Glucose-treated Cells Research Articles

Effect of miR-137 on the proliferation and apoptosis of endothelial cells regulated by AKT2 gene

To assess the effect of miR-137 on the proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) induced by high glucose and its mechanism. HUVECs cells were divided into low-glucose group (5.5 mmol/L glucose-treated cells), high-glucose group (33.36 mmol/L glucose-treated cells), anti-NC group (cells treated with 33.36 mmol/L glucose after anti-NC transfection) and anti-miR-137 group (cells treated with 33.36 mmol/L glucose after anti-miR-137 transfection). After 48 hours, qRT-PCR was used to determine the expression of miR-137. CCK-8 assay and flow cytometry were used to detect cell proliferation and apoptosis rate, respectively. The targeting relationship between miR-137 and AKT2 was validated by dual fluorescence reporter gene detection system and AKT2 protein expression after overexpression or inhibition of miR-137. High glucose could significantly up-regulate the expression of miR-137 in HUVECs cells, and the expression of miR-137 in HUVECs cells transfected with miR-137 inhibitor was significantly decreased (P<0.05). High glucose can significantly inhibit HUVECs cell proliferation and induce apoptosis, while inhibition of miR-137 expression can weaken the effect of high glucose on HUVECs cell proliferation inhibition and apoptosis promotion (P<0.05). Inhibiting AKT2 expression could weaken the inhibitory effect of miR-137 inhibitor on HUVECs cell proliferation and apoptosis (P<0.05). Inhibiting the expression of miR-137 gene can attenuate the proliferation inhibition and apoptosis promotion of HUVECs induced by high glucose, and the mechanism is related to activating the expression of AKT2.

MicroRNA-96 regulates pancreatic β cell function under the pathological condition of diabetes mellitus through targeting Foxo1 and Sox6

To elucidate the potential function of microRNA-96 in protecting pancreatic β cell function under the pathological condition of T2DM and the underlying mechanism. Relative levels of microRNA-96 and genes associated with β cell function in the in vivo and in vitro T2DM and obesity models were detected by qRT-PCR. Insulin functions, including fasting blood glucose, plasma insulin, HOMA-IR, HOMA-%b, glucose tolerance and insulin tolerance, were assessed in microRNA-96 KO mice and wild-type mice fed with normal diet or high-fat diet. Downstream targets of microRNA-96 were verified by dual-luciferase reporter gene assay. Finally, regulatory effects of microRNA-96 on proliferation and apoptosis of MIN6 cells were determined. MicroRNA-96 was upregulated in mice fed with high-fat diet, db/db mice, high-level glucose-treated cells, TNF-α-treated cells, pancreatic cells isolated from the obesity and T2DM patients. Increased fasting blood glucose and HOMA-IR, as well as decreased plasma insulin and HOMA-%b were observed in microRNA-96 KO mice. IPGTT and IPITT results indicated that knockout of microRNA-96 led to pancreatic β cell dysfunction under the pathological condition of T2DM. Dual-luciferase reporter gene assay confirmed that microRNA-96 could bind Foxo1 and Sox6. MicroRNA-96 negatively regulated Foxo1 and Sox6 levels. Moreover, overexpression of microRNA-96 promoted proliferative ability and inhibited apoptosis in MIN6 cells. Relative levels of Pdx1, Nkx6.1, Cyclin D1 and Cyclin E1 were upregulated in MIN6 cells overexpressing microRNA-96. Opposite results were obtained after knockdown of microRNA-96 in MIN6 cells. MicroRNA-96 is upregulated in pancreatic β cells under the pathological condition of T2DM. Overexpression of microRNA-96 promotes proliferative ability and inhibits apoptosis in pancreatic β cells through targeting Foxo1 and Sox6.

Effect of butein and glucose on oxidative stress and p38 activation marker in non-small cell lung cancer cell.

