In the Early Stages of Diabetes,Rat Retinal Mitochondria Undergo Mild Uncoupling due to UCP2 Activity

In order to maintain high transmembrane ionic gradients, retinal tissues require a large amount of energy probably provided by a high rate of both, glycolysis and oxidative phosphorylation. However, little information exists on retinal mitochondrial efficiency. We analyzed the retinal mitochondrial activity in ex vivo retinas and in isolated mitochondria from normal rat retina and from short-term streptozotocin-diabetic rats. In normal ex vivo retinas, increasing glucose concentrations from 5.6mM to 30mM caused a four-fold increase in glucose accumulation and CO₂ production. Retina from diabetic rats accumulated similar amounts of glucose. However, CO₂ production was not as high. Isolated mitochondria from normal rat retina exhibited a resting rate of oxygen consumption of 14.6 ± 1.1 natgO (min.mg prot)^-1 and a respiratory control of 4.0. Mitochondria from 7, 20 and 45 days diabetic rats increased the resting rate of oxygen consumption and the activity of the electron transport complexes; under these conditions the mitochondrial transmembrane potential decreased. In spite of this, the ATP synthesis was not modified. GDP, an UCP2 inhibitor, increased mitochondrial membrane potential and superoxide production in controls and at 45 days of diabetes. The role of UCP2 is discussed. The results suggest that at the early stage of diabetes we studied, retinal mitochondria undergo adaptations leading to maintain energetic requirements and prevent oxidative stress.     

Postnatal overfeeding in rats leads to moderate overweight and to cardiometabolic and oxidative alterations in adulthood

In contrast to the masses of data on obesity, few data are available concerning the cardiometabolic and oxidative consequences of moderate overweight. The model of postnatal overfeeding (OF) induces an increase in body weight at weaning that remains during adult life.Litters of Wistar rats were either maintained at 12 pups (normal-fed group, NF), or reduced to 3 pups at birth in order to induce OF. At 6 months of age, metabolic parameters, circulating oxidative stress and aortic and coronary vasoreactivity were assessed. Cardiac susceptibility to ischemia-reperfusion injury was also evaluated ex vivo as were markers of cardiac remodeling. OF led to an increase in body weight at weaning (+50%); the increase in body weight persisted throughout adult life, but was less marked (+10%). Significant increases in plasma levels of fasting glucose, insulin and leptin were found in OF rats. An increase in both plasma hydroperoxides and cardiac superoxide dismutase activity and a decrease in plasma ascorbate were found in OF rats. Vasoreactivity was not modified, but ex vivo, after 30 min of ischemia, isolated hearts from OF rats showed lower recovery of coronary flow along with a greater release of LDH. Studies on heart tissues showed an increase in collagen content and increased expression and activity of MMP-2.Our findings show that moderate overweight in adult rats, induced by postnatal overfeeding, leads to both metabolic and oxidative disturbances as well as a higher susceptibility to cardiac injury after ischemia ex vivo, which may be explained, at least in part, by ventricular remodeling.     

Divergent effects of different oxidants on glutathione homeostasis and protein damage in erythrocytes from diabetic patients: effects of high glucose.

Long-term complications of diabetes mellitus have been ascribed to both the effects of prolonged hyperglycemia and to increased oxidative stress. In an attempt to identify the mechanisms underlying the acute effects of hyperglycemia on oxidative stress, we investigated the hypothesis that high glucose might lead to an insufficiency in reducing equivalents (such as NADPH) and thus to a disruption in the glutathione-dependent antioxidant defences and to an incapacity to deal with oxidant attack. For this purpose, erythrocytes from diabetic patients were incubated for 0-90 min in 5.55 or 33.3 mM D-glucose containing tertbutyl hydroperoxide 0.5 and 1 mM, Menadione 100 microM, or glucose oxidase. The time course of the changes in non-protein bound glutathione (reduced and oxidised), lactate and pyruvate, alanine and fluorescent products of oxidative proteolysis, hemolysis and methemoglobin was monitored. The results show that although glucose utilisation was unaffected, all oxidants caused a persistent decrease in total non-protein-bound glutathione suggesting binding to proteins. However, changes in glutathione and redox status differed between the various oxidants and were not directly related to the extent of oxidative cellular damage. In these experimental conditions, with short incubations and using the erythrocyte as the simplest cellular model of glucose metabolism, neither high glucose nor the diabetic condition worsened the susceptibility of erythrocytes to acute in vitro oxidative damage.     

