some animal experiments suggest that glucosamine may mediate insulin resistance, it is not clear whether glucosamine is the mediator of vascular complications associated with hyperglycemia. Several processes may contribute to diabetic atherosclerosis including decreased vascular heparin sulfate proteoglycans (HSPG), increased endothelial permeability and increased smooth muscle cell (SMC) proliferation. In this study, we determined the effects of glucose and glucosamine on endothelial cells and SMCs in vitro and on atherosclerosis in apoE null mice. Incubation of endothelial cells with glucosamine, but not glucose, significantly increased matrix HSPG (perlecan) containing heparin-like sequences. Increased HSPG in endothelial cells was associated with decreased protein transport across endothelial cell monolayers and decreased monocyte binding to subendothelial matrix. Glucose increased SMC proliferation, whereas glucosamine significantly inhibited SMC growth. The antiproliferative effect of glucosamine was mediated via induction of perlecan HSPG. We tested if glucosamine affects atherosclerosis development in apoE-null mice. Glucosamine significantly reduced the atherosclerotic lesion in aortic root. (P < 0.05) These data suggest that macrovascular disease associated with hyperglycemia is unlikely due to glucosamine. In fact, glucosamine by increasing HSPG showed atheroprotective effects.     

Effects of cellular ATP depletion on glucose transport and insulin signaling in 3T3-L1 adipocytes

Glucosamine induced insulin resistance in 3T3-L1 adipocytes, which was associated with a 15% decrease in cellular ATP content. To study the role of ATP depletion in insulin resistance, we employed sodium azide (NaN3) and dinitrophenol (DNP), which affect mitochondrial oxidative phosphorylation, to achieve a similar 15% ATP depletion. Unlike glucosamine, NaN3 and DNP markedly increased basal glucose transport, and the increased basal glucose transport was associated with increased GLUT-1 content in the plasma membrane without changes in total GLUT-1 content. These agents, like glucosamine, did not affect the early insulin signaling that is implicated in insulin stimulation of glucose transport. In cells with a severe 40% ATP depletion, basal glucose transport was similarly elevated, and insulin-stimulated glucose transport was similar in cells with 15% ATP depletion. In these cells, however, early insulin signaling was severely diminished. These data suggest that cellular ATP depletion by glucosamine, NaN3, and DNP exerts differential effects on basal and insulin-stimulated glucose transport and that ATP depletion per se does not induce insulin resistance in 3T3-L1 adipocytes.     

Adoptive cellular immunotherapy: NK cells and bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) has been increasingly used for the treatment of both neoplastic and non-neoplastic disorders. However, serious obstacles currently limit the efficacy and thus more extensive use of BMT. These obstacles include: graft-versus-host disease (GVHD), relapse from the original tumor, and susceptibility of patients to opportunistic infections due to the immunosuppressive effects of the conditioning regimen.Overcoming these obstacles is complicated by dual outcome of existing regimens; attempts to reduce GVHD by depleting T cells from the graft, result in increased rates of tumor relapse and failure of engraftment. On the other hand, efforts to increase graft-versus-tumor (GVT) effects of the transplant also promote GVHD. In this review, the use of natural killer (NK) cells to overcome some of these obstacles of allogeneic BMT is evaluated. Adoptive immunotherapy using NK cells after allogeneic BMT has several potential advantages. First, NK cells can promote hematopoiesis and therefore engraftment by production of hematopoietic growth factors. Second, NK cells have been shown to prevent the incidence and severity of GVHD. This has been shown to be at least partially due to TGF-beta, an immunosuppressive cytokine. Third, NK cells have been shown to augment numerous anti-tumor effects in animals after BMT suggesting a vital role of NK cells in mediating GVT effects. Finally, NK cells have been demonstrated to affect B cell recovery and function in mice. Therefore, understanding the mechanisms of beneficial effects of NK cells after BMT may lead to significant increases in the efficacy of this procedure.     

Antibody-mediated immunosuppression of a cytotoxic cell response not involving a simple antigen-masking mechanism.