Tumor microenvironment is known to alter the anticancer drug efficiency. One of the factors that get altered in cancer microenvironment is glucose concentration. Butein, an active principle from plant, known to have anticancer effect against different types of tumor. The objective of the study is to determine the effect of butein on glucose exposed non-small cell lung cancer cells (NSCLCCs). The current study deals with the effect of butein (6.25-50μM) on NSCLCCs treated with different concentrations (0-40 mM) of glucose. Glucose concentration, 0 mM and 40 mM, was found to be lethal at 72 h. Viable cell numbers were statistically increased in 5-mM, 10-mM, and 20-mM glucose-treated cells. Butein at 12.5 µM inhibits (p < 0.05) glucose-induced cell proliferation. Butein inhibits glucose-induced proliferation through DNA damage and oxidative stress. Mitochondrial reactive oxygen species (ROS) level was elevated in 20-mM glucose-treated cells when compared to 5-mM glucose-treated cells, whereas butein treatment further increases glucose-induced mitochondrial ROS. Pharmacological inhibitor of glycolysis, such as 2-deoxy glucose (2-DG), was found to inhibit (p < 0.05) glucose-induced cells proliferation. Furthermore, 2-DG and butein showed synergistic anticancer effect. Butein treatment increases p38 phosphorylation. Inhibition of p38 phosphorylation and antioxidant pretreatment partially revert the glucose-induced cell proliferation. However, inhibition of p38 phosphorylation combined with antioxidant pretreatment completely reverts the anticancer effect of butein. The present study concludes through the evidence that butein could serve as a potential anticancer compound in tumor microenvironment.

Mitochondria-targetable red-emitting probe for real-time fluorescence monitoring of NAD(P)H in live cells

We have developed a mitochondria-targetable and red-emitting probe (1) that allows for the quantitative analysis of mitochondrial NAD(P)H in live human cells. Probe 1 is non-fluorescent and gets reduced in the presence of NAD(P)H to emit a strong fluorescence. This probe is highly selective to NAD(P)H over various potential biological interferants such as redox species, metals, anions, and other biomolecules. In addition, confocal microscopic images revealed the predominant accumulation of probe 1 into the mitochondria of human breast cancer cells (MDA-MB-231), as evident from the red fluorescence in response to NAD(P)H activity in glucose-treated cells as NAD(P)H induction model vs. cyanide 4-(trifluoromethoxy)phenylhydrazone-treated cells as NAD(P)H depletion model. Probe 1 can be used to differentiate between cancer (MDA-MB-231) and normal cells (NIH-3T3) based on mapping mitochondrial NAD(P)H-dependent fluorescence intensity. Probe 1 will be useful for various applications, ranging from elucidation of biological pathways to pathological diagnosis.

Activation of mammalian target of rapamycin signaling pathway in placental tissues of gravidas with gestational diabetes mellitus

Objective To investigate the activation of mammalian target of rapamycin (mTOR) signaling pathway in placental tissues of gestational diabetes mellitus (GDM) women and in JEG3 cells after high glucose treatment. Methods Placental tissues were collected from 60 singleton pregnant women at term, who underwent elective cesarean section in Peking University First Hospital from December 2016 to June 2017, including 30 GDM women (GDM group) and 30 normal glucose tolerance (NGT) women (NGT group). Expressions of mTOR, Rictor, Raptor, S6 kinase (S6K) and phosphorylated-S6K (p-S6K), which were mTOR signaling pathway-related molecules in those placental tissues were detected by real-time fluorescence quantitative polymerase chain reaction, Western blotting and immunohistochemistry. At the same time, JEG3 cells were treated with glucose of different concentrations (5.5, 10.0, 15.0, 25.0 and 50.0 mmol/L) for 24 h, and the expressions of mTOR, S6K and p-S6K were detected by Western blotting. Two independent samples t-test, one-way analysis of variance test and Spearman correlation analysis were used for statistical analysis. Results Compared with the NGT group, the pre-pregnancy body mass index(BMI), neonatal birth weight, fasting blood glucose(FBG), and 1 h and 2 h post-load blood glucose in oral glucose tolerance test (OGTT) of the GDM group were significantly increased [pre-pregnant BMI: (21.6±2.7) vs (23.4±3.5) kg/m2, t=-2.192; neonatal birth weight: (3 337.5±347.7) vs (3 618.3±580.9) g, t=-2.727; FBG: (4.6±0.4) vs (5.2±0.8) mmol/L, t=-3.947; 1 h glucose level: (7.4±1.2) vs (9.6±1.9) mmol/L, t=-5.332; 2 h glucose level: (6.3±1.0) vs (8.1±1.5) mmol/L, t=-5.314; all P<0.05]. The mRNA expressions of mTOR, Rictor and Raptor were significantly higher in the GDM group than in the NGT group (0.051±0.015 vs 0.031±0.011, t=-5.635; 0.038±0.017 vs 0.026±0.010, t=-3.485; 0.036±0.012 vs 0.025±0.011, t=-3.312; all P<0.05). The expression of mTOR, Rictor, Raptor and p-S6K protein in the GDM group were enhanced as compared with those in the NGT group (0.834±0.432 vs 0.386±0.361, t=-2.249; 0.589±0.236 vs 0.262±0.075, t=-3.726; 0.767±0.345 vs 0.323±0.109, t=-3.472; 1.847±1.025 vs 0.834±0.432, t=-2.575; all P<0.05). The results of immunohistochemistry also showed that the p-S6K in the placental tissues of the GDM group was higher than that of the NGT group (0.29±0.09 vs 0.18±0.08, t=-3.122, P=0.004). The expressions of mTOR protein (0.190±0.085, 0.301±0.089, 0.419±0.065, 0.562±0.108, 0.412±0.058, F=18.351, P<0.05) and p-S6K protein (0.753±0.150, 1.146±0.289, 2.148±0.188, 2.248±0.115, 2.134±0.214, F=66.242, P<0.05) in JEG3 cells treated with different concentrations of glucose (5.5, 10.0, 15.0, 25.0 and 50.0 mmol/L) were significantly different and increased with increasing concentrations of glucose (r=0.314, P=0.035; r=0.457, P=0.002). Conclusions The up-regulated expressions of mTOR signaling pathway-related molecules in GDM placenta and high glucose-treated trophoblast cells (JEG3 cells) indicate a possible correlation between mTOR signaling pathway and GDM. However, the specific mechanisms need further study. Key words: Diabetes, gestational; Placenta; Mammalian target of rapamycin, serine/threonine kinases; Signal transduction

STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

Schwann cells are the main supportive cells of the peripheral nerves. Schwann cells suffer inhibition of autophagy under hyperglycemia treatment in diabetic peripheral neuropathy (DPN). However, the exact mechanism is still not fully elucidated. We first observed the decrease of autophagy markers (LC3-II/LC3-I, P62) in the sciatic nerves of diabetic mice vs. normal mice, accompanied with the loss of myelinated nerve fibers and abnormal myelin sheath. In line with this, LC3-II/LC3-I and P62 were also significantly reduced in high glucose-treated rat Schwann cell 96 (RSC96) cells compared with normal glucose-treated cells. Furthermore, we found that trichostatin A [an inhibitor of histone deacetylase (HDAC)] evidently improved LC3-II/LC3-I in high glucose-treated RSC96 cells, without an effect on P62 expression. Again, HDAC1 and HDAC5 were revealed to be increased in RSC96 cells stimulated with high glucose. Inhibition of HDAC1 but not HDAC5 by small hairpin RNA vector enhanced LC3-II/LC3-I in high glucose-cultured RSC96 cells. In addition, LC3-II conversion regulators [autophagy-related protein (Atg)3, Atg5, and Atg7] were detected in high glucose-treated and HDAC1-knockdown RSC96 cells, and Atg3 was proven to be the key target of HDAC1. The presuppression of Atg3 offset the improvement of LC3-II/LC3-I resulting from HDAC1 inhibition in high glucose-treated RSC96 cells. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway was activated in RSC96 cells treated with high glucose, which was indicated by increased STAT3 phosphorylation. Blocking STAT3 phosphorylation by chemical inhibitor AG490 induced HDAC1 down-regulation followed by increases in Atg3 and LC3-II/LC3-I. Interestingly, we also found that AG490 treatment enhanced P62 expression in high glucose-stimulated RSC96 cells. Taken together, our findings demonstrate that hyperglycemia inhibits LC3-II/LC3-I in an HDAC1-Atg3-dependent manner and decreases P62 expression in an HDAC-independent manner via the JAK-STAT3 signaling pathway in the Schwann cells of DPN.-Du, W., Wang, N., Li, F. Jia, K., An, J., Liu, Y., Wang, Y., Zhu, L., Zhao, S. Hao, J. STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

Inhibition of autophagy promoted high glucose/ROS-mediated apoptosis in ADSCs

BackgroundIncreased apoptosis in adipose tissue-derived stem cells (ADSCs) limits their application in treating diabetes complications. Autophagy is a molecular process that allows cells to degrade and recover damaged macromolecules, and closely interacts with apoptosis. The aim of the present study was to investigate the potential role of autophagy in ADSC apoptosis induced by high glucose.MethodsHuman ADSCs were cultured in normal or high-glucose medium for 6 h, 12 h, or 24 h. The effects of high glucose on ADSC autophagy, reactive oxygen species (ROS) production, and apoptosis were evaluated. The impact of autophagy on ROS production and apoptosis was explored by treatment with rapamycin or 3-methyladenine (3-MA). The c-jun kinase (JNK) signaling pathway was investigated by pharmacological disruption of SP600125.ResultsADSCs subjected to high glucose stress showed an obvious induction of autophagy and apoptosis and a significant increase in intracellular ROS levels. The JNK signaling pathway was confirmed to be involved in high glucose-induced autophagy. Pre-treatment with SP600125 or N-acetylcysteine reversed the effects of high glucose on the JNK signaling pathway and autophagy-related proteins. Pretreatment of ADSCs with 3-MA under high glucose stress induced a further increase in ROS levels compared to those of high glucose-treated cells. Furthermore, ADSCs pretreated with 3-MA under high glucose stress showed a marked increase in apoptosis compared with that of the cells treated with high glucose. Conversely, pre-treatment with rapamycin inhibited the apoptosis of ADSCs.ConclusionsTaken together, our data suggest that autophagy may play a protective role in high glucose-induced apoptosis in ADSCs. ROS/JNK signaling is essential in upregulating high glucose-induced autophagy. This study provides new insights into the molecular mechanism of autophagy involved in high glucose-induced apoptosis in ADSCs.

