Zhe Dai

Intermittent high glucose enhances proliferation of vascular smooth muscle cells by upregulating osteopontin

OBJECTIVE Hyperglycemia induces vascular smooth muscle cells (VSMCs) proliferation and may thus contribute to the formation of atherosclerotic lesions. Glucose fluctuations are strong predictor of diabetic vascular complications. We investigate the effects of exposure to constant and intermittent high glucose concentrations on the proliferation and matrix metalloproteinase (MMP)-2 activity of rat aortic VSMCs in culture, as well as the expression of osteopontin (OPN). METHODS Rat aortic VSMCs were grown to confluence and then exposed to 5 mmol/L glucose, 25 mmol/L glucose, or 5 mmol/L alternating with 25 mmol/L glucose in the absence or presence of neutralizing antibodies to OPN, beta3 integrin receptor and beta5 integrin receptor. The cell proliferation, MMP-2 activity and the expression of OPN were assessed. RESULTS In cultured VSMCs, treatment with constant or intermittent high glucose significantly increased [(3)H]thymidine incorporation in a time-dependent manner. A modest increase was observed at 12h, and further deteriorated afterwards, and reached the maximum expression at 48h. However, [(3)H]thymidine incorporation was more pronounced in intermittent high glucose than in constant high glucose. Treatment with constant high glucose for 48 h significantly increase cell number, MMP-2 activation, OPN protein and mRNA expression compared with VSMCs treated with the cells normal glucose, and these effects were further enhanced when VSMCs were treated with intermittent high glucose. In addition, neutralizing antibodies to either OPN or its receptor beta3 integrin but not neutralizing antibodies to beta5 integrin significantly suppressed increase in [(3)H]thymidine incorporation and MMP-2 activity induced by constant or intermittent high glucose. CONCLUSIONS In cultured VSMCs, constant high glucose concentrations enhanced MMP-2 activity, cell proliferation and OPN expression. These effects are enhanced following intermittent exposure to high glucose, indicating that short lived excursions in glycaemic control have important pathological effects on the development of diabetic atherosclerosis, which is mediated by the stimulation of OPN expression and synthesis.

Intermittent high glucose exacerbates the aberrant production of adiponectin and resistin through mitochondrial superoxide overproduction in adipocytes

Hypoadiponectinemia and hyperresistinemia may be important in mediating signals from adipocytes to insulin-sensitive tissue and vasculature. However, the mechanism that mediates the aberrant production of adipokines remains poorly understood. In this study, we have investigated the effect of intermittent high glucose on the expression of adiponectin and resistin, and the production of 8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine in the adipocytes, either in the presence or in the absence of Mn(III) tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) or thenoyltrifluoroacetone (TTFA). 3T3-L1 adipocytes were incubated for 72 h in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l, 5 mmol/l alternating with 20 mmol/l glucose, with or without MnTBAP and TTFA. We measured the expression of resistin and adiponectin. The production of nitrotyrosine and 8-OHdG as oxidative stress parameter was measured. Both constant and intermittent high glucose significantly suppressed the expression and secretion of adiponectin, and increased expression and secretion of resistin in mature adipocytes compared to normal glucose conditions. However, these effects were significantly greater under intermittent high glucose conditions compared to constant high glucose. The levels of nitrotyrosine and 8-OHdG were significantly elevated under both intermittent and constant high glucose conditions, the effect being greater under intermittent high glucose. In addition, the antioxidants MnTBAP or TTFA reversed the aberrant production of adiponectin and resistin, as well as overproduction of nitrotyrosine and 8-OHdG in adipocytes induced by constant or intermittent high glucose. Intermittent high glucose exacerbates the aberrant production of adiponectin and resistin through reactive oxygen species overproduction at the mitochondrial transport chain level in adipocytes, indicating that glycemic variability has important pathological effects on the secretion of adipokines.