The diabetic rat kidney mediates inosituria and selective urinary partitioning of D-chiro-inositol

Diabetic nephropathy is a serious complication of diabetes mellitus with a pressing need for effective metabolic markers to detect renal impairment. Of potential significance are the inositol compounds, myo-inositol (MI), and the less abundant stereoisomer, D-chiro-inositol (DCI), which are excreted at increased levels in the urine in diabetes mellitus, a phenomenon known as inosituria. There is also a selective urinary excretion of DCI compared to MI. As the biological origins of altered inositol metabolism in diabetes mellitus are unknown, the aim of this study was to determine whether the diabetic kidney was directly responsible. Kidneys isolated from four-week streptozotocin-induced diabetic rats were characterized by a 3-fold reduction in glomerular filtration rate (GFR) compared to matched non-diabetic kidneys. When perfused with fixed quantities of MI (50 µM) and DCI (5 µM) under normoglycemic conditions (5 mM glucose), GFR-normalized urinary excretion of MI was increased by 1.7-fold in diabetic vs. non-diabetic kidneys. By comparison, GFR-normalized urinary excretion of DCI was increased by 4-fold. Perfusion conditions replicating hyperglycemia (20 mM glucose) potentiated DCI but not MI urinary excretion in both non-diabetic and diabetic kidneys. Overall, there was a 2.4-fold increase in DCI urinary excretion compared to MI in diabetic kidneys that was independent of glucose ambience. This increased urinary excretion of DCI and MI in diabetic kidneys occurred despite increased renal expression of the inositol transporters, sodium myo-inositol transporter subtype 1 and 2 (SMIT1 and SMIT2). These findings show that the diabetic kidney primarily mediates inosituria and altered urinary partitioning of MI and DCI. Urinary inositol levels might therefore serve as an indicator of impaired renal function in diabetes mellitus with wider implications for monitoring chronic kidney disease.     

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.     

Effects of Syzygium aromaticum-Derived Triterpenes on Postprandial Blood Glucose in Streptozotocin-Induced Diabetic Rats Following Carbohydrate Challenge

Purpose

Recent reports suggest that the hypoglycaemic effects of the triterpenes involve inhibition of glucose transport in the small intestine. Therefore, the effects of Syzygium spp-derived triterpenes oleanolic acid (OA) and maslinic acid (MA) were evaluated on carbohydrate hydrolyzing enzymes in STZ-induced diabetic rats and consequences on postprandial hyperglycaemia after carbohydrate loading.

Methods

We determined using Western blot analysis the expressions of α-amylase and α-glucosidase and glucose transporters SGLT1 and GLUT2 in the small intestine intestines isolated from diabetic rats treated with OA/MA for 5 weeks. In vitro assays were used to assess the inhibitory activities of OA and MA against α-amylase, α-glucosidase and sucrase.

Results

OA and MA ameliorated postprandial hyperglycemia in carbohydrate loaded diabetic rats as indicated by the significantly small glucose area under the curve (AUC) in treated diabetic animals compared with that in untreated diabetic rats. Western blotting showed that OA and MA treatment not only down-regulated the increase of SGLT1 and GLUT2 expressions in the small intestine of STZ-induced diabetic rats, but also inhibited small intestine α-amylase, sucrase and α-glucosidase activity. IC₅₀ values of OA against α-amylase (3.60 ± 0.18 mmol/L), α-glucosidase (12.40 ± 0.11 mmol/L) and sucrase (11.50 ± 0.13 mmol/L) did not significantly differ from those of OA and acarbose.

