Chronic High Glucose Research Articles

18F-FDGPET/CT: diabetes and hyperglycaemia

Some patients who undergo 18F-FDG PET/CTfor neoplastic or benign disease are also affected by diabetes or hyperglycaemia. We propose different preparation procedures inpatients (pts) with hyperglycaemia (acute, temporary or chronic)or diabetes (type 1 or 2) at the time of the 18F-FDG injection, in order to improve the diagnostic scheduling of 18F-FDG PET/CT. We evaluated a sample of 13,063 pts, examined in two different PET/CT centres, one with a stationary scanner (94.4%) and the other with a mobile device (5.6%). High blood sugar was present in 1,698 patients (13%) at the time of the 18F-FDG injection (hyperglycaemia was defined as fasting blood glucose > 11.1 mmol/l). We considered all 18F-FDG PET/CT tests performed over a period of 4 years (2006-2009). In the first 2 years (6,236 tests), scheduling was done directly by the administrative secretary. In the next two years, 6,827 pts underwent a preliminary visit to assess the test indications, medical history, and therapy as well as pre-test preparation. We evaluated different preparation protocols for hyperglycaemic or diabetic pts, especially those recommended in the guidelines of the European Association of Nuclear Medicine (EANM) and Society of Nuclear Medicine (SNM). In the four-year period, 713/13,063 patients (5.45%)were rescheduled; of these, 78.8% were rescheduled in the two years before the implementation of our preparation protocols and 21.2% in the next two years.Before the implementation of our preparation protocols, 562 patients (9%) presented occasional, acute or chronic hyperglycaemia (56.7%), or diabetes (43.3%), requiring postponement of the test to a later date. The test was not performed in 17 of 6,236 pts (0.27%) because of blood glucose levels above 11.1 mmol/l for several days, while in 16/6236 pts (0.26%) the18F-FDG injection was performed despite high blood glucose levels, in view of the clinical urgency.After the implementation of the preparation protocols, 2.2% of pts were rescheduled because of occasional, acute or chronic hyperglycaemia (79%), or diabetes (21%); 0.1% of pts did not undergo the test because of chronic high blood glucose levels. Although the administration of insulin is recommended in theEANM and SNM guidelines, in our new preparation procedures experience it was not necessary, because we reduced the numbers of hyperglycaemic pts thanks to screening at the preliminary visit and a subsequent good preparation of the patient before scheduling. The application of our preparation protocols improves the on-time performance and diagnostic accuracy,and increases patients' compliance.

Advances in molecular mechanisms of pancreatic beta-cell glucotoxicity

The deterioration of beta-cell function and survival caused by chronic high glucose exposure is termed glucotoxicity.The mechanisms of pancreatic beta-cell glucotoxicity involve in oxidative stress,endoplasmic reticulum stress,mitochrondrial dysfunction,inflammation,advanced glycation end products,hypoxia and activation of HIF-1 α,and loss of differentiation in the alteration of beta-cell phenotype.The role of oxidative stress,endoplasmic reticulum stress,and loss of differentiation in the alteration of beta-cell phenotype is well ascertained.Oxidative stress is the central mechanism for glucotoxicity in pancreatic beta cells in diabetes. Key words: Pancreatic beta-cell; Glucotoxicity; Molecular mechanism

Chronic ethanol and high glucose inducible CYP2E1 mediated oxidative stress leads to greater cellular injury in VL-17A cells: a potential mechanism for liver injury due to chronic alcohol consumption and hyperglycemia

Diabetes, characterized by the presence of inherent oxidative stress, may be further complicated by the additional oxidative stress generated due to the metabolism of alcohol. This study focuses on the roles of alcohol and high glucose inducible ADH and CYP2E1, both of which function as ethanol metabolizing enzymes, in promoting oxidative stress and cellular damage under chronic alcohol plus hyperglycemic conditions in VL-17A cells over-expressing ADH and CYP2E1. A specific CYP2E1 inhibitor, diallyl sulfide, proved to be more effective in decreasing the toxicity in VL-17A cells exposed to chronic alcohol plus high glucose than the specific ADH inhibitor pyrazole and the dual ADH and CYP2E1 inhibitor 4-methyl pyrazole. Furthermore, the greatest decrease in several parameters for oxidative stress such as ROS level, lipid peroxidation, protein carbonyl and protein aldehyde adduct formation and apoptosis was observed in the diallyl sulfide plus chronic alcohol plus high glucose treated VL-17A cells. In addition, specific inhibition of CYP2E1 with phenethyl isothiocyanate or CYP2E1 siRNA led to significant restoration of viability, and decrease in oxidative stress and apoptosis. Thus, the inducibility of CYP2E1 with both ethanol and high glucose leads to greater oxidative stress and cellular dysfunction in liver.

