Inhibitory Effect Of High Glucose Research Articles

Abstract 3112: Identifying NogoB Receptor As A Resilience Factor In Preventing Diabetes-associated Vasculature Complications Through The Regulation Of Histone Acetylation

The COVID-19 pandemic has underlined the link between cytokine storms and acute lung injury (ALI). Importantly, diabetic patients demonstrate a higher susceptibility to severe lung injury and increased mortality, indicating that hyperglycemia could compromise the host's resilience necessary to counteract cytokine-induced lung injury. Single-nucleus RNA-seq data from COVID-19 patients reveal a significant decline in endothelial cells expressing the Nogo-B receptor (NgBR). Our recent research confirms that NgBR plays a pivotal role in maintaining the structural integrity of blood vessels by regulating KAT7-mediated histone acetylation.To investigate NgBR's resilience role, we examined its expression in response to hyperglycemic stress. We found a reduction in NgBR expression levels in endothelial cells (ECs) isolated from db/db mice, mirroring observations in ECs from Streptozocin (STZ)-injected mice. Moreover, when human microvascular endothelial cells (HMECs) were exposed to high glucose levels, NgBR expression increased initially but subsequently declined with extended exposure. This inhibitory effect of high glucose on NgBR expression was both time- and dose-dependent and was mediated through the DNA methylation mechanism.We also examined the effects of NgBR loss on the integrity of pulmonary vasculature and the pathogenesis of lung injury. We induced genetic depletion of NgBR in ECs through tamoxifen administration and found severe lung hemorrhage in NgBR inducible endothelial cell-specific knockout (iecKO) mice. Impaired EC junctions were identified by reduced VE-cadherin immunostaining. Following exposure to a lipopolysaccharide (LPS), ALI model, we observed increased neutrophil accumulation in the alveolar or interstitial space, alveolar wall thickening, and edema in the lungs of either NgBR iecKO or diabetic mice. RNA-seq analysis revealed that NgBR knockdown resulted in decreased transcription of CDC42, a key gene regulating endothelial adherens junction through KAT7-mediated histone acetylation. Our findings indicate that NgBR is a resilience factor for maintaining vascular integrity, and epigenetic medicine can rescue NgBR loss in endothelial cells and the severity of diabetes-associated acute lung injury.

Study on the Regulation of Trophoblast Activity by Abnormally Expressed lncRNA CCDC144NL-AS1 in Patients with Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a common complication in pregnant women. The growth and differentiation of trophoblast cells determine the function of the placenta, and therefore further affect the transport of nutrients to the fetus. lncRNA Coiled-Coil Domain Containing 144 N-Terminal-Like antisense1 (CCDC144NL-AS1) was reported to be abnormally expressed in GDM, but its function and mechanism remain undefined. This study aimed to reveal the expression of CCDC144NL-AS1 in GDM and evaluate its significance in disease development. The expression of CCDC144NL-AS1 in serum and placenta tissues of GDM patients and healthy pregnant women was evaluated using PCR. The effect of CCDC144NL-AS1 on the proliferation, migration, and invasion of trophoblast cells was evaluated with CCK8 and Transwell assay. The mechanism of the interaction between CCDC144NL-AS1 and miR-143-3p was assessed by luciferase reporter assay and cell transfection. CCDC144NL-AS1 was upregulated in GDM patients, which discriminated GDM patients from healthy pregnant women with high sensitivity and specificity and was positively correlated with the insulin resistance indexes. In trophoblast cells, high glucose exposure induced increased CCDC144NL-AS1 and suppressed cell proliferation, migration, and invasion. Silencing CCDC144NL-AS1 could alleviate the inhibitory effect of high glucose, while the knockdown of miR-143-3p reversed the effect of CCDC144NL-AS1. In conclusion, upregulated CCDC144NL-AS1 served as a diagnostic biomarker of GDM and regulated the development of trophoblast cells via negatively modulating miR-143-3p.