Human chorionic villous mesenchymal stem/stromal cells protect endothelial cells from injury induced by high level of glucose

BackgroundMesenchymal stem/stromal cells derived from chorionic villi of human term placentae (pMSCs) protect human endothelial cells from injury induced by hydrogen peroxide (H2O2). In diabetes, elevated levels of glucose (hyperglycaemia) induce H2O2 production, which causes the endothelial dysfunction that underlies the enhanced immune responses and adverse complications associated with diabetes, which leads to thrombosis and atherosclerosis. In this study, we examined the ability of pMSCs to protect endothelial cell functions from the negative impact of high level of glucose.MethodspMSCs isolated from the chorionic villi of human term placentae were cultured with endothelial cells isolated from human umbilical cord veins in the presence of glucose. Endothelial cell functions were then determined. The effect of pMSCs on gene expression in glucose-treated endothelial cells was also determined.ResultspMSCs reversed the effect of glucose on key endothelial cell functions including proliferation, migration, angiogenesis, and permeability. In addition, pMSCs altered the expression of many genes that mediate important endothelial cell functions including survival, apoptosis, adhesion, permeability, and angiogenesis.ConclusionsThis is the first comprehensive study to provide evidence that pMSCs protect endothelial cells from glucose-induced damage. Therefore, pMSCs have potential therapeutic value as a stem cell-based therapy to repair glucose-induced vascular injury and prevent the adverse complications associated with diabetes and cardiovascular disease. However, further studies are necessary to reveal more detailed aspects of the mechanism of action of pMSCs on glucose-induced endothelial damage in vitro and in vivo.

Cyanidin-3-rutinoside protects INS-1 pancreatic β cells against high glucose-induced glucotoxicity by apoptosis.

Exposure to high levels of glucose may cause glucotoxicity, leading to pancreatic β cell dysfunction, including cell apoptosis and impaired glucose-stimulated insulin secretion. The aim of this study was to explore the effect of cyanidin-3-rutinoside (C3R), a derivative of anthocyanin, on glucotoxicity-induced apoptosis in INS-1 pancreatic β cells. Glucose (30 mM) treatment induced INS-1 pancreatic β cell death, but glucotoxicity and apoptosis significantly decreased in cells treated with 50 μM C3R compared to that observed in 30 mM glucose-treated cells. Furthermore, hyperglycemia increased intracellular reactive oxygen species (ROS), lipid peroxidation, and nitric oxide (NO) levels, while C3R treatment reduced these in a dose-dependent manner. C3R also increased the activity of antioxidant enzymes, markedly reduced the expression of pro-apoptotic proteins (such as Bax, cytochrome c, caspase 9 and caspase 3), and increased the expression of the anti-apoptotic protein, Bcl-2, in hyperglycemia-exposed cells. Finally, cell death was examined using annexin V/propidium iodide staining, which revealed that C3R significantly reduced high glucose-induced apoptosis. In conclusion, C3R may have therapeutic effects against hyperglycemia-induced β cell damage in diabetes.

O‐GlcNAc Transferase Regulates Phenotypic Transformation of Vascular Smooth Muscle Cells in Response to Hyperglycemia