Calcium Efflux From the Endoplasmic Reticulum Leads to β-Cell Death

It has been established that intracellular calcium homeostasis is critical for survival and function of pancreatic β-cells. However, the role of endoplasmic reticulum (ER) calcium homeostasis in β-cell survival and death is not clear. Here we show that ER calcium depletion plays a critical role in β-cell death. Various pathological conditions associated with β-cell death, including ER stress, oxidative stress, palmitate, and chronic high glucose, decreased ER calcium levels and sarcoendoplasmic reticulum Ca(2+)-ATPase 2b expression, leading to β-cell death. Ectopic expression of mutant insulin and genetic ablation of WFS1, a causative gene for Wolfram syndrome, also decreased ER calcium levels and induced β-cell death. Hyperactivation of calpain-2, a calcium-dependent proapoptotic protease, was detected in β-cells undergoing ER calcium depletion. Ectopic expression of sarcoendoplasmic reticulum Ca(2+)-ATPase 2b, as well as pioglitazone and rapamycin treatment, could prevent calcium efflux from the ER and mitigate β-cell death under various stress conditions. Our results reveal a critical role of ER calcium depletion in β-cell death and indicate that identification of pathways and chemical compounds restoring ER calcium levels will lead to novel therapeutic modalities and pharmacological interventions for type 1 and type 2 diabetes and other ER-related diseases including Wolfram syndrome.

P 12: Chronic and oscillating high glucose induce, through oxidative stress, A “memory” effect for endoglin and NRF-2-downstream genes in endothelial cells

Background and aims: Hyperglycaemia produces endothelial dysfunction, and the so called “Metabolic Memory”, convincingly through the generation of oxidative stress. Endoglin (Eng), the TGF-β co-receptor, may drive angiogenesis and vascular remodelling in the endothelial cells, regulating two divergent effects of the TGF-β signalling: proliferative effects (through Alk1-smad1/5) or pro-apoptotic signals (through Alk5-smad2/3). Many studies have shown this role for Eng but little is known in the diabetes-associated pathologies. Recently, a correlation between the soluble form of Eng (s-Eng) and glycaemia and HbA1c has been reported as well as its involvement in diabetic nephropathy and in diabetes-associated hypertension. The goal of the present study was to evaluate if high glucose (chronic and oscillating) may induce Eng over-expression in cultured endothelial cells, and if it could be also accompanied by a “Memory” effect. Furthermore, the possible role of oxidative damage has been studied, evaluating the antioxidant intracellular defence mediated by the detoxification enzyme NQO-1, and by the oxidative stress-related protein HO-1. The effect of the antioxidant α-lipoic acid on them was also evaluated. Primary Human Umbilical Vein Endothelial cells (HUVEC) were incubated in different conditions: for 3 weeks in normal glucose (NG, 5 mmol/l), chronic high glucose (HG, 25 mmol/l), and oscillating glucose (OG, 5/25 mmol/l); for 1 week in constant 5 mmol/l glucose after being exposed for 2 weeks to continuous high glucose (25 mmol/l, HG-M) and oscillating glucose (5/25 mmol/l every 24 h, OsG-M). The impact of α-lipoic acid (ALA, 62.5 μM) was also measured. mRNA and protein levels of Nrf2-dependent ARE-responsive genes (hmox1 and nqo1) and Endoglin were measured using real-time PCR, Western blot and quantified in the standard method. All analyses were evaluated for statistical significance. Eng mRNA expression was more elevated in oscillating than in high glucose (NG-OG, p=0.006; NG-HG, p=0.01). Endoglin protein level was found to be upregulated in oscillating glucose (3.5-fold) respect to basal conditions. These increases in Endoglin were reduced with α-lipoic acid treatment both in mRNA (HG-HG+, r=0.98, p=0.001) and protein levels. Elevation in mRNA expression of cytoprotective genes hmox-1 (NG-OG, p=0,005) and nqo1 (NG-OG, p=0,007) were found, but the ALA-treatment induced a more distinct expression of HO-1 (2-fold) and NQO1 (2.5-fold) gene levels, in oscillating glucose. The similar trend was also assessed for protein expression. A “memory” effect was also detected for nqo-1 (NG-OsG-M, p=0,01), hmox-1 (NG-OsG-M, p=0,05) and Eng (NG-OsG-M, p=0,04). These data show that both oscillating and high glucose induce expression of Eng, suggesting the TGF-β signalling implication on endothelial cells regulation. The memory effect was assessed for Eng, hmox1 and nqo1, being oscillating glucose more dangerous. Moreover, the ALA treatment induced expression of Nrf-2 related genes suggesting a role for oxidative stress in such phenomenon. Finally, the findings establish Eng as a critical regulator of endothelial cells in oscillating and high glucose, and that Lipoic Acid positively influences Nrf2-driven gene expression of defence mechanisms against ROS.