Expression of human insulin gene wrapped with chitosan nanoparticles in NIH3T3 cells and diabetic rats

AbstractAim:To study the expression of human insulin gene wrapped with chitosan nanoparticles in NIH3T3 cells and diabetic rats.Methods:pCMV.Ins, an expression plasmid of the human insulin gene, was constructed. In total, 100 μg pCMV.Ins wrapped with chitosan nanoparticles (chitosan–pCMV.Ins) was transfected to NIH3T3 cells and diabetes rats through lav age and coloclysis, respectively. The transfected cells were grown in Dulbeccos modified Eagles medium, containing G418, for 72 h after transfection. The clones were selected and continued to grow in G418 medium for 24 d. The expression of human insulin was detected by immunohistochemistry. Human insulin in the culture medium of transfected cells was measured. Fasting blood glucose and plasma human insulin of diabetic rats were measured for 5 d after transfection. RT–PCR and Western blotting were performed to confirm the expression of the human insulin gene in diabetic rats.Results:Approximately 10% of NIH3T3 cells transfected by chitosan–pCMV.Ins expressed human insulin. Human insulin in the culture medium of NIH3T3 cells transfected by chitosan–pCMV.Ins significantly increased compared with that of the control group (P<0.01). Fasting blood glucose levels of the lavage group and the coloclysis group decreased significantly in 5 d (P<0.01) in comparison, while plasma insulin levels were much higher (P<0.01). The human insulin gene mRNA and human insulin were only detected in the lavage and the coloclysis groups.Conclusion:The human insulin gene can be transfected and expressed successfully by chitosan–pCMV.Ins in NIH3T3 cells and diabetes rats, which indicates that chitosan is a promising, non-viral vector for gene expression.

RIP140 is Associated with Subclinical Inflammation in Type 2 Diabetic Patients

AIMS To evaluate the expression level of RIP140 (receptor interaction protein 140) and its correlation with inflammatory cytokine production and free fatty acids (FFAs) in type 2 diabetes. METHODS Plasma and peripheral blood mononuclear cells (PBMCs) were collected from 24 diabetic and 30 healthy individuals. The levels of FFAs, TC, TG, HDL-C, LDL-C, FIN, and FBG were measured. The insulin resistance index was calculated using the homeostasis model assessment (HOMA). Additionally, PBMCs from control group were cultured alone or with 500 μmol/L palmitic acid (PA). Levels of RIP140 TNF-α, and IL-6 in PBMCs were analyzed using real-time RT-PCR, Western blots and ELISA. The relationship between RIP140 and other variables was performed using SPSS 11.5 software. RESULTS TG, LDL-C, FIN, FBG, HOMA, and HDL-C were significantly different between diabetic patients and the control group. Levels of RIP140, TNF-α, and IL-6 were higher in the diabetic group compared to control. RIP140 expression was positively correlated with FFAs, HDL-c, TNF-α, IL-6, FIN, FBG, and HOMA. Finally, 500 μmol/L PA treatment increased RIP140 expression and the secretion of inflammatory cytokines in cultured control PBMCs. CONCLUSIONS Increased RIP140 level may be closely associated with inflammation and disorder of lipid and glucose metabolism in diabetic patients.

Involvement of osteopontin upregulation on mesangial cells growth and collagen synthesis induced by intermittent high glucose

