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.     

Knockdown of thioredoxin interacting protein attenuates high glucose-induced apoptosis and activation of ASK1 in mouse mesangial cells

Mesangial cell apoptosis contributes to the pathogenesis of diabetic nephropathy. Here we show that thioredoxin interacting protein (TXNIP) is involved in high glucose (HG)-induced mouse mesangial cell (MMC) apoptosis. HG induced activation of apoptosis signal regulating kinase-1 (ASK1) in a time-dependent manner in MMCs. Treatment with antioxidant, tempol, or knockdown of TXNIP in MMCs reduced HG-mediated apoptosis, expression of cleaved caspase-3, Bax/Bcl-2 ratio and activation of ASK1. These data suggest that knockdown of TXNIP prevented HG-induced cell apoptosis and activation of ASK1 may be via reduction of oxidative stress in MMCs.     

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.     

p38 MAPK pathway is involved in high glucose-induced thioredoxin interacting protein induction in mouse mesangial cells

Excessive reactive oxygen species (ROS) play a key role in the pathogenesis of diabetic nephropathy. The thioredoxin (TRX) system, a major thiol antioxidant system, regulates the reduction of intracellular ROS. Here we show that high glucose (HG) inhibits TRX ROS-scavenging function through p38 mitogen-activated protein kinase (MAPK)-mediated induction of thioredoxin interacting protein (TXNIP) in mouse mesangial cells (MMCs). Knockdown of TXNIP in MMCs reversed HG-induced reduction of TRX activity and inhibited HG-induced activation of p38 MAPK and increased synthesis of TGF-β1 and fibronectin. These data suggest that HG-induced overexpression of TXNIP in MMCs, which may be via the p38 MAPK pathway.     

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.     

Insulin resistance of glycogen synthase mediated by o-linked N-acetylglucosamine

We have investigated the mechanism by which high concentrations of glucose inhibit insulin stimulation of glycogen synthase. In NIH-3T3-L1 adipocytes cultured in low glucose (LG; 2.5 mm), the half-maximal activation concentration (A(0.5)) of glucose 6-phosphate was 162 +/- 15 microm. Exposure to either high glucose (HG; 20 mm) or glucosamine (GlcN; 10 mm) increased the A(0.5) to 558 +/- 61 or 612 +/- 34 microm. Insulin treatment with LG reduced the A(0.5) to 96 +/- 10 microm, but cells cultured with HG or GlcN were insulin-resistant (A(0.5) = 287 +/- 27 or 561 +/- 77 microm). Insulin resistance was not explained by increased phosphorylation of synthase. In fact, culture with GlcN decreased phosphorylation to 61% of the levels seen in cells cultured in LG. Hexosamine flux and subsequent enzymatic protein O-glycosylation have been postulated to mediate nutrient sensing and insulin resistance. Glycogen synthase is modified by O-linked N-acetylglucosamine, and the level of glycosylation increased in cells treated with HG or GlcN. Treatment of synthase in vitro with protein phosphatase 1 increased basal synthase activity from cells cultured in LG to 54% of total activity but was less effective with synthase from cells cultured in HG or GlcN, increasing basal activity to only 13 or 16%. After enzymatic removal of O-GlcNAc, however, subsequent digestion with phosphatase increased basal activity to over 73% for LG, HG, and GlcN. We conclude that O-GlcNAc modification of glycogen synthase results in the retention of the enzyme in a glucose 6-phosphate-dependent state and contributes to the reduced activation of the enzyme in insulin resistance.     

Upstream stimulatory factor (USF) proteins induce human TGF-beta1 gene activation via the glucose-response element-1013/-1002 in mesangial cells: up-regulation of USF activity by the hexosamine biosynthetic pathway