Ghrelin regulates the proliferation and apoptosis of high glucose‐induced islet cells through the PI3K–Akt signaling pathway

Ghrelin may have therapeutic value in mitigating insulin resistance and type 2 diabetes, based on which we further explore the action mechanism of ghrelin on islet cells in this research. In the course of experiments, MIN6 cells were induced by glucose and then treated with acylated or unacylated ghrelin. The effects of ghrelin on the viability, proliferation, apoptosis, and insulin release of high glucose-induced islet cells were detected by CellCounting Kit-8, 5-ethynyl-2'-deoxyuridine, flow cytometry, and enzyme-linked immunosorbent assays, respectively. Meanwhile, cells were treated with LY294002 to explore whether and how the inhibited phosphoinositide 3-kinase-protein kinase B(PI3K-AKT) signaling pathway participated in the internal mechanism of ghrelin-regulating islet cells. Western blotting was performed to quantify the expression levels of Bcl-2, Bax, Cleaved caspase-3, PI3K, and AKT. As a result, ghrelin alleviated high glucose-induced suppression of viability and proliferation and promotion on apoptosis of MIN6 cells. Ghrelin also attenuated the inhibitory effects of high glucose on expression levels of PI3K-Akt signaling axis-related proteins and insulin release in MIN6 cells. Besides, ghrelin weakened the impacts of high glucose on boosting MIN6 cell apoptosis and hindering proliferation through thePI3K-Akt signaling axis. Collectively, ghrelin regulates the proliferation and apoptosis of high glucose-induced islet cells through the PI3K-Akt signaling pathway.

Uncarboxylated osteocalcin alleviates the inhibitory effect of high glucose on osteogenic differentiation of mouse bone marrow\u2013derived mesenchymal stem cells by regulating TP63

BackgroundProgressive population aging has contributed to the increased global prevalence of diabetes and osteoporosis. Inhibition of osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by hyperglycemia is a potential pathogenetic mechanism of osteoporosis in diabetic patients. Uncarboxylated osteocalcin (GluOC), a protein secreted by mature osteoblasts, regulates bone development as well as glucose and lipid metabolism. In our previous studies, GluOC was shown to promote osteoblastic differentiation of BMSCs; however, the underlying mechanisms are not well characterized. Tumor protein 63 (TP63), as a transcription factor, is closely related to bone development and glucose metabolism.ResultsIn this study, we verified that high glucose suppressed osteogenesis and upregulated adipogenesis in BMSCs, while GluOC alleviated this phenomenon. In addition, high glucose enhanced TP63 expression while GluOC diminished it. Knock-down of TP63 by siRNA transfection restored the inhibitory effect of high glucose on osteogenic differentiation. Furthermore, we detected the downstream signaling pathway PTEN/Akt/GSK3β. We found that diminishing TP63 decreased PTEN expression and promoted the phosphorylation of Akt and GSK3β. We then applied the activator and inhibitor of Akt, and concluded that PTEN/Akt/GSK3β participated in regulating the differentiation of BMSCs.ConclusionsOur results indicate that GluOC reduces the inhibitory effect of high glucose on osteoblast differentiation by regulating the TP63/PTEN/Akt/GSK3β pathway. TP63 is a potential novel target for the prevention and treatment of diabetic osteoporosis.

Activating transcription factor 4 is required for high glucose inhibits proliferation and differentiation of MC3T3-E1 cells

Activating transcription factor 4 (ATF4) promotes bone formation in human bone marrow mesenchymal stem cells. However, the underlying mechanisms of ATF4 in high glucose-induced injury of osteoblast still remain unclear. Small interfering RNA and plasmid targeting ATF4 were used to transfect MC3T3-E1 cells to knock down and overexpress ATF4 using Lipofectamin 3000. Cell viability, alkaline phosphatase (ALP) activity and levels were determined by MTT, ALP kit assay, quantitative real-time (qRT)-PCR and Western blot. Osteocalcin (OCN) expression was determined by ELISA, PCR and Western blot. The mRNA and protein levels of ATF4, glucose regulated protein 78 kDa (GRP78) and C/EBP homologous protein (CHOP) were detected by PCR and Western blot. In the current study, viabilities of MC3T3-E1 cells were inhibited by high glucose. Meanwhile, the mRNA and protein levels of ATF4 were effectively up-regulated in high glucose-incubated MC3T3-E1 cells. By conducting functional experiments, silencing ATF4 induced by small interfering RNA partially reversed the inhibitory effects of high glucose on viabilities of MC3T3-E1 cells. We also found that the expressions of ER stress-related proteins (ATF4, GRP78 and CHOP) were higher in high glucose-treated MC3T3-E1 cells but were inhibited by siATF4. However, overexpression of AFT4 had opposite results, and high glucose attenuated the protein levels of osteogenic marker genes ALP and OCN, which were further inhibited by ATF4 knockout gene. Thus, ATF4 was a necessary gene for high glucose to inhibit the proliferation and differentiation of MC3T3-E1 cells.