Conclusions

The results of suggest that OA and MA may be used as potential supplements for treating postprandial hyperglycemia.

Novelty of the Work

The present observations indicate that besides improving glucose homeostasis in diabetes, OA and MA suppress postprandial hyperglycaemia mediated in part via inhibition of carbohydrate hydrolysis and reduction of glucose transporters in the gastrointestinal tract. Inhibition of α-glucosidase and α-amylase can significantly decrease the postprandial hyperglycaemia after a mixed carbohydrate diet and therefore can be an important strategy in the management of postprandial blood glucose levels in NIDDM patients.     

Short-term exposure of high glucose concentration induces generation of reactive oxygen species in endothelial cells: implication for the oxidative stress associated with postprandial hyperglycemia

Recent studies demonstrating a close relationship between postprandial hyperglycemia and the incidence of atherosclerotic cardiovascular disease prompted us to investigate the generation and source of reactive oxygen species (ROS) in endothelial cells stimulated by short-term exposure to a high glucose concentration. In addition, we investigated the effect of insulin on ROS production induced by high glucose concentration. Cultured bovine aortic endothelial cells demonstrated a significant increase in intracellular ROS generation after a 3-h exposure to 25 mM glucose (131.4% versus 5 mM glucose). This increased generation of ROS was suppressed by an inhibitor of NAD(P)H oxidase. Intracellular ROS production in cells exposed to 3 h of high glucose concentration was increased significantly by the presence of a physiological concentration of insulin. However, after a 1-h exposure to high glucose levels, ROS generation in cells incubated with insulin was only about 80% of that measured in cells incubated without insulin. The generation of intracellular nitric oxide (NO) resulting from an acute insulin effect may account for this difference. In conclusion, acute hyperglycemia itself may possibly cause endothelial oxidative stress in patients with postprandial hyperglycemia. Endothelial oxidative stress may be determined by the interaction between NO and superoxide generation.     

Reducing oxidative stress in patients with type 2 diabetes mellitus: A primary care call to action

Background: Oxidative stress is believed to be the primary cause of the microvascular and macrovascular complications of type 2 diabetes mellitus (DM).Objective: This paper examines the evidence linking oxidative stress with long-term complications of type 2 DM and explores methods to minimize its effect.Methods: A literature search was performed to identify relevant studies for this review. Articles published in English from 2000 to 2008 were identified through searches of PubMed, Diabetes Care, and Google using the search terms oxidative stress, postprandial hyperglycemia, ACCORD Trial, and endothelial cell dysfunction.Results: The literature search identified 423 articles. Although chronic hyperglycemia can be effectively monitored and targeted using glycosylated hemoglobin concentrations, postprandial glucose levels are also important. Postprandial glucose excursions are exhibited by almost all patients with type 2 DM and are independent risk factors for cardiovascular morbidity and mortality. Furthermore, glucose fluctuations during the postprandial period elicit more oxidative stress than chronic, sustained hyperglycemia and can lead to endothelial dysfunction, vascular inflammation, and microvascular complications. In turn, endothelial dysfunction has been implicated in the development of vascular pathologies such as atherosclerosis. Pharmacologic interventions (eg, rapid-acting insulin analogues that target post-prandial glucose excursions) reduce oxidative stress and vascular inflammation and improve endothelial dysfunction.Conclusions: Given the important role of oxidative stress in the development of complications of type 2 DM, physi-cians should consider methods to reduce oxidative stress that may occur during both acute (postprandial) and chronic hyperglycemia. One critical aspect is to reduce postprandial glucose levels to <180 mg/dL while lowering fasting glucose levels to <110 mg/dL. By coaching patients to reach these goals, physicians and other health care professionals can minimize the risk of long-term complications of type 2 DM.     