Glucose fluctuations are strong predictor of diabetic vascular complications. We explored the effects of constant and intermittent high glucose on the proliferation and collagen synthesis of cultured rat mesangial cells. Furthermore, the possible involvement of osteopontin (OPN) was assessed. In rat mesangial cells cultured in 5, 25, or 5 mmol/L alternating with 25 mmol/L glucose in the absence or presence of neutralizing antibodies to OPN, β3 integrin receptor and β5 integrin receptor, the cell proliferation, collagen synthesis, and the expression of OPN and type IV collagen were assessed. In cultured mesangial cells, treatment with constant or intermittent high glucose significantly increased [3H]thymidine incorporation in a time‐dependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum incorporation at 48 h. Treatment with constant high glucose for 48 h resulted in significant increases in [3H]thymidine incorporation, cell number, [3H]proline incorporation, mRNA, and protein levels of type IV collagen and OPN compared with mesangial cells treated with the normal glucose, which were markedly enhanced in cells exposed to intermittent high glucose medium. In addition, neutralizing antibodies to either OPN or its receptor β3 integrin but not neutralizing antibodies to β5 integrin can effectively prevented proliferation and collagen synthesis of mesangial cells induced by constant or intermittent high glucose. Intermittent high glucose exacerbates mesangial cells growth and collagen synthesis by upregulation of OPN expression, indicating that glycemic variability have important pathological effects on the development of diabetic nephropathy, which is mediated by the stimulation of OPN expression and synthesis. J. Cell. Biochem. 109: 1210–1221, 2010.

Nr2e1 Deficiency Augments Palmitate-Induced Oxidative Stress in Beta Cells

Nuclear receptor subfamily 2 group E member 1 (Nr2e1) has been regarded as an essential regulator of the growth of neural stem cells. However, its function elsewhere is unknown. In the present study, we generated Nr2e1 knockdown MIN6 cells and studied whether Nr2e1 knockdown affected basal beta cell functions such as proliferation, cell death, and insulin secretion. We showed that knockdown of Nr2e1 in MIN6 cells resulted in increased sensitivity to lipotoxicity, decreased proliferation, a partial G0/G1 cell-cycle arrest, and higher rates of apoptosis. Moreover, Nr2e1 deficiency exaggerates palmitate-induced impairment in insulin secretion. At the molecular level, Nr2e1 deficiency augments palmitate-induced oxidative stress. Nr2e1 deficiency also resulted in decreases in antioxidant enzymes and expression level of Nrf2. Together, this study indicated a potential protective effect of Nr2e1 on beta cells, which may serve as a target for the development of novel therapies for diabetes.

NR4A1 is associated with chronic low‑grade inflammation in patients with type 2 diabetes

Type 2 diabetes (T2D) is a common disorder characterized by chronic low-grade inflammation. In the present study, the expression levels of nuclear receptor subfamily 4 group A member 1 (NR4A1) and the correlation with inflammatory cytokine production and free fatty acids (FFAs) in patients with T2D and healthy participants were investigated. NR4A1 expression levels in peripheral blood mononuclear cells (PBMCs) from patients with T2D (n=30) and healthy controls (n=34) were analyzed. In addition, the levels of fasting blood glucose (FBG), fasting plasma insulin (FIN), FFAs, total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) were analyzed, and the homeostasis model assessment (HOMA) was used to estimate the insulin resistance (IR). Additionally, PBMCs from healthy subjects were cultured with or without 250 μM palmitic acid (PA). Levels of NR4A1, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the PBMCs were also analyzed. The basal expression levels of NR4A1, TNF-α and IL-6 were higher in the T2D patients when compared with the controls. In addition, the levels of FFAs, TG and LDL-C, as well as the HOMA-IR, were higher in T2D patients. Furthermore, NR4A1 expression was demonstrated to positively correlate with the HOMA-IR and the levels of FFAs, TNF-α, IL-6, FIN and FBG. Furthermore, 250 μM PA stimulation was shown to increase NR4A1 expression and the secretion of inflammatory cytokines in the cultured PBMCs. Therefore, increased NR4A1 expression levels are correlated with a chronic low-grade inflammatory state and the disorder of lipid metabolism in patients with T2D.