Deregulation of phytoene-\u03b2-carotene synthase results in derepression of astaxanthin synthesis at high glucose concentration in Phaffia rhodozyma astaxanthin-overproducing strain MK19

BackgroundA major obstacle to industrial-scale astaxanthin production by the yeast Phaffia rhodozyma is the strong inhibitory effect of high glucose concentration on astaxanthin synthesis. We investigated, for the first time, the mechanism of the regulatory effect of high glucose (> 100 g/L) at the metabolite and transcription levels.ResultsTotal carotenoid, β-carotene, and astaxanthin contents were greatly reduced in wild-type JCM9042 at high (110 g/L) glucose; in particular, β-carotene content at 24–72 h was only 14–17% of that at low (40 g/L) glucose. The inhibitory effect of high glucose on astaxanthin synthesis appeared to be due mainly to repression of lycopene-to-β-carotene and β-carotene-to-astaxanthin steps in the pathway. Expression of carotenogenic genes crtE, pbs, and ast was also strongly inhibited by high glucose; such inhibition was mediated by creA, a global negative regulator of carotenogenic genes which is strongly induced by glucose. In contrast, astaxanthin-overproducing, glucose metabolic derepression mutant strain MK19 displayed de-inhibition of astaxanthin synthesis at 110 g/L glucose; this de-inhibition was due mainly to deregulation of pbs and ast expression, which in turn resulted from low creA expression. Failure of glucose to induce the genes reg1 and hxk2, which maintain CreA activity, also accounts for the fact that astaxanthin synthesis in MK19 was not repressed at high glucose.ConclusionWe conclude that astaxanthin synthesis in MK19 at high glucose is enhanced primarily through derepression of carotenogenic genes (particularly pbs), and that this process is mediated by CreA, Reg1, and Hxk2 in the glucose signaling pathway.

MicroRNA-590-5p antagonizes the inhibitory effect of high glucose on osteoblast differentiation by suppressing Smad7 in MC3T3-E1 cells.

ObjectiveMicroRNA-590-5p (miR-590-5p) has been reported to stimulate osteoblast differentiation; however, its effect in diabetic osteoporosis remains unknown. This study investigated the effect of miR-590-5p on high glucose (HG)-suppressed osteoblast differentiation.MethodsThe effect of HG on MC3T3-E1 cell survival was assessed using the MTT assay. The expression levels and activities of osteoblastic proteins were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR), alkaline phosphatase (ALP) assay, and immunoblotting assay. Tumor growth factor-β (TGF-β) signaling in MC3T3-E1 cells was assessed using luciferase assay, qRT-PCR, and immunoblotting. Mineralized nodule formation in MC3T3-E1 cells was examined by using the mineralization assay.ResultsWhen MC3T3-E1 cells were exposed to HG conditions, there was significant downregulation of miR-590-5p and osteoblastic proteins (e.g., collagen I, Runx2, and ALP); in contrast, Smad7 was upregulated. Furthermore, miR-590-5p targeted Smad7 and inhibited its expression. Additionally, overexpression of miR-590-5p significantly promoted osteoblast growth and differentiation by upregulating TGF-β signaling in HG-treated MC3T3-E1 cells.ConclusionsCollectively, the results showed that miR-590-5p was involved in osteogenesis; moreover, miR-590-5p may represent a potential target for the treatment of diabetic osteoporosis.

Celastrol antagonizes high glucose-evoked podocyte injury, inflammation and insulin resistance by restoring the HO-1-mediated autophagy pathway

Diabetic nephropathy (DN) contributes to end-stage renal disease and kidney dysfunction with a proverbial feature of podocyte injury. Inflammation and insulin resistance is recently implicated in the pathogenesis of diabetic kidney injury. Celastrol exerts critical roles in inflammatory diseases and injury progression. However, its function and mechanism in DN remains elusive. Here, celastrol dose-dependently restored podocyte viability under high glucose (HG) conditions, but with little cytotoxicity in podocyte. Preconditioning with celastrol counteracted HG-evoked cell apoptosis, LDH release, ROS production and podocyte depletion. Additionally, HG-elevated high transcripts and secretions of pro-inflammatory cytokines were reversed following celastrol treatment, including IL-1β, TNF-α, IL-6. Simultaneously, the inhibitory effects of HG on insulin-triggered glucose uptake and nephrin expression were overturned after celastrol exposure. Intriguingly, celastrol restored HG-induced deficiency of autophagy pathway. Nevertheless, blocking the autophagy signaling by its antagonist 3-MA muted celastrol-protected against HG-evoked cell injury, inflammation and insulin resistance. Importantly, celastrol enhanced heme oxygenase-1 (HO-1) expression in HG-stimulated podocytes. Notably, HO-1 cessation depressed autophagy pathway activation and subsequently blunted beneficial effects of celastrol on HG-exposed podocytes. These finding suggest that celastrol may protect against HG-induced podocyte injury, inflammation and insulin resistance by restoring HO-1-mediated autophagy pathway, implying a promising therapeutic strategy against DN.

