An analysis of high glucose and glucosamine-induced gene expression and oxidative stress in renal mesangial cells

Renal mesangial cells play an important role in the development of diabetic kidney disease. We have previously demonstrated that some of the effects of high glucose on mesangial extracellular matrix (ECM) protein expression are mediated by the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). Using Affymetrix murine expression U430 2.0 oligochips, we examined the global effects of high glucose (HG) and glucosamine (GlcN) on mRNA expression of a mouse mesangial cell line (MES-13). We sought to determine the portion of mRNA expression in MES-13 cells, which is mediated both by high glucose and glucosamine, i.e., via the HBP. Of the 34,000 genes on the chip, approximately 55.7 - 60.8% genes are detected in MES-13 cells. Culturing MES-13 cells for 48 h with HG alters the expression of approximately 389 genes at our preset threshold levels (at least 2-fold change) where 263 genes are up-regulated and 126 genes are down-regulated. GlcN also increases the expression of 106 genes and decreases 94 genes during the same period of incubation. Seventy-two genes in the chip are commonly regulated by HG and GlcN, in which 33 genes are up and 39 genes are down. The mRNA level of thioredoxin interacting protein (TXNIP), an inhibitor of thioredoxin activity, is maximally increased approximately 18.8 and 9.9-fold respectively by HG and GlcN. The differential expression of several genes found in the microarray analysis is further validated by real-time quantitative PCR. Significant biological processes commonly targeted by HG and GlcN are the TXNIP-thioredoxin system, oxidative stress, endoplasmic reticulum (ER) stress, extracellular matrix genes, and interferon-inducible genes. Stable overexpression of TXNIP in MES-13 cells increases glucose and glucosamine-mediated ECM gene expression and oxidative stress. We conclude from these results that the HBP mediates several effects of high glucose on mesangial cell metabolism, which promotes reactive oxygen species generation to cause cellular oxidative stress, ECM gene expression and apoptosis.     

Hexosamine induction of oxidative stress, hypertrophy and laminin expression in renal mesangial cells: effect of the anti-oxidant alpha-lipoic acid

We have previously shown that one of the potential mediators of the deleterious effects of high glucose on extracellular matrix protein (ECM) expression in renal mesangial cells is its metabolic flux through the hexosamine biosynthesis pathway (HBP). Here, we investigate further whether the hexosamines induce oxidative stress, cell-cycle arrest and ECM expression using SV-40-transformed rat mesangial (MES) cells and whether the anti-oxidant alpha-lipoic acid will reverse some of these effects. Culturing renal MES cells with high glucose (HG, 25 mM) or glucosamine (GlcN, 1.5 mM) for 48 h stimulates laminin gamma1 subunit expression significantly approximately 1.5 +/- 0.2- and 1.9 +/- 0.3-fold, respectively, when compared to low glucose (LG, 5 mM). Similarly, HG and GlcN increase the level of G0/G1 cell-cycle progression factor cyclin D1 significantly approximately 1.7 +/- 0.2- and 1.4 +/- 0.04-fold, respectively, versus LG (p < 0.01 for both). Azaserine, an inhibitor of glutamine:fruc-6-PO(4) amidotransferase (GFAT) in the HBP, blocks the HG-induced expression of laminin gamma1 and cyclin D1, but not GlcN's effect because it exerts its metabolic function distal to GFAT. HG and GlcN also elevate reactive oxygen species (ROS) generation, pro-apoptotic caspase-3 activity, and lead to mesangial cell death as revealed by TUNEL and Live/Dead assays. FACS analysis of cell-cycle progression shows that the cells are arrested at G1 phase; however, they undergo cell growth and hypertrophy as the RNA/DNA ratio is significantly (p < 0.05) increased in HG or GlcN-treated cells relative to LG. The anti-oxidant alpha-lipoic acid (150 microM) reverses ROS generation and mesangial cell death induced by HG and GlcN. Alpha-lipoic acid also reduces HG and GlcN-induced laminin gamma1 and cyclin D1 expression in MES cells. In addition, induction of diabetes in rats by streptozotocin (STZ) increases both laminin gamma1 and cyclin D1 expression in the renal cortex and treatment of the diabetic rats with alpha-lipoic acid (400 mg kg(-1) body weight) reduces the level of both proteins significantly (p < 0.05) when compared to untreated diabetic rats. These results support the hypothesis that the hexosamine pathway mediates mesangial cell oxidative stress, ECM expression and apoptosis. Anti-oxidant alpha-lipoic acid reverses the effects of high glucose, hexosamine and diabetes on oxidative stress and ECM expression in mesangial cells and rat kidney.     