Risks of vascular complications are enhanced two‐to‐four fold in diabetes, accounting for increased morbidity and mortality in these patients. Hyperglycemia is an important risk factor for macrovascular complications associated with diabetes. Diabetic patients exhibit an increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation, a hallmark of VSMC phenotypic switching from the quiescent contractile state to a synthetic proliferative phenotype. Hyperglycemia increases glucose flux through the hexosamine biosynthetic pathway triggering an enhanced signaling via O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT), the key enzyme catalyzing addition of O‐GlcNAc moieties to proteins. Protein O‐GlcNAcylation is a unique post‐translational protein modification implicated in diabetes and related cardiovascular complications. Previous studies have suggested both cardio‐protective and cardio‐detrimental effects of protein O‐GlcNAcylation in diabetes. However, the precise role of OGT in the etiology of diabetic atherogenesis remains incompletely understood. The goal of the current study was to investigate whether OGT modulates VSMC transition to a de‐differentiated phenotype under hyperglycemic conditions. Immunoblotting experiments revealed that in primary cultures of human aortic smooth muscle cells (HASMC) in vitro, benzyl 2‐deoxy α‐D‐galactopyranoside (BG), a pharmacological inhibitor of OGT, decreased high glucose‐induced OGT and O‐GlcNAc protein expression, and this was accompanied with attenuated PCNA (proliferation marker), reduced vimentin (SM synthetic marker) and increased SM‐MHC (SM contractile marker) expression. Consistent with these findings, deletion of OGT using siRNA gene silencing significantly lowered protein O‐GlcNAc and PCNA expression in glucose‐stimulated HASMC compared with glucose‐treated cells transfected with control siRNA. Notably, under glucose‐stimulated conditions, OGT knockdown enhanced calponin (SM contractile marker) expression in HASMC transfected with OGT siRNA vs cells transfected with control siRNA. Finally, electrophoretic mobility shift assay (EMSA) revealed that specific OGT inhibitors (BG and ST045849) attenuated high glucose‐induced DNA‐binding activity of YY1 and Elk1 (transcriptional repressors of SM contractile phenotype) in HASMC compared to cells treated with glucose alone. Taken together, these data clearly demonstrate that loss of OGT inhibits VSMC phenotypic de‐differentiation in response to hyperglycemia, suggesting OGT as a potential therapeutic target in diabetic atherosclerosis.Support or Funding InformationAmerican Heart Association ‐ Grant‐in‐Aid 16GRNT31200034This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of metabolic disorders in vivo and in vitro by main constituent of Coreopsis tinctoria

ObjectiveTo investigate the effects of the ethyl acetate extract of Coreopsis tinctoria (EAEC) on insulin resistance (IR) in rats fed a high-fat diet. MethodsMale Sprague-Dawley (SD) rats were fed a HFD (60% fat) supplemented with EAEC for 8 weeks. The administration of EAEC to the rats with HFD-induced insulin resistance reduced hyperglycemia, plasma levels of insulin, and steatosis in the liver. Metabolomic study was used to analyze the metabolic levels of the high glucose-treated cells, control cells and marein-treated cells. ResultsHigh glucose and high fat conditions caused a significant increase in blood glucose, insulin, serum TC, TG and LDL-C levels, leading to abnormal IR in rats. However, treatment with EAEC protects against HFD-induced IR by improving the fasting serum glucose homeostasis and lipid homeostasis. The high glucose conditions significantly decreased glycogen synthesis and increased PEPCK, G6Pase and Krebs cycle-related enzyme protein levels, leading to an abnormal metabolic state in HepG2 cells. However, treatment with marein improved IR by increasing glucose uptake and glycogen synthesis and by downregulating PEPCK and G6Pase protein levels. The statistical analysis of the HPLC/MS data demonstrated that marein could restore the normal metabolic state. ConclusionThe results revealed that EAEC ameliorates IR in rats, and marein has the potential to improve IR by ameliorating glucose metabolism disorders.

Hyperglycemia and advanced glycation end products (AGEs) suppress the differentiation of 3T3-L1 preadipocytes.

Aging is characterized by mild hyperglycemia and accumulation of advanced glycation end products (AGEs). Effects of chronic exposure to hyperglycemia or AGEs on the adipogenic differentiation of 3T3-L1 preadipocytes remain unclear. We examined the chronic effect of AGEs and high glucose on the differentiation of 3T3-L1 cells by culturing 3T3-L1 cells in the presence of AGEs or 25 mM glucose for 1 month. Chronic incubation of 3T3-L1 cells with AGEs or high glucose blocked their differentiation into mature adipocytes as evidenced by reduced levels of adipocyte markers such as accumulated oil droplets, GPDH, aP2, adiponectin and of adipogenesis regulators PPARγ and C/EBPα. Levels or activities of Src, PDK1, Akt, and NF-κB were higher in AGEs- and high glucose-treated cells than those in 3T3-L1 cells. Levels of Bcl-2 were elevated in AGEs- and high glucose-treated cells, and were attenuated by inhibitors of PI3-kinase, Akt and NF-κB. Moreover, adipogenesis was attenuated in 3T3-L1 cells stably expressing Bcl-2 or YAP. These results suggest that chronic AGEs and high glucose treatments up-regulate Bcl-2 and YAP via the Akt-NF-κB pathway and impair adipogenesis.