PROFIBROTIC INFLUENCE OF HIGH GLUCOSE ON HUMAN CARDIAC FIBROBLAST FUNCTIONS: EFFECTS OF 606A AND IMIDAPRILAT

ObjectivesRecent studies have demonstrated an important role of chronic high glucose concentration for collagen deposition in fibroblasts. However, little is known about the action of angiotensin II type 1 receptor...

The influence of high glucose on the aerobic metabolism of endothelial EA.hy926 cells

The endothelium is considered to be relatively independent of the mitochondrial energy supply. The goals of this study were to examine mitochondrial respiratory functions in endothelial cells and isolated mitochondria and to assess the influence of chronic high glucose exposure on the aerobic metabolism of these cells. A procedure to isolate of bioenergetically active endothelial mitochondria was elaborated. Human umbilical vein endothelial cells (EA.hy926 line) were grown in medium containing either 5.5 or 25 mM glucose. The respiratory response to elevated glucose was observed in cells grown in 25 mM glucose for at least 6 days or longer. In EA.hy926 cells, growth in high glucose induced considerably lower mitochondrial respiration with glycolytic fuels, less pronounced with glutamine, and higher respiration with palmitate. The Crabtree effect was observed in both types of cells. High glucose conditions produced elevated levels of cellular Q10, increased ROS generation, increased hexokinase I, lactate dehydrogenase, acyl-CoA dehydrogenase, uncoupling protein 2 (UCP2), and superoxide dismutase 2 expression, and decreased E3-binding protein of pyruvate dehydrogenase expression. In isolated mitochondria, hyperglycaemia induced an increase in the oxidation of palmitoylcarnitine and glycerol-3-phosphate (lipid-derived fuels) and a decrease in the oxidation of pyruvate (a mitochondrial fuel); in addition, increased UCP2 activity was observed. Our results demonstrate that primarily glycolytic endothelial cells possess highly active mitochondria with a functioning energy-dissipating pathway (UCP2). High-glucose exposure induces a shift of the endothelial aerobic metabolism towards the oxidation of lipids and amino acids.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-012-1156-1) contains supplementary material, which is available to authorized users.

Neferine Inhibits the Upregulation of CCL5 and CCR5 in Vascular Endothelial Cells During Chronic High Glucose Treatment

We investigated whether the expressions of CCL5 and CCR5 participate in dysfunctional changes in human umbilical vein endothelial cells (HUVECs) induced by chronic high glucose treatment and examined whether neferine exerts its therapeutic effects by blocking the development of dysfunctional vascular endothelium. HUVECs were cultured with control or high concentrations of glucose in the absence or presence of neferine for 5days. Nitric acid reductase method was used to detect the concentration of nitric oxide (NO) released into culture media. The level of intracellular reactive oxygen species (ROS) was measured by fluorescent DCFH-DA probe. The expressions of 84 genes related to endothelial cell biology were assessed by Human Endothelial Cell Biology RT(2) Profiler PCR Array. The expressions of the chemokine CCL5 and its receptor CCR5 were further determined by real-time RT-PCR and western blotting. PCR array indicated that CCL5 was the most significantly upregulated when HUVECs were exposed to chronic high glucose; the intracellular ROS level and the expressions of CCL5 and CCR5 at both mRNA and protein levels were significantly increased, whereas NO production was decreased simultaneously. The increased level of ROS and elevated expressions of CCL5 and CCR5 at high glucose were significantly inhibited by neferine; meanwhile the decreased NO production upon chronic high glucose treatment was relieved. An antioxidant (vitamin E) exerted similar beneficial effects. These data indicate that neferine can reduce the upregulation of CCL5 and CCR5 of vascular endothelium exposure to chronic high glucose and prevent or inhibit subsequent occurrence of inflammation in blood vessels possibly through antioxidation.