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.     

Glutathione stability and oxidative stress in P. falciparum infection in vitro: Responses of normal and G6PD deficient cells

Red cell oxidative stress in P. falciparum infection in vitro was investigated in relation to the G6PD-Malaria hypothesis. Glutathione stability was enhanced in infected red cells; glucose consumption and pentose pathway activity were not different in normal and G6PD deficient cells, although parasite growth was impaired in G6PD deficiency. Evidence for a response to oxidative stress was not found. Infected red cells have glutamate dehydrogenase activity which was not found in uninfected cells. This enzyme provides a separate pathway for the generation of NADPH independent from the pentose shunt. The data suggest that a significant oxidative stress is not present in falciparum malaria and that another mechanism may be operative in G6PD deficiency.     

Glutathione and K(+) channel remodeling in postinfarction rat heart

Electrical remodeling of the diseased ventricle is characterized by downregulation of K(+) channels that control action potential repolarization. Recent studies suggest that this shift in electrophysiological phenotype involves oxidative stress and changes in intracellular glutathione (GSH), a key regulator of redox-sensitive cell functions. This study examined the role of GSH in regulating K(+) currents in ventricular myocytes from rat hearts 8 wk after myocardial infarction (MI). Colorimetric analysis of tissue extracts showed that endogenous GSH levels were significantly less in post-MI hearts compared with controls, which is indicative of oxidative stress. This change in GSH status correlated with significant decreases in activities of glutathione reductase and gamma-glutamylcysteine synthetase. Voltage-clamp studies of isolated myocytes from post-MI hearts demonstrated that downregulation of the transient outward K(+) current (I(to)) could be reversed by pretreatment with exogenous GSH or N-acetylcysteine, a precursor of GSH. Upregulation of I(to) was also elicited by dichloroacetate, which increases glycolytic flux through the GSH-related pentose pathway. This metabolic effect was blocked by inhibitors of glutathione reductase and the pentose pathway. These data indicate that oxidative stress-induced alteration in the GSH redox state plays an important role in I(to) channel remodeling and that GSH homeostasis is influenced by pathways of glucose metabolism.     

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.     

The hexosamine biosynthetic pathway and glucose-induced down regulation of glucose transport in L6 myotubes

Based on experiments in cultured adipocytes, it has been proposed that glucose-induced down regulation of glucose transport is mediated by the conversion of fructose-6-phosphate to glucosamine-6-phosphate via the first and rate-determining enzyme of the hexasamine biosynthetic pathway, glutamine: fructose-6-phosphate amidotransferase (glutamine hexosephosphate aminotransferase). Evidence for this assertion was: (a) l-glutamine, the provider group for the aminotransferase was essential; (b) two inhibitors of glutamine hexosephosphate aminotransferase, 6-diazo-5-oxonorleucine (l form) and azaserine, blocked glucose-induced down regulation of glucose transport; (c) azaserine inhibited the activity of the aminotransferase, (d) glucosamine, which enters the hexosamine pathway distal to this enzyme was 40-times more potent than glucose; and (e) azaserine was unable to block the effect of glucosmaine. Since muscle is quantitatively much more important than adipose tissue for whole body glucose utilization, we sought to determine if the hexosamine pathway was involved in glucose-induced down regulation of glucose transport in L₆ myotubes. Glucose was effective, both in the presence and absence of glutamine in the incubation media. Glucosamine was also effective but was as equipotent as glucose. Small amounts of glutamine hexosephosphate aminotransferase were present in the L₆ myotubes and although the leucine derivative (20 μM)_ inhibited the enzyme, it did not impair glucose-induced down regulation of glucose transport. Total GLUT-1 levels were similar when the cells were incubated in the absence or presence of 5 mM glucose or glucosamine although glucosamine was associated with a marked increase in a lower molecular weight band. These results do not suggest that the hexosamine biosynthetic pathway is involved in glucose-induced down regulation of glucose transport in L₆ myotubes. Thus, this phenomenon is regulated differently in muscle and fat.     