Role of Toll - like receptor 4 in higher sensitivity of kidney tubular cells to actue injury in high glucose

Objective To study the role of Toll-like receptor 4 (TLR4) in higher sensitivity of renal proximal tubular cells (RPTCs) to actue injury in high glucose in vitro. Methods The RPTCs were cultured for 2 days in a medium containing 30 mmol/L glucose (high glucose), where as the cells in the control groups were grown with 5.5 mmol/L glucose (normal glucose) or 5.5 mmol/L glucose with 24.5 mmol/L mannitol, and infected with lentivirus containing TLR4-shRNA or control shRNA. These cells were then subjected to azide treatment for adenosine-triphosphate (ATP) depletion, the apoptosis was measured by staining, and TLR4 protein level was qualified by Western blotting. Results TLR4-shRNA lentivirus was constructed sucessesfully. TLR4 protein level was significantly higher in high glucose-treated RPTCs, and the apoptosis rate was significantly higher in high glucose-treated RPTCs [(65.4±8.1)%] than in low glucose-treated RPTCs [(20.5±9.9)%] after azide treatment (P<0.05). TLR4-shRNA decreased the TLR4 protein level in high glucose-treated RPTCs, and significantly inhibited the apoptosis in high glucose-treated RPTCs after azide treatment [(68.5±9.3)% vs. (30.2±7.8)%, P<0.05]. Conclusion High glucose-treated RPTCs showed markedly higher TLR4 induction and higher apoptosis after azide treatment. The suppression of TLR4 diminished the sensitivity of high glucose-treated cells to acute injury in vitro. These results suggest the involvement of TLR4 in the susceptibility of diabetic models to acute kidney injury in vitro. Key words: High glaucose; Toll-like receptor 4; Renal proximal tubular cells; Acute kidney injury

High glucose concentration induces endothelial cell proliferation by regulating cyclin‐D2‐related miR‐98

Cyclin D2 is involved in the pathology of vascular complications of type 2 diabetes mellitus (T2DM). This study investigated the role of cyclin‐D2‐regulated miRNAs in endothelial cell proliferation of T2DM. Results showed that higher glucose concentration (4.5 g/l) significantly promoted the proliferation of rat aortic endothelial cells (RAOECs), and significantly increased the expression of cyclin D2 and phosphorylation of retinoblastoma 1 (p‐RB1) in RAOECs compared with those under low glucose concentration. The cyclin D2‐3′ untranslated region is targeted by miR‐98, as demonstrated by miRNA analysis software. Western blot also confirmed that cyclin D2 and p‐RB1 expression was regulated by miR‐98. The results indicated that miR‐98 treatment can induce RAOEC apoptosis. The suppression of RAOEC growth by miR‐98 might be related to regulation of Bcl‐2, Bax and Caspase 9 expression. Furthermore, the expression levels of miR‐98 decreased in 4.5 g/l glucose‐treated cells compared with those treated by low glucose concentration. Similarly, the expression of miR‐98 significantly decreased in aortas of established streptozotocin (STZ)‐induced diabetic rat model compared with that in control rats; but cyclin D2 and p‐RB1 levels remarkably increased in aortas of STZ‐induced diabetic rats compared with those in healthy control rats. In conclusion, this study demonstrated that high glucose concentration induces cyclin D2 up‐regulation and miR‐98 down‐regulation in the RAOECs. By regulating cyclin D2, miR‐98 can inhibit human endothelial cell growth, thereby providing novel therapeutic targets for vascular complication of T2DM.

Inhibition of microRNA195 attenuates high-glucose induced neonatal cardiomyocytes hypertrophy in vitro