The hyperglycemia-enhanced flux through the hexosamine biosynthetic pathway (HBP) has been implicated in the up-regulated gene expression of transforming growth factor-beta1 (TGF-beta1) in mesangial cells, thus leading to mesangial matrix expansion and diabetic glomerulosclerosis. Since the -1013 to -1002 region of the TGF-beta1 promoter shows high homology to glucose-response elements (GlRE) formerly described in genes involved in glucose metabolism, we studied the function of the GlRE in the high glucose-induced TGF-beta1 gene activation in mesangial cells. We found that high glucose concentrations enhanced the nuclear amount of upstream stimulatory factors (USF) and their binding to this sequence. Fusion of the GlRE to the thymidine kinase promoter resulted in glucose responsiveness of this promoter construct. Overexpression of either USF-1 or USF-2 increased TGF-beta1 promoter activity 2-fold, which was prevented by mutation or deletion of the GlRE. The high glucose-induced activation of the GlRE is mediated by the HBP; increased flux through the HBP induced by high glucose concentrations, by glutamine, or by overexpression of the rate-limiting enzyme glutamine:fructose-6-phosphate aminotransferase (GFAT) particularly activated USF-2 expression. GFAT-overexpressing cells showed higher USF binding activity to the GlRE and enhanced promoter activation via the GlRE. Increasing O-GlcNAc modification of proteins by streptozotocin, thereby mimicking HBP activation, also resulted in increased mRNA and nuclear protein levels of USF-2, leading to enhanced DNA binding activity to the GlRE. USF proteins themselves were not found to be O-GlcNAc-modified. Thus, we have provided evidence for a new molecular mechanism linking high glucose-enhanced HBP activity with increased nuclear USF protein levels and DNA binding activity and with up-regulated TGF-beta1 promoter activity.     

Glucosamine causes overproduction of reactive oxygen species, leading to repression of hypocotyl elongation through a hexokinase-mediated mechanism in Arabidopsis

Glucosamine (GlcN) is a naturally occurring amino-sugar that is synthesized by amidation of fructose-6-phosphate. Although a number of reports have examined the biological effects of GlcN on insulin resistance in mammalian systems, little is known about its effects on plant growth. In this study, we have shown that exogenous GlcN inhibits hypocotyl elongation in Arabidopsis, whereas glucose and its analogs alleviate this inhibitory effect. The hexokinase (HXK)-specific inhibitor mannoheptulose also restored hypocotyl elongation. The gin2-1 mutants with an alteration in AtHXK1 exhibited higher tolerance to GlcN. We also found that GlcN induces a significant increase in the production of reactive oxygen species (ROS). In addition, the GlcN-mediated inhibition of hypocotyl elongation was relieved by reducing agents such as ascorbic acid and glutathione. GlcN treatment resulted in significant induction of expression of GST1, GST2 and GST6, which are marker genes for ROS production. The gin2 mutation also represses the ROS production and the GST2 induction by GlcN treatment. Taken together, these results provide evidence that GlcN induces HXK-mediated induction of oxidative stress, leading to growth repression in Arabidopsis thaliana.     

circLRP6 regulates high glucose-induced proliferation,oxidative stress,ECM accumulation,and inflammation in mesangial cells

Aberrant regulation in mesangial cell proliferation, extracellular matrix (ECM) accumulation, oxidative stress, and inflammation under hyperglycemic condition contributes significantly to the occurrence and development of diabetic nephropathy (DN). However, the mechanisms underlying the hyperglycemia-induced dysregulations have not been clearly elucidated. Here, we reported that high mobility group box 1 (HMGB1) was highly elevated in high glucose (HG)-treated mesangial cells, and induced the phosphorylation, nuclear translocation, and DNA binding activity of NF-κB via toll-like receptor 4 (TLR4). Function assays showed that inhibition of HMGB1 mitigated HG-induced proliferation, oxidative stress, ECM accumulation, and inflammation in mesangial cells via TLR4/NF-κB pathway. Increasing evidence has shown that circRNA, a large class of noncoding RNAs, functions by binding with miRNAs and terminating regulation of their target genes. We further investigated whether HMGB1 is involved in circRNA–miRNA–mRNA regulatory network. First, HMGB1 was identified and confirmed to be the target of miR-205, and miR-205 played a protective role against HG-induced cell injure via targeting HMGB1. Then circLRP6 was found to be upregulated in HG-treated mesangial cells, and regulate HG-induced mesangial cell injure via sponging miR-205. Besides, overexpression of miR-205 or knockdown of circLRP6 inhibited the NF-κB signaling pathway. Collectively, these data suggest that circLRP6 regulates HG-induced proliferation, oxidative stress, ECM accumulation, and inflammation in mesangial cells via sponging miR-205, upregulating HMGB1 and activating TLR4/NF-κB pathway. These findings provide a better understanding for the pathogenesis of DN.     