High Glucose Predisposes Gene Expression and ERK Phosphorylation to Apoptosis and Impaired Glucose-Stimulated Insulin Secretion via the Cytoskeleton

Chronic high glucose (HG) inflicts glucotoxicity on vulnerable cell types such as pancreatic β cells and contributes to insulin resistance and impaired insulin secretion in diabetic patients. To identify HG-induced cellular aberrations that are candidate mediators of glucotoxicity in pancreatic β cells, we analyzed gene expression in ERoSHK6, a mouse insulin-secreting cell line after chronic HG exposure (six-day exposure to 33.3 mM glucose). Chronic HG exposure which reduced glucose-stimulated insulin secretion (GSIS) increased transcript levels of 185 genes that clustered primarily in 5 processes namely cellular growth and proliferation; cell death; cellular assembly and organization; cell morphology; and cell-to-cell signaling and interaction. The former two were validated by increased apoptosis of ERoSHK6 cells after chronic HG exposure and reaffirmed the vulnerability of β cells to glucotoxicity. The three remaining processes were partially substantiated by changes in cellular morphology and structure, and instigated an investigation of the cytoskeleton and cell-cell adhesion. These studies revealed a depolymerized actin cytoskeleton that lacked actin stress fibers anchored at vinculin-containing focal adhesion sites as well as loss of E-cadherin-mediated cell-cell adherence after exposure to chronic HG, and were concomitant with constitutive ERK1/2 phosphorylation that was refractory to serum and glucose deprivation. Although inhibition of ERK phosphorylation by PD98059 promoted actin polymerization, it increased apoptosis and GSIS impairment. These findings suggest that ERK phosphorylation is a proximate regulator of cellular processes targeted by chronic HG-induced gene expression and that dynamic actin polymerization and depolymerization is important in β cell survival and function. Therefore, chronic HG alters gene expression and signal transduction to predispose the cytoskeleton towards apoptosis and GSIS impairment.

Adaptive metabolic response to 4 weeks of sugar-sweetened beverage consumption in healthy, lightly active individuals and chronic high glucose availability in primary human myotubes

Chronic sugar-sweetened beverage (SSB) consumption is associated with obesity and type 2 diabetes mellitus (T2DM). Hyperglycaemia contributes to metabolic alterations observed in T2DM, such as reduced oxidative capacity and elevated glycolytic and lipogenic enzyme expressions in skeletal muscle tissue. We aimed to investigate the metabolic alterations induced by SSB supplementation in healthy individuals and to compare these with the effects of chronic hyperglycaemia on primary muscle cell cultures. Lightly active, healthy, lean subjects (n = 11) with sporadic soft drink consumption underwent a 4-week SSB supplementation (140 ± 15 g/day, ~2 g glucose/kg body weight/day, glucose syrup). Before and after the intervention, body composition, respiratory exchange ratio (RER), insulin sensitivity, muscle metabolic gene and protein expression were assessed. Adaptive responses to hyperglycaemia (7 days, 15 mM) were tested in primary human myotubes. SSB supplementation increased fat mass (+1.0 kg, P < 0.05), fasting RER (+0.12, P < 0.05), fasting glucose (+0.3 mmol/L, P < 0.05) and muscle GAPDH mRNA expressions (+0.94 AU, P < 0.05). PGC1α mRNA was reduced (-0.20 AU, P < 0.05). Trends were found for insulin resistance (+0.16 mU/L, P = 0.09), and MondoA protein levels (+1.58 AU, P = 0.08). Primary myotubes showed elevations in GAPDH, ACC, MondoA and TXNIP protein expressions (P < 0.05). Four weeks of SSB supplementation in healthy individuals shifted substrate metabolism towards carbohydrates, increasing glycolytic and lipogenic gene expression and reducing mitochondrial markers. Glucose-sensing protein MondoA might contribute to this shift, although further in vivo evidence is needed to corroborate this.