Enhanced drought tolerance in Arabidopsis via genetic manipulation aimed at the reduction of glucosamine-induced ROS generation

In animals, high glucose exerts some of its deleterious effects by activation of the hexosamine biosynthesis pathway (HBP), a branch of the glycolytic pathway that produces amino sugars (Daniels et al. in Mol Endocrinol 7:1041-1048, 1993; Du et al. in Proc Natl Acad Sci USA 97:12222-12226, 2000). Glucosamine (GlcN) is a naturally occurring amino sugar produced by amidation of fructose-6-phosphate. Previously, we observed that glucosamine (GlcN) inhibits hypocotyl elongation in Arabidopsis thaliana by a process involving the significant increase of reactive oxygen species. The present study investigated the relationship between GlcN-induced ROS generation and abiotic stress responses in Arabidopsis by generating two types of transgenic plant. Scavenging of endogenous GlcN by ectopic expression of E. coli glucosamine-6-phosphate deaminase (NagB) was observed to confer enhanced tolerance to oxidative, drought, and cold stress. Consistent with this result, overproduction of GlcN by the ectopic expression of E. coli glucosamine-6-phosphate synthase (GlmS) induced cell death at an early stage. Taken together, these data suggest that genetic manipulation of endogenous GlcN level can effectively lead to the generation of abiotic stress-tolerant transgenic crop plants.     

Increased efflux of glutathione conjugate in acutely diabetic cardiomyocytes

In diabetes, cell death and resultant cardiomyopathy have been linked to oxidative stress and depletion of antioxidants like glutathione (GSH). Although the de novo synthesis and recycling of GSH have been extensively studied in the chronically diabetic heart, their contribution in modulating cardiac oxidative stress in acute diabetes has been largely ignored. Additionally, the possible contribution of cellular efflux in regulating GSH levels during diabetes is unknown. We used streptozotocin to make Wistar rats acutely diabetic and after 4 days examined the different processes that regulate cardiac GSH. Reduction in myocyte GSH in diabetic rats was accompanied by increased oxidative stress, excessive reactive oxygen species, and an elevated apoptotic cell death. The effect on GSH was not associated with any change in either synthesis or recycling, as both gamma-glutamylcysteine synthetase gene expression (responsible for bio syn thesis) and glutathione reductase activity (involved with GSH recycling) remained unchanged. However, gene expression of multidrug resistance protein 1, a transporter implicated in effluxing GSH during oxidative stress, was elevated. GSH conjugate efflux mediated by multidrug resistance protein 1 also increased in diabetic cardiomyocytes, an effect that was blocked using MK-571, a specific inhibitor of this transporter. As MK-571 also decreased oxidative stress in diabetic cardiomyocytes, an important role can be proposed for this transporter in GSH and reactive oxygen species homeostasis in the acutely diabetic heart.     

Role of hexosamine biosynthesis in glucose-mediated up-regulation of lipogenic enzyme mRNA levels: effects of glucose,glutamine, and glucosamine on glycerophosphate dehydrogenase,fatty acid synthase,and acetyl-CoA carboxylase mRNA levels