An in vitro cytotoxic cell response against alloantigens was inhibited by antibody directed against the alloantigens and also by anti-trinitrophenyl antibody provided that the allogeneic stimulator cells were modified with trinitrophenyl. Inhibition of an anti-allogeneic cytotoxic cell response against trinitrophenyl-coupled allogeneic stimulator cells by anti-trinitrophenyl antibody is dependent upon the intact Fc portion of inhibitory antibody. The magnitude of the difference between intact and F(ab')2 anti-trinitrophenyl antibody in immunosuppressive activity suggests that the Fc portion emits negative signals rather than simply adding to a steric hindrance effect.     

Activation of the hexosamine pathway causes oxidative stress and abnormal embryo gene expression: involvement in diabetic teratogenesis

BACKGROUND: Oxidative stress is critical to the teratogenic effects of diabetic pregnancy, yet the specific biochemical pathways responsible for oxidative stress have not been fully elucidated. The hexosamine pathway is activated in many tissues during diabetes and could contribute to oxidative stress by inhibiting the pentose shunt pathway, thereby diminishing production of the cellular antioxidant, reduced glutathione (GSH). METHODS: To test the hypothesis that activation of the hexosamine pathway might contribute to the teratogenic effects of diabetic pregnancy, pregnant mice were injected with glucose, to induce hyperglycemia, or glucosamine, to directly activate the hexosamine pathway. Embryo tissue fragments were also cultured in physiological glucose, high glucose, or physiological glucose plus glucosamine, to test effects on oxidative stress and embryo gene expression. RESULTS: Glucosamine increased hexosamine synthesis and inhibited pentose shunt activity. There was a trend for transient hyperglycemia to have the same effects, but they did not reach statistical significance. However, both glucose and glucosamine significantly decreased GSH, and increased oxidative stress, as indicated by 2',7'-dichloro-dihydrofluorescein fluorescence. Glucose and glucosamine inhibited expression of Pax-3, a gene required for neural tube closure both in vivo and in vitro, and increased neural tube defects (NTDs) in vivo; these effects were prevented by GSH ethyl ester. High glucose and glucosamine inhibited Pax-3 expression by embryo culture, but culture in glutamine-free media to block the hexosamine pathway prevented the inhibition of Pax-3 expression by high glucose. CONCLUSIONS: Activation of the hexosamine pathway causes oxidative stress through depletion of GSH and consequent disruption of embryo gene expression. Activation of this pathway may contribute to diabetic teratogenesis.     

Effects of glucosamine on insulin and glucagon secretion in dogs and ducks.

The effects of infusion of glucosamine on immunoreactive glucagon (IRG) and insulin (IRI) secretion were studied in dogs and ducks. During systemic infusion of glucosamine, hyperglycemia developed and insulin secretion was inhibited in both species. An immediate and sustained elevation of peripheral IRG levels was induced in ducks but a transient rise, detectable only in the pancreatic vein blood, was provoked in dogs. Suppression of insulin release and stimulation of glucagon release may be mediated by the inhibition of glucose utilization in beta- and alpha-cells. The very prompt response of IRG in ducks may imply that glucosamine has a specific stimulating effect on the alpha-cells of ducks. Intrapancreatic administration of glucosamine in dogs, however, failed to elicit the rise of IRG, although insulin secretion was inhibited. Thus, it is suggested that the systemic administration of glucosamine in dogs may stimulate IRG secretion by some indirect effect. In one dog, however, a sustained rise of the pancreatic vein IRG was observed. Thus, the possibility cannot be ruled out that the difference in IRG response to glucosamine in dogs and ducks is quantitative rather than qualitative. Glucagon release by glucosamine may provide an additional factor to the hyperglycemic effect of glucosamine, in addition to its effect to suppress insulin release as well as its direct inhibitory effect on glucose utilization in tissues.     

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.     

The role of glucose metabolites in the activation and translocation of glycogen synthase by insulin in 3T3-L1 adipocytes.