In Vitro Evidence for Chronic Alcohol and High Glucose Mediated Increased Oxidative Stress and Hepatotoxicity

Hyperglycemia or alcoholism can lead to impaired liver functions. Cytochrome P450 2E1 (CYP2E1) is elevated in hyperglycemia or alcoholism and plays a critical role in generating oxidative stress in the cell. In the present study, we have used VL-17A cells that overexpress the alcohol metabolizing enzymes [alcohol dehydrogenase (ADH) and CYP2E1] to investigate the toxicity due to ethanol (EtOH) plus high glucose. Toxicity was assessed through viability assay and amount of acetaldehyde adduct formation. Oxidative stress parameters included measuring reactive oxygen species (ROS) levels and malondialdehyde adduct formation. Apoptosis was determined through caspase-3 activity, Annexin V- Propidium iodide staining, and changes in mitochondrial membrane potential. The effects of antioxidants and specific inhibitors of ADH and CYP2E1 on cell viability and ROS levels were also studied. When present together, EtOH plus high glucose-treated VL-17A cells exhibited greater oxidative stress and toxicity than other groups. Apoptosis was observed in liver cells treated with the toxins, and the EtOH plus high glucose-treated VL-17A cells exhibited apoptosis to the largest extent. A distinct and graded increase in CYP2E1 level occurred in the different groups of VL-17A cells. Further, antioxidants or inhibitors of ADH and CYP2E1 were effective in decreasing the observed oxidative stress and toxicity. The combined oxidative insult due to alcohol plus high glucose leads to greater liver injury, which may prove to be a timely warning for the injurious effects of alcohol consumption in diabetics.

Chronic high glucose downregulates mitochondrial calpain 10 and contributes to renal cell death and diabetes-induced renal injury

Whereas most calpains are cytosolic proteases, calpain 10 is resident in mitochondria and is important in mitochondrial homeostasis. Because calpain 10 has been implicated in type 2 diabetes, we studied its possible role in diabetes-induced renal dysfunction. We treated renal proximal tubular cells with high glucose (17 mmol/l) and found decreased mitochondrial calpain 10 mRNA and protein at 96 h compared with cells incubated with 0 or 5 mmol/l glucose or 17 mmol/l D-mannitol. High glucose increased mitochondrial calpain 10 substrates (NDUFB8 and ATP synthase β), decreased basal and uncoupled respiration, and initiated cell apoptosis as indicated by cleaved caspase 3 and nuclear condensation. Renal calpain 10 protein and mRNA were specifically decreased in streptozotocin-induced diabetic rats with kidney dysfunction, and in diabetic ob/ob mice. In agreement with our in vitro data, the kidneys of streptozotocin-induced diabetic rats had elevated calpain 10 substrates and cleaved caspase 3. Finally, specific siRNA-induced knockdown of calpain 10 in the proximal tubules of control rats resulted in decreased renal function as evidenced by increased serum creatinine, and increased caspase 3 cleavage compared with rats receiving scrambled siRNA. Thus, the glucose-induced loss of calpain 10 in vivo results in renal cell apoptosis and organ failure through accumulation of mitochondrial calpain 10 substrates and mitochondrial dysfunction. Whether this is a major cause of the decreased renal function in diabetic nephropathy will require further studies.