Diabetes and mitochondrial oxidative stress: A study using heart mitochondria from the diabetic Goto-Kakizaki rat

Increasing evidence shows that the overproduction of reactive oxygen species, induced by diabetic hyperglycemia, contributes to the development of several cardiopathologies. The susceptibility of diabetic hearts to oxidative stress, induced in vitro by ADP-Fe²⁺ in mitochondria, was studied in 12-month-old Goto-Kakizaki rats, a model of non-insulin dependent diabetes mellitus, and normal (non-diabetic) Wistar rats. In terms of lipid peroxidation the oxidative damage was evaluated on heart mitochondria by measuring both the O₂ consumption and the concentrations of thiobarbituric acid reactive substances. Diabetic rats display a more intense formation of thiobarbituric acid reactive substances and a higher O₂ consumption than non-diabetic rats. The oxidative damage, assessed by electron microscopy, was followed by an extensive effect on the volume of diabetic heart mitochondria, as compared with control heart mitochondria. An increase in the susceptibility of diabetic heart mitochondria to oxidative stress can be explained by reduced levels of endogenous antioxidants, so we proceeded in determinating -tocopherol, GSH and coenzyme Q content. Although no difference of -tocopherol levels was found in diabetic rats as compared with control rat mitochondria, a significant reduction in GSH (21.5% reduction in diabetic rats) and coenzyme Q levels of diabetic rats was observed. The data suggest that a significant decrease of coenzyme Q₉, a potent antioxidant involved in the elimination of mitochondria-generated reactive oxygen species, may be responsible for an increased susceptibility of diabetic heart mitochondria to oxidative damage.     

Effect of vitamin E on the response to ischemia-reperfusion of Langendorff heart preparations from hyperthyroid rats

Hyperthyroidism has been reported to decrease heart antioxidant capacity and increase its susceptibility to in vitro oxidative stress. This may affect the heart response to ischemia-reperfusion, a condition that increases free radical production. We compared the functional recovery from in vitro ischemia-reperfusion (Langendorff) of hearts from euthyroid (E), hyperthyroid (H, ten daily intraperitoneal injections of T3, 10 microg/100g body weight), vitamin E-treated (VE, ten daily intramuscular injections, 20 mg/100g body weight) and hyperthyroid vitamin E-treated (HVE) rats. We also determined lipid peroxidation, tissue antioxidant capacity and the tissue capability to face an oxidative stress in vitro. A significant tachycardia was displayed during reperfusion following 20 min ischemia by the hyperthyroid hearts, together with a low recovery of left ventricular developed pressure (LVDP) and left ventricular dP/dt(max). When H hearts were paced at 300 beats/min, the functional recovery (LVDP and dP/dt(max)) was close to 100% and significantly higher than in E paced hearts. At the end of the ischemia-reperfusion protocol, myocardium antioxidant capacity was significantly lower, whereas lipid peroxidation and the susceptibility to in vitro oxidative stress were higher in the T3 treated (H) than in euthyroid rats. The in vitro tachycardic response, the reduction in the antioxidant capacity and the increase in lipid peroxidation were prevented by treatment of hyperthyroid rats with vitamin E (HVE). These results suggest that the tachycardic response to reperfusion following chronic T3 pretreatment was associated with the reduced capability of the heart to face oxidative stresses in hyperthyroidism.     