The role of increased glucose transport in the hormonal regulation of glycogen synthase by insulin was investigated in 3T3-L1 adipocytes. Insulin treatment stimulated glycogen synthase activity 4-5-fold in these cells. Cytosolic glycogen synthase levels decreased by 75% in response to insulin, whereas, conversely, the glycogenolytic agent isoproterenol increased cytosolic enzyme levels by 200%. Removal of extracellular glucose reduced glycogen synthase activation by 40% and completely blocked enzymatic translocation. Addition of 5 mM 2-deoxyglucose did not restore glycogen synthase translocation but did augment dephosphorylation of the protein by insulin. The translocation event could be reconstituted in vitro only by the addition of UDP-glucose to basal cell lysates. Amylase pretreatment of the extracts suppressed glycogen synthase translocation, indicating that the enzyme was binding to glycogen. Incubation of 3T3-L1 adipocytes with 10 mM glucosamine induced a state of insulin resistance, blocked the translocation of glycogen synthase, and inhibited insulin-stimulated glycogen synthesis by 50%. Surprisingly, glycogen synthase activation by insulin was enhanced 4-fold, in part due to allosteric activation by a glucosamine metabolite. In vitro, glucosamine 6-phosphate and glucose 6-phosphate stimulated glycogen synthase activity with similar concentration curves. These results indicate that glucose metabolites have an impact on the regulation of glycogen synthase activation and localization by insulin.     

UDP-N-acetylglucosamine pyrophosphorylase, a key enzyme in encysting Giardia, is allosterically regulated

Giardia synthesizes UDP-GalNAc during cyst wall formation (encystment) via a pathway of inducible enzymes similar to that used to synthesize chitin or peptidoglycan and that includes the UTP-requiring UDP-N-acetylglucosamine pyrophosphorylase. Although it has never been reported as a regulatory enzyme in any system studied to date, kinetic data including Hill plots demonstrate clearly that UDP-N-acetylglucosamine pyrophosphorylase activity, purified from encysting Giardia, is allosterically activated anabolically by physiological levels of glucosamine 6-phosphate (3 microm). Capillary electrophoresis demonstrates that within 24 h after trophozoites are induced to encyst, the level of glucosamine 6-phosphate increases 3-fold over that of non-encysting cells and that by 48 h into encystment the level of glucosamine 6-phosphate has decreased to non-encysting levels or below. UDP-N-acetylglucosamine pyrophosphorylase protein is present constitutively in encysting as well as non-encysting cells. UDP-N-acetylglucosamine pyrophosphorylase immunoaffinity purified from encysting and non-encysting cells exhibited the same molecular weight, amino acid composition, and circular dichroism spectra. Moreover, regardless of whether the enzyme came from encysting or non-encysting cells, the change in its circular dichroism spectra and up to a 6-fold increase in its specific activity anabolically were due to its activation with glucosamine 6-phosphate. Thus, the data support the idea that UDP-N-acetylglucosamine pyrophosphorylase is a major regulatory point in amino sugar synthesis in encysting Giardia and that its allosteric anabolic activation may shift the equilibrium of this pathway toward UDP-GalNAc synthesis.     

Arterial smooth muscle cell proteoglycans synthesized in the presence of glucosamine demonstrate reduced binding to LDL.

Atherosclerosis is the main cause of morbidity and mortality in diabetes, yet the underlying mechanisms remain unclear. Retention of atherogenic lipoproteins by vascular proteoglycans is thought to play a key role in the development of atherosclerotic lesions. High glucose levels cause a variety of diabetic complications by several mechanisms, including upregulation of the hexosamine pathway. Glucosamine, a component of the hexosamine pathway, is a precursor for the synthesis of glycosaminoglycan components of proteoglycans. This study evaluated whether high glucose or glucosamine supplementation of vascular smooth muscle cells would increase proteoglycan synthesis, leading to increased lipoprotein retention. Aortic smooth muscle cells were exposed to physiologic (5.6 mM) or high (25 mM) glucose levels, such as seen in diabetes, or to glucosamine (12 mM). Extracellular proteoglycans were characterized by sulfate incorporation, molecular sieve chromatography, and SDS-PAGE. LDL interactions were assessed by affinity chromatography and gel mobility shift assay. Proteoglycans synthesized in the presence of high glucose demonstrated no differences in size, sulfate incorporation, or LDL binding affinity compared with proteoglycans synthesized under physiological glucose conditions. However, proteoglycans synthesized in the presence of glucosamine had smaller glycosaminoglycan chains than control proteoglycans with a corresponding decrease in lipoprotein retention.Thus, glucose and glucosamine have different effects on proteoglycan biosynthesis and different effects on lipoprotein retention.