To investigate the effects of micro(mi) RNA-195 on high-glucose induced neonatal cardiomyocyte hypertrophy and to explore the related mechanism. The potential target gene of miRNA-195 (Smad7) was predicted by TargetScan5. 1 software. Cardiomyocytes were isolated from neonatal SD rats and cells were then randomly divided into three groups: cells treated by culture medium containing 5 mmol/L glucose (control group) , by culture medium containing 25 mmol/L glucose (high glucose group) and treated by culture medium containing 25 mmol/L glucose and miRNA-195 inhibitor transfection (miRNA-195 inhibitor group). After 24, 48, or 72 h of in vitro culture, the morphology of cardiomyocytes was examined under phase contrast microscope. Micrographs were captured and the cell surface was calculated. The mRNA expressions of miRNA-195 and myosin heavy chain β (β-MHC), a biomarker for cardiomyocyte hypertrophy, in cardiomyocytes were detected by RT-PCR. The protein expression of Smad7 was determined by Western blot. The concentration of transforming growth factor-β1 (TGF-β1) in the supernatant of culture medium was measured by ELISA. Cross-sectional area of cardiomyocytes, expression of miRNA-195 and β-MHC and secretion of TGF-β1 were significantly increased in high glucose-treated cells (P < 0.05 vs. normal control). The protein expression of Smad7 was significantly downregulated in cells exposed to high glucose for 48 h (P < 0.05 vs. normal control). Downregulation of miRNA-195 partly reversed the high glucose-induced effects. The expression of Smad7 was negatively correlated with miRNA-195 in high glucose control group (correlation coefficient: -0.945, P < 0.05). Our results demonstrate that Smad7 could be the target gene of miRNA-195. miRNA-195 might play a crucial role in the development and progression of diabetic cardiomyopathy possibly through downregulating the expression of Smad7 and modulating TGF-β/Smad pathways.

Activation of hepatic CREBH and Insig signaling in the anti-hypertriglyceridemic mechanism of R-α-lipoic acid

The activation of sterol regulatory element binding proteins (SREBPs) is regulated by insulin-induced genes 1 and 2 (Insig-1 and Insig-2) and SCAP. We previously reported that feeding R-α-lipoic acid (LA) to Zucker diabetic fatty (ZDF) rats improves severe hypertriglyceridemia. In this study, we investigated the role of cyclic AMP-responsive element binding protein H (CREBH) in the lipid-lowering mechanism of LA and its involvement in the SREBP-1c and Insig pathway. Incubation of McA cells with LA (0.2 mM) or glucose (6 mM) stimulated activation of CREBH. LA treatment further induced mRNA expression of Insig-1 and Insig-2a, but not Insig-2b, in glucose-treated cells. In vivo, feeding LA to obesity-induced hyperlipidemic ZDF rats activated hepatic CREBH and stimulated transcription and translation of Insig-1 and Insig-2a. Activation of CREBH and Insigs induced by LA suppressed processing of SREBP-1c precursor into nuclear SREBP-1c, which subsequently inhibited expression of genes involved in fatty acid synthesis, including FASN, ACC and SCD-1, and reduced triglyceride (TG) contents in both glucose-treated cells and ZDF rat livers. Additionally, LA treatment also decreased abundances of very low density lipoprotein (VLDL)-associated apolipoproteins, apoB100 and apoE, in glucose-treated cells and livers of ZDF rats, leading to decreased secretion of VLDL and improvement of hypertriglyceridemia. This study unveils a novel molecular mechanism whereby LA lowers TG via activation of hepatic CREBH and increased expression of Insig-1 and Insig-2a to inhibit de novo lipogenesis and VLDL secretion. These findings provide novel insight into the therapeutic potential of LA as an anti-hypertriglyceridemia dietary molecule.

Increased activity of mitochondrial uncoupling protein 2 improves stress resistance in cultured endothelial cells exposed in vitro to high glucose levels.

The endothelium is relatively independent of the mitochondrial energy supply, but mitochondria-derived ROS may play an important role in the development of many cardiovascular diseases. Energy-dissipating uncoupling proteins (UCPs) mediate free fatty acid-activated, purine nucleotide-inhibited proton conductance (uncoupling) in the inner mitochondrial membrane. We have described a functional characteristic and an antioxidative role for UCP2 in endothelial cells and isolated mitochondria and how this function is altered by long-term growth in high concentrations of glucose. Human umbilical vein endothelial cells (EA.hy926 line) were grown in media with either high (25 mM) or normal (5.5 mM) glucose concentrations. Under nonphosphorylating and phosphorylating conditions, UCP activity was significantly higher in mitochondria isolated from high glucose-treated cells. More pronounced control of the respiratory rate, membrane potential, and ROS by UCP2 was observed in these mitochondria. A greater UCP2-mediated decrease in ROS generation indicates an improved antioxidative role for UCP2 under high glucose conditions. Mitochondrial and nonmitochondrial ROS generations were significantly higher in high glucose-treated cells independent of UCP2 expression. UCP2 gene silencing led to elevated mitochondrial ROS formation and ICAM1 expression, especially in high glucose-cultured cells. UCP2 influenced endothelial cell viability and resistance to oxidative stress. Endothelial cells exposed to high glucose concentrations were significantly more resistant to peroxide. In these cells, the increased activity of UCP2 led to improved stress resistance and protection against acute oxidative stress. Our results indicate that endothelial UCP2 may function as a sensor and negative regulator of mitochondrial ROS production in response to hyperglycemia.