Activation of the hexosamine biosynthesis pathway and protein <Emphasis Type="Italic">O</Emphasis>-GlcNAcylation modulate hypertrophic and cell signaling pathways in cardiomyocytes from diabetic mice

Patients with diabetes have a much greater risk of developing heart failure than non-diabetic patients, particularly in response to an additional hemodynamic stress such as hypertension or infarction. Previous studies have shown that increased glucose metabolism via the hexosamine biosynthesis pathway (HBP) and associated increase in O-linked-β-N-acetylglucosamine (O-GlcNAc) levels on proteins contributed to the adverse effects of diabetes on the heart. Therefore, in this study we tested the hypothesis that diabetes leads to impaired cardiomyocyte hypertrophic and cell signaling pathways due to increased HBP flux and O-GlcNAc modification on proteins. Cardiomyocytes isolated from type 2 diabetic db/db mice and non-diabetic controls were treated with 1 μM ANG angiotensin II (ANG) and 10 μM phenylephrine (PE) for 24 h. Activation of hypertrophic and cell signaling pathways was determined by assessing protein expression levels of atrial natriuretic peptide (ANP), α-sarcomeric actin, p53, Bax and Bcl-2 and phosphorylation of p38, ERK and Akt. ANG II and PE significantly increased levels of ANP and α-actin and phosphorylation of p38 and ERK in the non-diabetic but not in the diabetic group; phosphorylation of Akt was unchanged irrespective of group or treatment. Constitutive Bcl-2 levels were lower in diabetic hearts, while there was no difference in p53 and Bax. Activation of the HBP and increased protein O-GlcNAcylation in non-diabetic cardiomyocytes exhibited a significantly decreased hypertrophic signaling response to ANG or PE compared to control cells. Inhibition of the HBP partially restored the hypertrophic signaling response of diabetic cardiomyocytes. These results suggest that activation of the HBP and protein O-GlcNAcylation modulates hypertrophic and cell signaling pathways in type 2 diabetes.     

Chronic hyperglycemia reduces substrate oxidation and impairs metabolic switching of human myotubes

Skeletal muscle of insulin resistant individuals is characterized by lower fasting lipid oxidation and reduced ability to switch between lipid and glucose oxidation. The purpose of the present study was to examine if chronic hyperglycemia would impair metabolic switching of myotubes. Human myotubes were treated with or without chronic hyperglycemia (20 mmol/l glucose for 4 days), and metabolism of [¹⁴C]oleic acid (OA) and [¹⁴C]glucose was studied. Myotubes exposed to chronic hyperglycemia showed a significantly reduced OA uptake and oxidation to CO₂, whereas acid-soluble metabolites were increased compared to normoglycemic cells (5.5 mmol/l glucose). Glucose suppressibility, the ability of acute glucose (5 mmol/l) to suppress lipid oxidation, was 50% in normoglycemic cells and reduced to 21% by hyperglycemia. Adaptability, the capacity to increase lipid oxidation with increasing fatty acid availability, was not affected by hyperglycemia. Glucose uptake and oxidation were reduced by about 40% after hyperglycemia, and oxidation of glucose in presence of mitochondrial uncouplers showed that net and maximal oxidative capacities were significantly reduced. Hyperglycemia also abolished insulin-stimulated glucose uptake. Moreover, ATP concentration was reduced by 25% after hyperglycemia. However, none of the measured mitochondrial genes were downregulated nor was mitochondrial DNA content. Microarray and real-time RT-PCR showed that no genes were significantly regulated by chronic hyperglycemia. Addition of chronic lactate reduced both glucose and OA oxidation to the same extent as hyperglycemia. In conclusion, chronic hyperglycemia reduced substrate oxidation in skeletal muscle cells and impaired metabolic switching. The effect is most likely due to an induced mitochondrial dysfunction.     

Ursolic acid regulates high glucose-induced apoptosis

A high concentration of glucose has been implicated as a causal factor in initiation and progression of diabetic complications and there is evidence to suggest that hyperglycemia increases the production of free radicals and oxidative stress. Therefore, compounds that scavenge reactive oxygen species (ROS) may confer regulatory effects on high glucose-induced apoptosis. Ursolic acid (UA), a pentacyclic triterpene, is reported to have an antioxidant activity. We investigated the effect of UA on high glucose-induced apoptosis in U937 cells. Upon exposure to 35 mM glucose for two days, there was a distinct difference between untreated cells and cells pre-treated with 50 nM UA for 2 h in regard to cellular redox status and oxidative DNA damage to cells. UA pre-treated cells showed significant suppression of apoptotic features such as DNA fragmentation, damage to mitochondrial function and modulation of apoptotic marker proteins upon exposure to high glucose. This study indicates that UA may play an important role in regulating the apoptosis induced by high glucose presumably through scavenging of ROS.     