Polymorphism of apolipoprotein A5 is a risk factor for cerebral infarction in type 2 diabetes

SummaryThis study investigated the association of apolipoprotein A5 (apoA5) gene polymorphism at position −1131T>C with cerebral infarction in patients with type 2 diabetes. A total of 256 type 2 diabetic patients without cerebral infarction (T2DM), 220 type 2 diabetic patients with cerebral infarction (T2DMCI) and 340 healthy subjects were recruited from the same region (Hubei province, China). The genotype of apoA5 −1131T〉C was analyzed by polymerase chain reaction, followed by restriction fragment length polymorphism (PCR-RFLP). Total cholesterol, HDL cholesterol, LDL-cholesterol and triglycerides were quantitatively detected by using standard enzymatic techniques. The results showed that the prevalence of the apoA5 −1131C allele was significantly higher in T2DMCI group than that in control group (42.7% versus 31.2%, P<0.01). The carriers of rare C allele had higher TG levels as compared with carriers of common allele in the three groups (P<0.01). Logistic regression models, which were adjusted for age, gender, blood pressure, BMI, FBS, smoking, LDL-C and HDL-C, revealed that patients carrying the apoA5 −1131C allele and CC homozygotes were at high risk for T2DMCI. It was concluded that the apoA5 −1131C allele variant is an independent genetic risk factor for T2DMCI.This study investigated the association of apolipoprotein A5 (apoA5) gene polymorphism at position −1131T>C with cerebral infarction in patients with type 2 diabetes. A total of 256 type 2 diabetic patients without cerebral infarction (T2DM), 220 type 2 diabetic patients with cerebral infarction (T2DMCI) and 340 healthy subjects were recruited from the same region (Hubei province, China). The genotype of apoA5 −1131T〉C was analyzed by polymerase chain reaction, followed by restriction fragment length polymorphism (PCR-RFLP). Total cholesterol, HDL cholesterol, LDL-cholesterol and triglycerides were quantitatively detected by using standard enzymatic techniques. The results showed that the prevalence of the apoA5 −1131C allele was significantly higher in T2DMCI group than that in control group (42.7% versus 31.2%, P<0.01). The carriers of rare C allele had higher TG levels as compared with carriers of common allele in the three groups (P<0.01). Logistic regression models, which were adjusted for age, gender, blood pressure, BMI, FBS, smoking, LDL-C and HDL-C, revealed that patients carrying the apoA5 −1131C allele and CC homozygotes were at high risk for T2DMCI. It was concluded that the apoA5 −1131C allele variant is an independent genetic risk factor for T2DMCI.

Reduction of insulin resistance in HepG2 cells by knockdown of LITAF expression in human THP-1 macrophages

SummaryThe molecular mechanism by which obesity induces insulin resistance is not completely understood. The aim of this study was to determine how lipopolysaccharide-induced tumor necrosis-α factor (LITAF) influenced obesity-induced insulin resistance using a cellular co-culture system. The cells were divided into 3 groups: palmitic acid (PA) stimulation group, LITAF small interfering RNA (siRNA) group and untreated (NC) group. The LITAF siRNA was used for knockdown of LITAF expression in human THP-1 macrophages. The expression levels of LITAF, IRS-2, IRS-2Tyr465, PI3K, and GLUT2 in each group were measured by using quantitative reverse transcriptase real-time polymerase chain reaction and Western blotting. The expression of LITAF was much higher in the PA group than in the siRNA and NC groups (*P<0.05); meanwhile, the expression of IRS-2, IRS-2Tyr465, PI3K, and GLUT2 was much lower in the PA group than in the NC group (*P<0.05); however, IRS-2, IRS-2Tyr465, PI3K, and GLUT2 had much higher expression in the siRNA group than in the PA group (*P<0.05). It is concluded that PA can induce insulin resistance in liver cells and knockdown of LITAF expression can reduce insulin resistance in liver cells, suggesting LITAF may regulate the insulin signal transduction pathway involved in obesity-induced insulin resistance.

WITHDRAWN: RIP140 mediates hyperglycemia-induced glucotoxicity in β-cells via the activation of JNK and ERK1/2 signaling pathways.

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