硫氧还蛋白相互作用蛋白在糖尿病及其并发症中的作用

硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,TXNIP)又称维生素D₃上调蛋白1,因其能够与硫氧还蛋白(thioredoxin,Trx)结合并抑制其活性和表达而得名。本文概述了TXNIP的发现与结构,及其自身通过发挥调节糖脂代谢的作用进而影响糖尿病前期的发生发展。并在此基础上总结了TXNIP参与糖尿病发生发展的2条主要途径:TXNIP通过拮抗Trx的抗凋亡作用来激发细胞凋亡信号导致胰岛细胞凋亡;TXNIP过表达促使胰岛细胞磷酸化,进而使抑癌相关蛋白质表达增加,最终引起胰岛细胞衰老。进一步重点阐述了TXNIP在糖尿病心肌病、糖尿病肾病、糖尿病性视网膜病等糖尿病并发症中的作用:TXNIP能通过各种间接途径干预信号通路,进一步参与氧化应激、细胞凋亡、激活炎症、细胞自噬及糖脂代谢等生理生化过程。TXNIP具有极其重要的生物学功能,深入了解TXNIP在糖尿病及其并发症中的影响机制,对糖尿病及其并发症的治疗具有重要意义。最后对TXNIP的研究进行了展望,未来可进一步着手研究TXNIP基因是如何与其他基因或危险因素协同作用,进而共同参与糖尿病及其并发症的发生发展,且TXNIP单个基因甲基化尚不能全面揭示糖尿病及其并发症发生的分子机制,这些后续的深入研究,将为在糖尿病及其并发症的诊断与治疗中作为靶标分子的应用奠定基础。     

All‐trans retinoic acid ameliorates hepatic stellate cell activation via suppression of thioredoxin interacting protein expression

Activation of hepatic stellate cells (HSCs) is the effector factor of hepatic fibrosis and hepatocellular carcinoma (HCC) development. Accumulating evidence suggests that retinoic acids (RAs), derivatives of vitamin A, contribute to prevention of liver fibrosis and carcinogenesis, however, regulatory mechanisms of RAs still remain exclusive. To elucidate RA signaling pathway, we previously performed a genome‐wide screening of RA‐responsive genes by in silico analysis of RA‐response elements, and identified 26 RA‐responsive genes. We found that thioredoxin interacting protein (TXNIP), which inhibits antioxidant activity of thioredoxin (TRX), was downregulated by all‐trans retinoic acid (ATRA). In the present study, we demonstrate that ATRA ameliorates activation of HSCs through TXNIP suppression. HSC activation was attenuated by TXNIP downregulation, whereas potentiated by TXNIP upregulation, indicating that TXNIP plays a crucial role in activation of HSCs. Notably, we showed that TXNIP‐mediated HSC activation was suppressed by antioxidant N‐acetylcysteine. In addition, ATRA treatment or downregulation of TXNIP clearly declined oxidative stress levels in activated HSCs. These data suggest that ATRA plays a key role in inhibition of HSC activation via suppressing TXNIP expression, which reduces oxidative stress levels.     

Thioredoxin-interacting protein deficiency alleviates phenotypic alterations of podocytes via inhibition of mTOR activation in diabetic nephropathy

Thioredoxin-interacting protein (TXNIP) is induced by high glucose (HG), whereupon it acts to inhibit thioredoxin, thereby promoting oxidative stress. We have found that TXNIP knockdown in human renal tubular cells helped prevent the epithelial-to-mesenchymal transition (EMT). Here, we studied the potential effect of TXNIP on podocyte phenotypic alterations in diabetic nephropathy (DN) in vivo and in vitro. In conditionally immortalized mouse podocytes under HG conditions, knocking down TXNIP disrupted EMT, reactive oxygen species (ROS) production, and mammalian target of rapamycin (mTOR) pathway activation. Further, Raptor short hairpin RNA (shRNA), Rictor shRNA, and mTOR specific inhibitor KU-0063794 were used to assess if the mTOR signal pathway is involved in HG-induced EMT in podocytes. We found that Raptor shRNA, Rictor shRNA, and KU-0063794 could all restrain HG-induced EMT and ROS production in podocytes. In addition, antioxidant Tempol or N-acetylcysteine presented a prohibitive effect on HG-induced EMT in podocytes. Streptozotocin was utilized to render equally diabetic in wild-type (WT) control and TXNIP ^−/− (TKO) mice. Diabetes did not increase levels of 24-hr urinary protein, serum creatinine, blood urea nitrogen, and triglyceride in TXNIP ^−/− mice. Podocyte phenotypic alterations and podocyte loss were detected in WT but not in TKO diabetic mice. Oxidative stress was also suppressed in diabetic TKO mice relative to WT controls. Also, TXNIP deficiency suppresses the activation of mTOR in glomeruli of streptozotocin-induced diabetic mice. Moreover, TXNIP expression, mTOR activation, Nox1, and Nox4 could be detected in renal biopsy tissues of patients with DN. This suggests that decreased TXNIP could ameliorate phenotypic alterations of podocytes via inhibition of mTOR in DN, highlighting TXNIP as a promising therapeutic target.     