Naringenin alleviates hyperglycemia-induced renal toxicity by regulating activating transcription factor 4–C/EBP homologous protein mediated apoptosis

Endoplasmic reticulum (ER) dysfunction plays a prominent role in the pathophysiology of diabetic nephropathy (DN). This study aimed to investigate the novel role of Naringenin (a flavanone mainly found in citrus fruits) in modulating ER stress in hyperglycemic NRK 52E cells and STZ/nicotinamide induced diabetes in Wistar rats. The results demonstrated that Naringenin supplementation downregulated the expression of ER stress marker proteins, including p-PERK, p-eIF2α, XBP1s, ATF4 and CHOP during hyperglycemic renal toxicity in vitro and in vivo. Naringenin abrogated hyperglycemia-induced ultrastructural changes in ER, evidencing its anti-ER stress effects. Interestingly, treatment of Naringenin prevented nuclear translocation of ATF4 and CHOP in hyperglycemic renal cells and diabetic kidneys. Naringenin prevented apoptosis in hyperglycemic renal cells and diabetic kidney tissues by downregulating expression of apoptotic marker proteins. Further, photomicrographs of TEM confirmed anti-apoptotic potential of Naringenin as it prevented membrane blebbing and formation of apoptotic bodies in hyperglycemic renal cells. Naringenin improved glucose tolerance, restored serum insulin level and reduced serum glucose level in diabetic rats evidencing its anti-hyperglycemic effects. Histopathological examination of kidney tissues also confirmed prevention of damage after 28 days of Naringenin treatment in diabetic rats. Additionally, Naringenin diminished oxidative stress and improved antioxidant defense response during hyperglycemic renal toxicity. Taken together, our study revealed a novel role of Naringenin in ameliorating ER stress during hyperglycemic renal toxicity along with prevention of apoptosis, cellular and tissue damage. The findings suggest that prevention of ER stress can be exploited as a novel approach for the management of hyperglycemic nephrotoxicity. Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00644-0.     

Acute glucose fluctuations and chronic sustained hyperglycemia as risk factors for cardiovascular diseases in patients with type 2 diabetes.

Chronic hyperglycemia, usually assessed from HbA1c determinations, results in excessive glycation and generation of oxidative stress. As a consequence, chronic hyperglycemia has been identified as a risk factor for diabetes complications leading to accelerated atherosclerosis. Both fasting and postprandial hyperglycemia contribute to this process. However the acute glucose fluctuations that occur in diabetes have been recently described as an additional factor that activates the oxidative stress. As a consequence, acute glucose swings, including upward (postprandial) and downward (interprandial) fluctuations can be considered as risk factors for cardiovascular events and should be included in the "dysglycemia" of diabetes in combination with fasting and postprandial hyperglycemia. As postprandial glucose is a contributor of both acute glucose fluctuations and chronic sustained hyperglycemia, it remains difficult to know whether these 2 mechanisms are equivalent or not equivalent risk factors for cardiovascular disease.     

Oxidative,Metabolic, and Apoptotic Responses of Schwann Cells to High Glucose Levels

The specific response of murine Schwann cells IMS32 to acute and chronic hyperglycemia conditions was evaluated. The pathophysiological alterations were studied to deepening the role of Schwann cells in diabetes‐related neurotoxicity and to assess a model to screen new protective molecules. IMS32 were incubated with 30 and 56 mM glucose for 48 h and 7 and 14 days, and markers of oxidative stress, apoptosis, and polyol pathway were evaluated. High glucose induced O²‐production and lipid peroxidation at all time point whereas Caspase 3 activity was induced only after 14 days. Aldose reductase activity and expression were significantly increased after 48 h and 14 days, respectively. Our results describe the response of Schwann cells to high glucose conditions and suggest the use of IMS32 for the screening of protective molecules in diabetes‐induced neuropathy.     