Glucose uptake into adipose and liver cells is known to up-regulate mRNA levels for various lipogenic enzymes such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). To determine whether the hexosamine biosynthesis pathway (HBP) mediates glucose regulation of mRNA expression, we treated primary cultured adipocytes for 18 h with insulin (25 ng/ml) and either glucose (20 mm) or glucosamine (2 mm). A ribonuclease protection assay was used to quantitate mRNA levels for FAS, ACC, and glycerol-3-P dehydrogenase (GPDH). Treatment with insulin and various concentrations of d-glucose increased mRNA levels for FAS (280%), ACC (93%), and GPDH (633%) in a dose-dependent manner (ED50 8-16 mm). Mannose similarly elevated mRNA levels, but galactose and fructose were only partially effective. l-glucose had no effect. Omission of glutamine from the culture medium markedly diminished the stimulatory effect of glucose on mRNA expression. Since glutamine is a crucial amide donor in hexosamine biosynthesis, we interpret these data to mean that glucose flux through the HBP is linked to regulation of lipogenesis through control of gene expression. Further evidence for hexosamine regulation was obtained using glucosamine, which is readily transported into adipocytes where it directly enters the HBP. Glucosamine was 15-30 times more potent than glucose in elevating FAS, ACC, and GPDH mRNA levels (ED50 approximately 0.5 mm). In summary: 1) GPDH, FAS, and ACC mRNA levels are upregulated by glucose; 2) glucose-induced up-regulation requires glutamine; and 3) mRNA levels for lipogenic enzymes are up-regulated by glucosamine. Hyperglycemia is the hallmark of diabetes mellitus and leads to insulin resistance, impaired glucose metabolism, and dyslipidemia. We postulate that disease pathophysiology may have a common underlying factor, excessive glucose flux through the HBP.     

Oxidative stress stimulates the synthesis of the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid by inflammatory cells

5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that acts through a selective G-protein coupled receptor. It is formed by oxidation of the 5-lipoxygenase product 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH). Although leukocytes and platelets display high microsomal 5-HEDH activity, unstimulated intact cells do not convert 5-HETE to appreciable amounts of 5-oxo-ETE. To attempt to resolve this dilemma we explored the possibility that 5-oxo-ETE synthesis could be enhanced by oxidative stress. We found that hydrogen peroxide and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U937 monocytic cells. This was dependent on the GSH redox cycle, as it was blocked by depletion of GSH or inhibition of glutathione reductase and mimicked by oxidation of GSH to GSSG by diamide. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, as its effect was reversed by the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-butyl hydroperoxide. Oxidative stress could act by depleting NADPH, resulting in the formation NADP+, the cofactor for 5-HEDH. This is opposed by the pentose phosphate pathway, which converts NADP+ back to NADPH. Oxidative stress could be an important mechanism for stimulating 5-oxo-ETE production in inflammation, promoting further infiltration of granulocytes into inflammatory sites.     

Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance

Based on our previous finding that desensitization of the insulin-responsive glucose transport system (GTS) requires three components, glucose, insulin, and glutamine, we postulated that the routing of incoming glucose through the hexosamine biosynthesis pathway plays a key role in the development of insulin resistance in primary cultured adipocytes. Two approaches were used to test this hypothesis. First, we assessed whether glucose-induced desensitization of the GTS could be prevented by glutamine analogs that irreversibly inactivate glutamine-requiring enzymes, such as glutamine:fructose-6-phosphate amidotransferase (GFAT) the first and the rate-limiting enzyme in hexosamine biosynthesis. Both O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxonorleucine inhibited desensitization in 18-h treated cells without affecting maximal insulin responsiveness in control cells. Moreover, close agreement was seen between the ability of azaserine to prevent desensitization of the GTS in intact adipocytes (70% inhibition, ED50 = 1.1 microM), its ability to inactivate GFAT in intact adipocytes (64% inhibition, ED50 = 1.0 microM) and its ability to inactivate GFAT activity in a cytosolic adipocyte preparation (ED50 = 1.3 microM). From these results we concluded that a glutamine amidotransferase is involved in the induction of insulin resistance. As a second approach, we determined whether glucosamine, an agent known to preferentially enter the hexosamine pathway at a point distal to enzymatic amidation by GFAT, could induce cellular insulin resistance. When adipocytes were exposed to various concentrations of glucosamine for 5 h, progressive desensitization of the GTS was observed (ED50 = 0.36 mM) that culminated in a 40-50% loss of insulin responsiveness. Moreover, we estimated that glucosamine is at least 40 times more potent than glucose in mediating desensitization, since glucosamine entered adipocytes at only one-quarter of the glucose uptake rate, yet induced desensitization at an extra-cellular dose 10 times lower than glucose. In addition, we found that glucosamine-induced desensitization did not require glutamine and was unaffected by azaserine treatment. Thus, we conclude that glucosamine enters the hexosamine-desensitization pathway at a point distal to GFAT amidation. Overall, these studies indicate that a unique metabolic pathway exists in adipocytes that mediates desensitization of the insulin-responsive GTS, and reveal that an early step in this pathway involves the conversion of fructose 6-phosphate to glucosamine 6-phosphate by the first and rate-limiting enzyme of the hexosamine pathway, glutamine:fructose-6-phosphate amidotransferase.     