The hexosamine biosynthesis pathway negatively regulates IL-2 production by Jurkat T cells

To test the hypothesis that the hexosamine biosynthesis pathway (HBP) affects cytokine production, we studied IL-2 production by Jurkat cells in response to PHA. We found that the HBP activator glucosamine (GlcN), but not glucose (Glc), dose-dependently reduced IL-2 production. Importantly, GlcN blocked trafficking of a GFP-NFAT chimeric protein to the nucleus of stimulated transfectants. Not surprisingly, changes in O-GlcNAc protein modifications were noted during cell activation with and without GlcN addition. These findings could not be explained by some non-specific change in cell metabolism because ATP concentrations did not significantly change. We speculate that HBP-active compounds may contribute to patient care in certain inflammatory and autoimmune diseases.     

Thioredoxin interacting protein (TXNIP) induces inflammation through chromatin modification in retinal capillary endothelial cells under diabetic conditions

Chronic hyperglycemia and activation of receptor for advanced glycation end products (RAGE) are known risk factors for microvascular disease development in diabetic retinopathy. Thioredoxin‐interacting protein (TXNIP), an endogenous inhibitor of antioxidant thioredoxin (TRX), plays a causative role in diabetes and its vascular complications. Herein we investigate whether HG and RAGE induce inflammation in rat retinal endothelial cells (EC) under diabetic conditions in culture through TXNIP activation and whether epigenetic mechanisms play a role in inflammatory gene expression. We show that RAGE activation by its ligand S100B or HG treatment of retinal EC induces the expression of TXNIP and inflammatory genes such as Cox2, VEGF‐A, and ICAM1. TXNIP silencing by siRNA impedes RAGE and HG effects while stable over‐expression of a cDNA for human TXNIP in EC elevates inflammation. p38 MAPK‐NF‐κB signaling pathway and histone H3 lysine (K) nine modifications are involved in TXNIP‐induced inflammation. Chromatin immunoprecipitation (ChIP) assays reveal that TXNIP over‐expression in EC abolishes H3K9 tri‐methylation, a marker for gene inactivation, and increases H3K9 acetylation, an indicator of gene induction, at proximal Cox2 promoter bearing the NF‐κB‐binding site. These findings have important implications toward understanding the molecular mechanisms of ocular inflammation and endothelial dysfunction in diabetic retinopathy. J. Cell. Physiol. 221: 262–272, 2009. © 2009 Wiley‐Liss, Inc     

Comparative effects of fructose and glucose on lipogenic gene expression and intermediary metabolism in HepG2 liver cells