Inhibition of Autophagic Turnover in β-Cells by Fatty Acids and Glucose Leads to Apoptotic Cell Death

Autophagy, a cellular recycling process responsible for turnover of cytoplasmic contents, is critical for maintenance of health. Defects in this process have been linked to diabetes. Diabetes-associated glucotoxicity/lipotoxicity contribute to impaired β-cell function and have been implicated as contributing factors to this disease. We tested the hypothesis that these two conditions affect β-cell function by modulating autophagy. We report that exposure of β-cell lines and human pancreatic islets to high levels of glucose and lipids blocks autophagic flux and leads to apoptotic cell death. EM analysis showed accumulation of autophagy intermediates (autophagosomes), with abundant engulfed cargo in palmitic acid (PA)- or glucose-treated cells, indicating suppressed autophagic turnover. EM studies also showed accumulation of damaged mitochondria, endoplasmic reticulum distention, and vacuolar changes in PA-treated cells. Pulse-chase experiments indicated decreased protein turnover in β-cells treated with PA/glucose. Expression of mTORC1, an inhibitor of autophagy, was elevated in β-cells treated with PA/glucose. mTORC1 inhibition, by treatment with rapamycin, reversed changes in autophagic flux, and cell death induced by glucose/PA. Our results indicate that nutrient toxicity-induced cell death occurs via impaired autophagy and is mediated by activation of mTORC1 in β-cells, contributing to β-cell failure in the presence of metabolic stress.

Characterization and Antioxidant Properties of OJP2, a Polysaccharide Isolated from &amp;lt;i&amp;gt;Ophiopogon japonicus&amp;lt;/i&amp;gt;

A water-soluble polysaccharide (OJP2) obtained from the roots of Ophiopogon japonicas, was precipitated with 95% ethanol and purified by DEAE-52 cellulose anion-exchange and Sephadex G-100 gel filtration chromatography. The characteristics of OJP2 were determined by chemical analysis, high performance gel permeation chromatography (HPGPC), and gas chromatography-mass spectrometry (GC-MS). The results showed that the average molecular weight (Mw) of OJP2 was 35.2 kDa, and five kinds of monosaccharides including rhamnose, arabinose, xylose, glucose and galactose in a molar ratio of 0.5:5:4:1:10. Furthermore, the antioxidant activity of OJP2 was evaluated in H2O2-treated HaCaT cells and glucose-treated LO2 cells. The results show that OJP2 can increase the activity of SOD and NO production, and decrease the level of MDA in these two kinds of injury cells. OJP2 should be explored as a novel and potential natural antioxidant agent for use in functional foods or medicine.

The Effects of High Glucose Levels on Reactive Oxygen Species-Induced Apoptosis and Involved Signaling in Human Vascular Endothelial Cells

This study investigated the effects of high glucose levels on human vascular endothelial cells and the expression of apoptosis-associated signaling molecules. Cell proliferation of human umbilical vein endothelial cells (HUVECs) was analyzed by colorimetric assay and cell number counting. Apoptosis was measured by Annexin V/FITC staining and flow cytometry. Gene knockdown was established by transfection of synthesized small interfering RNA. Caspase-3 activation was inhibited by a caspase-3 inhibitor. Protein expression of signaling molecules was measured by Western blot. Glucose significantly decreased HUVEC viability, induced cell apoptosis, and elevated levels of intracellular reactive oxygen species in a concentration and time-dependent manner. Glucose significantly upregulated the Nox4 isoform of nicotinamide adenine dinucleotide phosphate oxidase and phosphatase and tensin homolog (PTEN) protein expression, increased PTEN phosphorylation, and activated caspase-3 in a concentration-dependent manner. Silencing Nox4 and PTEN gene expression and inhibiting caspase-3 activation significantly protected HUVECs from glucose-induced cell apoptosis. Silencing Nox4 significantly normalized the levels of reactive oxygen species in glucose-treated cells; 20 mM glucose obviously upregulated Nox4, PTEN, phosphor-PTEN, and Bax levels, but significantly reduced integrin-linked kinase (ILK) activity, Bcl-2 (B cell lymphoma 2) expression, and protein kinase B (Akt) phosphorylation at serine 473. High glucose levels can reduce cell viability and induce apoptosis in HUVECs through Nox4-produced reactive oxygen species. Elevated levels of reactive oxygen species decreased Bcl-2 expression and increased PTEN expression and phosphorylation, which lead to the subsequent inhibition of ILK-Akt signaling, elevation of Bax expression, and activation of caspase-3.