Impaired endothelium-dependent and -independent relaxation of aorta from diabetic rats

Vascular complication in diabetes has been reported to be due to the effects of chronic high blood glucose on the vascular system. Different relaxation mechanisms exist in the vasculature and effect of chronic high glucose on vascular relaxation mechanisms is not clearly understood. We assessed the effect of streptozotocin (STZ, 70 mg/kg, for 12 wks)-induced diabetes on vascular reactivity to isoproterenol (Isop, 10-9-10-5 M), a cAMP-dependent agent, acetylcholine (ACh, 10-9-10-5 M), a stimulant of NO (nitric oxide) synthase, sodium nitroprusside (SNP, 10-10-10-5 M), NO donor, or bradykinin (BK, 10-9-10-5 M) in the rat isolated aortic ring. Isop, ACh, SNP, or BK dose-dependently relaxed phenylephrine (PE, 10-7 M) pre-constricted ring producing a maximum relaxation of 82 % for Isop (10-5 M), 85 % for ACh (10-5 M), 100 % for SNP (10-6 M), and 30 % for BK (10-5 M) respectively. STZ attenuated Isop, ACh, and BK-induced relaxation by 45 % (n=7, pn (Fig. 5, Ref. 24).

Astrogliopathy as a loss of astroglial protective function against glycoxidative stress under hyperglycemia

Reactive oxygen species (ROS) derived from mitochondria play an essential role in stroke as well as in neurodegenerative disorders. Although hyperglycemia associated with diabetes mellitus is well known to enhance ROS production in vascular endothelial cells, the effects of either acute or chronic high glucose environments on neurons and glial cells remain unclear. Astroglia play a pivotal role in glucose metabolism. Thus, the astroglial metabolic response to high glucose environments is an interesting subject. In particular, the glutathione/pentose phosphate pathway (PPP) system, which is a major defense mechanism against ROS in the brain, contributes to glucose metabolism and is more active in astroglia. We propose that high glucose environments activate PPP through an increased flux to the hexosamine biosynthetic pathway (HBP). HBP is known to induce endoplasmic reticulum (ER) stress under hyperglycemia, resulting in the nuclear translocation of nuclear factor-erythroid-2-related factor 2 (Nrf2), a master regulator of phase 2 detoxifying enzymes including glucose-6-phosphate dehydrogenase that regulates PPP activity, as Nrf2 is reported to be a direct substrate of protein kinase RNA (PKR)-like ER kinase (PERK), a transducer of ER stress. Therefore, the phosphorylation of Nrf2 by hyperglycemia-induced ER stress facilitates Nrf2 translocation through PERK, thus activating the PPP. If acute or chronic hyperglycemia induces PPP activation in astroglia to reduce ROS, reducing the glucose concentration may be accompanied by a risk, which may explain the lack of evidence that strict glycemic control during the acute phase of stroke conveys no beneficial effect.

Increased glyoxalase I levels inhibit accumulation of oxidative stress and an advanced glycation end product in mouse mesangial cells cultured in high glucose

Chronic high glucose levels lead to the formation of advanced glycation end-products (AGEs) as well as AGE precursors, such as methylglyoxal (MG) and glyoxal, via non-enzymatic glycation reactions in patients with diabetic mellitus. Glyoxalase 1 (GLO-1) detoxifies reactive dicarbonyls that form AGEs. To investigate the interaction between AGEs and GLO-1 in mesangial cells (MCs) under diabetic conditions, AGE levels and markers of oxidative stress were measured in GLO-1-overexpressing MCs (GLO-1-MCs) cultured in high glucose. Furthermore, we also examined levels of high glucose-induced apoptosis in GLO-1-MCs. In glomerular MCs, high glucose levels increased the formation of both MG and argpyrimidine (an MG-derived adduct) as well as GLO-1 expression. GLO-1-MCs had lower intracellular levels of MG accumulation, 8-hydroxy-deoxyguanosine (an oxidative DNA damage marker), 4-hydroxyl-2-nonenal (a lipid peroxidation product), and nitrosylated protein (a marker of oxidative-nitrosative stress) compared to control cells. Expression of mitochondrial oxidative phosphorylation complexes I, II, and III was also decreased in GLO-1-MCs. Furthermore, fewer GLO-1-MCs showed evidence of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling assay, and activation of both poly (ADP-ribose) polymerase 1 cleavage and caspase-3 was lower in GLO-1-MCs than in control cells cultured in high glucose. These results suggest that GLO-1 plays a role in high glucose-mediated signaling by reducing MG accumulation and oxidative stress in diabetes mellitus.

Profibrotic influence of high glucose on human cardiac fibroblast functions

AimsIn this article, we determined the influence of chronic high glucose concentration on human cardiac fibroblasts functions and the effects of 606A and imidaprilat in these responses.MethodsHuman cardiac fibroblasts were...