Consumption of large amounts of fructose or sucrose increases lipogenesis and circulating triglycerides in humans. Although the underlying molecular mechanisms responsible for this effect are not completely understood, it is possible that as reported for rodents, high fructose exposure increases expression of the lipogenic enzymes fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC-1) in human liver. Since activation of the hexosamine biosynthesis pathway (HBP) is associated with increases in the expression of FAS and ACC-1, it raises the possibility that HBP-related metabolites would contribute to any increase in hepatic expression of these enzymes following fructose exposure. Thus, we compared lipogenic gene expression in human-derived HepG2 cells after incubation in culture medium containing glucose alone or glucose plus 5 mM fructose, using the HBP precursor 10 mM glucosamine (GlcN) as a positive control. Cellular metabolite profiling was conducted to analyze differences between glucose and fructose metabolism. Despite evidence for the active uptake and metabolism of fructose by HepG2 cells, expression of FAS or ACC-1 did not increase in these cells compared with those incubated with glucose alone. Levels of UDP-N-acetylglucosamine (UDP-GlcNAc), the end-product of the HBP, did not differ significantly between the glucose and fructose conditions. Exposure to 10 mM GlcN for 10 minutes to 24 hours resulted in 8-fold elevated levels of intracellular UDP-GlcNAc (P<0.001), as well as a 74-126% increase in FAS (P<0.05) and 49-95% increase in ACC-1 (P<0.01) expression above controls. It is concluded that in HepG2 liver cells cultured under standard conditions, sustained exposure to fructose does not result in an activation of the HBP or increased lipogenic gene expression. Should this scenario manifest in human liver in vivo, it would suggest that high fructose consumption promotes triglyceride synthesis primarily through its action to provide lipid precursor carbon and not by activating lipogenic gene expression.     

Excessive hexosamines block the neuroprotective effect of insulin and induce apoptosis in retinal neurons

In addition to microvascular abnormalities, neuronal apoptosis occurs early in diabetic retinopathy, but the mechanism is unknown. Insulin may act as a neurotrophic factor in the retina via the phosphoinositide 3-kinase/Akt pathway. Excessive glucose flux through the hexosamine biosynthetic pathway (HBP) is implicated in the development of insulin resistance in peripheral tissues and diabetic complications such as nephropathy. We tested whether increased glucose flux through the HBP perturbs insulin action and induces apoptosis in retinal neuronal cells. Exposure of R28 cells, a model of retinal neurons, to 20 mm glucose for 24 h attenuated the ability of 10 nm insulin to rescue them from serum deprivation-induced apoptosis and to phosphorylate Akt compared with 5 mm glucose. Glucosamine not only impaired the neuroprotective effect of insulin but also induced apoptosis in R28 cells in a dose-dependent fashion. UDP-N-acetylhexosamines (UDP-HexNAc), end products of the HBP, were increased approximately 2- and 15-fold after a 24-h incubation in 20 mm glucose and 1.5 mm glucosamine, respectively. Azaserine, a glutamine:fructose-6-phosphate amidotransferase inhibitor, reversed the effect of 20 mm glucose, but not that of 1.5 mm glucosamine, on attenuation of the ability of insulin to promote cell survival and phosphorylate Akt as well as accumulation of UDP-HexNAc. Glucosamine also impaired insulin receptor processing in a dose-dependent manner but did not decrease ATP content. By contrast, in L6 muscle cells, glucosamine impaired insulin receptor processing but did not induce apoptosis. These results suggest that the excessive glucose flux through the HBP may direct retinal neurons to undergo apoptosis in a bimodal fashion; i.e. via perturbation of the neuroprotective effect of insulin mediated by Akt and via induction of apoptosis possibly by altered glycosylation of proteins. The HBP may be involved in retinal neurodegeneration in diabetes.     

High glucose induces renal mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which is inhibited by resveratrol

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. Hyperglycemia-induced oxidative stress is implicated in the etiology of diabetic nephropathy. Resveratrol has potent antioxidative and protective effects on diabetic nephropathy. We aimed to examine whether high glucose (HG)-induced NADPH oxidase activation and reactive oxygen species (ROS) production contribute to glomerular mesangial cell proliferation and fibronectin expression and the effect of resveratrol on HG action in mesangial cells. By using rat mesangial cell line and primary mesangial cells, we found that NADPH oxidase inhibitor (apocynin) and ROS inhibitor (N-acetyl cysteine) both inhibited HG-induced mesangial cell proliferation and fibronectin expression. HG-induced elevation of NADPH oxidase activity and production of ROS in mesangial cells was inhibited by apocynin. These results suggest that HG induces mesangial cell proliferation and fibronectin expression through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunits p22(phox) and p47(phox) expression through JNK/NF-κB pathway, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced mesangial cell proliferation and fibronectin expression through inhibiting HG-induced JNK and NF-κB activation, NADPH oxidase activity elevation and ROS production. These results demonstrate that HG enhances mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide novel therapeutic targets for diabetic nephropathy.     

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.