High glucose-induced oxidative stress impairs proliferation and migration of human gingival fibroblasts.

Delayed gingival wound healing is widely observed in periodontal patients with diabetes. However, the molecular mechanisms of the impaired function of gingival fibroblasts in diabetes remain unclear. The purpose of this study was to investigate changes in the properties of human gingival fibroblasts (HGFs) under high-glucose conditions. Primary HGFs were isolated from healthy gingiva and cultured with 5.5, 25, 50, and 75 mM glucose for 72 h. In vitro wound healing, 5-ethynyl-2′-deoxyuridine (EdU), and water-soluble tetrazolium salt (WST-8) assays were performed to examine cell migration and proliferation. Lactase dehydrogenase (LDH) levels were measured to determine cytotoxicity. The mRNA expression levels of oxidative stress markers were quantified by real-time PCR. Intracellular reactive oxygen species (ROS) were also measured in live cells. The antioxidant N-acetyl-l-cysteine (NAC, 1 mM) was added to evaluate the involvement of ROS in the glucose effect on HGFs. As a result, the in vitro wound healing assay showed that high glucose levels significantly reduced fibroblast migration and proliferation at 6, 12, 24, 36, and 48 h. The numbers of cells positive for EdU staining were decreased, as was cell viability, at 50 and 75 mM glucose. A significant increase in LDH was proportional to the glucose concentration. The mRNA levels of heme oxygenase-1 and superoxide dismutase-1 and ROS levels were significantly increased in HGFs after 72 h of exposure to 50 mM glucose concentration. The addition of NAC diminished the inhibitory effect of high glucose in the in vitro wound healing assay. The results of the present study show that high glucose impairs the proliferation and migration of HGFs. Fibroblast dysfunction may therefore be caused by high glucose-induced oxidative stress and may explain the delayed gingival wound healing in diabetic patients.

High Glucose Inhibits Neural Stem Cell Differentiation Through Oxidative Stress and Endoplasmic Reticulum Stress.

Maternal diabetes induces neural tube defects by suppressing neurogenesis in the developing neuroepithelium. Our recent study further revealed that high glucose inhibited embryonic stem cell differentiation into neural lineage cells. However, the mechanism whereby high glucose suppresses neural differentiation is unclear. To investigate whether high glucose-induced oxidative stress and endoplasmic reticulum (ER) stress lead to the inhibition of neural differentiation, the effect of high glucose on neural stem cell (the C17.2 cell line) differentiation was examined. Neural stem cells were cultured in normal glucose (5 mM) or high glucose (25 mM) differentiation medium for 3, 5, and 7 days. High glucose suppressed neural stem cell differentiation by significantly decreasing the expression of the neuron marker Tuj1 and the glial cell marker GFAP and the numbers of Tuj1+ and GFAP+ cells. The antioxidant enzyme superoxide dismutase mimetic Tempol reversed high glucose-decreased Tuj1 and GFAP expression and restored the numbers of neurons and glial cells differentiated from neural stem cells. Hydrogen peroxide treatment imitated the inhibitory effect of high glucose on neural stem cell differentiation. Both high glucose and hydrogen peroxide triggered ER stress, whereas Tempol blocked high glucose-induced ER stress. The ER stress inhibitor, 4-phenylbutyrate, abolished the inhibition of high glucose or hydrogen peroxide on neural stem cell differentiation. Thus, oxidative stress and its resultant ER stress mediate the inhibitory effect of high glucose on neural stem cell differentiation.