Interrelations between glycolysis and the hexose monophosphate shunt in erythrocytes as studied on the basis of a mathematical model

A mathematical model is presented which comprises the reactions of glycolysis, the hexose monophosphate shunt (HMS) and the glutathione system in erythrocytes. The model is used to calculate stationary and time-dependent metabolic states of the cell in vitro and in vivo. The model properly accounts for the following metabolic features observed in vitro: (a) stimulation of the oxidative pentose pathway after addition of pyruvate due to a NADP-dependent lactate dehydrogenase as coupling enzyme between glycolysis and the oxidative pentose pathway, (b) relative share of the oxidative pentose pathway in the total consumption of glucose amounting to approximately 10% in the normal case and to approximately 90% under conditions of oxidative stress excreted by methylene blue. From the application of the model to in vivo conditions it is predicted that (c) under normal conditions glycolysis and the HMS are independently regulated by the energetic and oxidative load, respectively, (d) under conditions of enhanced energetic or oxidative load both glycolysis and the HMS are mainly controlled by the hexokinase; in this situation the highest possible values of the energetic and oxidative load which are compatible with cell integrity are strongly coupled and considerably restricted in comparison with the normal case, (e) the stationary states possess bifurcation points at high and low values of the energetic load.     

Insulin regulation of glutathione and contractile phenotype in diabetic rat ventricular myocytes

Cardiovascular complications of diabetes mellitus involve oxidative stress and profound changes in reduced glutathione (GSH), an essential tripeptide that controls many redox-sensitive cell functions. This study examined regulation of GSH by insulin to identify mechanisms controlling cardiac redox state and to define the functional impact of GSH depletion. GSH was measured by fluorescence microscopy in ventricular myocytes isolated from Sprague-Dawley rats made diabetic by streptozotocin, and video and confocal microscopy were used to measure mechanical properties and Ca(2+) transients, respectively. Spectrophotometric assays of tissue extracts were also done to measure the activities of enzymes that control GSH levels. Four weeks after injection of streptozotocin, mean GSH concentration ([GSH]) in isolated diabetic rat myocytes was approximately 36% less than in control, correlating with decreased activities of two major enzymes regulating GSH levels: glutathione reductase and gamma-glutamylcysteine synthetase. Treatment of diabetic rat myocytes with insulin normalized [GSH] after a delay of 3-4 h. A more rapid but transient upregulation of [GSH] occurred in myocytes treated with dichloroacetate, an activator of pyruvate dehydrogenase. Inhibitor experiments indicated that insulin normalized [GSH] via the pentose pathway and gamma-glutamylcysteine synthetase, although the basal activity of glucose-6-phosphate dehydrogenase was not different between diabetic and control hearts. Diabetic rat myocytes were characterized by significant mechanical dysfunction that correlated with diminished and prolonged Ca(2+) transients. This phenotype was reversed by in vitro treatment with insulin and also by exogenous GSH or N-acetylcysteine, a precursor of GSH. Our data suggest that insulin regulates GSH through pathways involving de novo GSH synthesis and reduction of its oxidized form. It is proposed that a key function of glucose metabolism in heart is to supply reducing equivalents required to maintain adequate GSH levels for the redox control of Ca(2+) handling proteins and contraction.