Hyperthermia-induced Hsp90·eNOS Preserves Mitochondrial Respiration in Hyperglycemic Endothelial Cells by Down-regulating Glut-1 and Up-regulating G6PD Activity

Uncoupling of NO production from NADPH oxidation by endothelial nitric-oxide synthase (eNOS) is enhanced in hyperglycemic endothelium, potentially due to dissociation of heat shock proteins 90 (Hsp90), and cellular glucose homeostasis is enhanced by a ROS-induced positive feed back mechanism. In this study we investigated how such an uncoupling impacts oxygen metabolism and how the oxidative phosphorylation can be preserved by heat shock (42 °C for 2 h, hyperthermia) in bovine aortic endothelial cells. Normal and heat-shocked bovine aortic endothelial cells were exposed to normoglycemia (NG, 5.0 mm) or hyperglycemia (30 mm). With hyperglycemia treatment, O₂ consumption rate was reduced (from V_O2max = 7.51 ± 0.54 to 2.35 ± 0.27 mm Hg/min/10⁶ cells), whereas in heat-shocked cells, O₂ consumption rate remained unaltered (8.19 ± 1.01 mm Hg/min/10 × 10⁶ cells). Heat shock was found to enhance Hsp90/endothelial NOS interactions and produce higher NO. Moreover, ROS generation in the hyperglycemic condition was also reduced in heat-shocked cells. Interestingly, glucose uptake was reduced in heat-shocked cells as a result of decrease in Glut-1 protein level. Glucose phosphate dehydrogenase activity that gives rise to NADPH generation was increased by hyperthermia, and mitochondrial oxidative metabolism was preserved. In conclusion, the present study provides a novel mechanism wherein the reduced oxidative stress in heat-shocked hyperglycemic cells down-regulates Glut-1 and glucose uptake, and fine-tuning of this pathway may be a potential approach to use for therapeutic benefit of diabetes mellitus.     

N-acetylcysteine and taurine prevent hyperglycemia-induced insulin resistance in vivo: possible role of oxidative stress

Exposure to high concentrations of glucose and insulin results in insulin resistance of metabolic target tissues, a characteristic feature of type 2 diabetes. High glucose has also been associated with oxidative stress, and increased levels of reactive oxygen species have been proposed to cause insulin resistance. To determine whether oxidative stress contributes to insulin resistance induced by hyperglycemia in vivo, nondiabetic rats were infused with glucose for 6 h to maintain a circulating glucose concentration of 15 mM with and without coinfusion of the antioxidant N-acetylcysteine (NAC), followed by a 2-h hyperinsulinemic-euglycemic clamp. High glucose (HG) induced a significant decrease in insulin-stimulated glucose uptake [tracer-determined disappearance rate (Rd), control 41.2 +/- 1.7 vs. HG 32.4 +/- 1.9 mg. kg-1. min-1, P < 0.05], which was prevented by NAC (HG + NAC 45.9 +/- 3.5 mg. kg-1. min-1). Similar results were obtained with the antioxidant taurine. Neither NAC nor taurine alone altered Rd. HG caused a significant (5-fold) increase in soleus muscle protein carbonyl content, a marker of oxidative stress that was blocked by NAC, as well as elevated levels of malondialdehyde and 4-hydroxynonenal, markers of lipid peroxidation, which were reduced by taurine. In contrast to findings after long-term hyperglycemia, there was no membrane translocation of novel isoforms of protein kinase C in skeletal muscle after 6 h. These data support the concept that oxidative stress contributes to the pathogenesis of hyperglycemia-induced insulin resistance.