Flavonol kaempferol improves chronic hyperglycemia-impaired pancreatic beta-cell viability and insulin secretory function

Considerable evidence shows that chronic hyperglycemia can cause pancreatic beta-cell dysfunction, which contributes to progressive deterioration of glucose homeostasis and overt diabetes. In the present study, we found that kaempferol, a flavonol compound present in various Chinese medicinal herbs, has cytoprotective effects on cultured clonal beta-cells and pancreatic human islets. Kaempferol treatment dose-dependently promoted viability, inhibited cellular apoptosis, and reduced caspase-3 activity in beta-cells and human islets exposed to chronic high glucose, with 10 μM kaempferol exerting the maximum effect. In addition, kaempferol treatment improved the expression of anti-apoptotic proteins Akt and Bcl-2 that was significantly reduced in beta-cells and human islets chronically exposed to hyperglycemia. Furthermore, exposure of beta-cells and human islets to kaempferol restored high glucose-attenuated intracellular cAMP and ATP production. Inhibition of protein kinase A or Akt activation ablated the anti-apoptotic effect of kaempferol. These cytoprotective effects of kaempferol were associated with improved insulin secretory function and synthesis in beta-cells and human islets. These findings provide evidence that kaempferol may be a naturally occurring anti-diabetic compound by protecting pancreatic beta-cell survival and function in a hostile environment that would otherwise lead to type 2 diabetes.

Astroglial protective mechanisms against ROS under brain ischemia

Reactive oxygen species (ROS) derived from mitochondria in neural cells play an essential role in the pathophysiology of stroke. Hyperglycemia is also known to enhance ROS production, resulting in oxidative stress. We reported that both acute and chronic high glucose environments enhance the pentose phosphate pathway (PPP) in astroglia, reducing ROS production and thereby providing a neuroprotective role. In particular, chronic hyperglycemia elicits PPP activation through the Keap1/Nrf2 system, which is induced by endoplasmic (ER) stress via an increase in hexosamine biosynthetic pathway flux. We examined the effects of hypoxia with or without glucoprivation on PPP in cultured neurons and astroglia. Hypoxia without glucoprivation for 12 or 24 hours induced PPP activation in astroglia (126% and 177%, respectively) but not in neurons. PPP activation by hypoxia was accompanied by Nrf2 translocation to the nucleus but not by Bip expression in the ER. Re-oxygenation supplemented with glucose after 12 hours of hypoxia with or without glucoprivation markedly enhanced PPP in astroglia (231% and 178%, respectively). Hypoxia induced PPP activation in astroglia, exerting a neuroprotective role. While the Keap1/Nrf2 system seems to be involved, ER stress is not necessarily required.

E0358 Profibrotic influence of high glucose on human cardiac fibroblast functions

AimsRecent studies have demonstrated an important role of chronic high glucose concentration for collagen deposition in fibroblasts. However, little is known about the action of angiotensin II type 1 recepter...

Activation of autophagy through modulation of 5′-AMP-activated protein kinase protects pancreatic β-cells from high glucose

Chronic hyperglycaemia is detrimental to pancreatic beta-cells by causing impaired insulin secretion and diminished beta-cell function through glucotoxicity. Understanding the mechanisms underlying beta-cell survival is crucial for the prevention of beta-cell failure associated with glucotoxicity. Autophagy is a dynamic lysosomal degradation process that protects organisms against metabolic stress. To date, little is known about the physiological function of autophagy in the pathogenesis of diabetes. In the present study, we explored the roles of autophagy in the survival of pancreatic beta-cells exposed to high glucose using pharmacological and genetic manipulation of autophagy. We demonstrated that chronic high glucose increases autophagy in rat INS-1 (832/13) cells and pancreatic islets, and that this increase is enhanced by inhibition of 5'-AMP-activated protein kinase. Our results also indicate that stimulation of autophagy rescues pancreatic beta-cells from high-glucose-induced cell death and inhibition of autophagy augments caspase-3 activation, suggesting that autophagy plays a protective role in the survival of pancreatic beta-cells. Greater knowledge of the molecular mechanisms linking autophagy and beta-cell survival may unveil novel therapeutic targets needed to preserve beta-cell function.