Exendin-4 relieves the inhibitory effects of high glucose on the proliferation and osteoblastic differentiation of periodontal ligament stem cells

BackgroundWith the impaired regenerative potential in patients with diabetes mellitus (DM), Periodontal ligament stem cells (PDLSCs) are regarded as an attractive source of stem cells for periodontal cytotherapy. Recent studies have shown that Exendin-4 (Ex-4) exerts cell-protective effects and bone remodeling ability on many types of cells. The aim of this study was to investigate whether Ex-4 alleviates the inhibition of high glucose on the proliferation and osteogenic differentiation of PDLSCs. MethodsPDLSCs were incubated in medium supplemented with 5.5 mM d-glucose (NG), 30 mM d-glucose (HG), NG plus Ex-4, and HG plus different concentration (1, 10, 20, 100 nM) of Ex-4 respectively. Cell proliferation was detected by CCK-8 assay and cell cycle analysis. Osteogenesis was assessed by Alizarin Red S staining and evaluation of the mRNA expression of Runx2, ALP and Osx at day 7, 14 and 21. Intracellular level of reactive oxygen species (ROS) was detected using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (CMH2DCF-DA). ResultsThe proliferation ability, mineralized nodules forming capacity and the mRNA expression of Runx2, ALP and Osx of PDLSCs in HG group were decreased, the ROS level was increased compared to NG group. With the treatment of Ex-4, the HG-inhibited proliferation ability and osteogenic differentiation ability of PDLSCs were significantly reversed, the HG-increased ROS level could be down-regulated. Moreover, Ex-4 enhanced the osteogenic differentiation of normal PDLSCs. ConclusionsEx-4 alleviates the inhibitory effect of HG on the proliferation and osteoblastic differentiation of PDLSCs, and has a significant enhance in the osteoblastic differentiation of normal PDLSCs, giving new insights into the possible therapeutic method of diabetic periodontitis.

Oxidative stress-induced miR-27a targets the redox gene nuclear factor erythroid 2-related factor 2 in diabetic embryopathy

Maternal diabetes induces neural tube defects, and oxidative stress is a causal factor for maternal diabetes-induced neural tube defects. The redox gene nuclear factor erythroid 2-related factor 2 is the master regulator of the cellular antioxidant system. In this study, we aimed to determine whether maternal diabetes inhibits nuclear factor erythroid 2-related factor 2 expression and nuclear factor erythroid 2-related factor 2-controlled antioxidant genes through the redox-sensitive miR-27a. We used a well-established type 1 diabetic embryopathy mouse model induced by streptozotocin for our invivo studies. Embryos at embryonic day 8.5 were harvested for analysis of nuclear factor erythroid 2-related factor 2, nuclear factor erythroid 2-related factor 2-controlled antioxidant genes, and miR-27a expression. To determine if mitigating oxidative stress inhibits the increase of miR-27a and the decrease of nuclear factor erythroid 2-related factor 2 expression, we induced diabetic embryopathy in superoxide dismutase 2 (mitochondrial-associated antioxidant gene)-overexpressing mice. This model exhibits reduced mitochondria reactive oxygen species even in the presence of hyperglycemia. To investigate the causal relationship between miR-27a and nuclear factor erythroid 2-related factor 2 invitro, we examined C17.2 neural stem cells under normal and high-glucose conditions. We observed that the messenger RNA and protein levels of nuclear factor erythroid 2-related factor 2 were significantly decreased in embryos on embryonic day 8.5 from diabetic dams compared to thosefrom nondiabetic dams. High-glucose also significantly decreased nuclear factor erythroid 2-related factor 2 expression in a dose- and time-dependent manner in cultured neural stem cells. Our data revealed that miR-27a was up-regulated in embryos on embryonic day 8.5 exposed to diabetes, and that high glucose increased miR-27a levels in a dose- and time-dependent manner in cultured neural stem cells. In addition, we found that a miR-27a inhibitor abrogated the inhibitory effect of high glucose on nuclear factor erythroid 2-related factor 2 expression, and a miR-27a mimic suppressed nuclear factor erythroid 2-related factor 2 expression in cultured neural stem cells. Furthermore, our data indicated that the nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1 were down-regulated by maternal diabetes in embryos on embryonic day 8.5 and high glucose in cultured neural stem cells. Inhibiting miR-27a restored expression of glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1. Overexpressing superoxide dismutase 2 reversed the maternal diabetes-induced increase of miR-27a and suppression of nuclear factor erythroid 2-related factor 2 and nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes. Our study demonstrates that maternal diabetes-induced oxidative stress increases miR-27a, which, in turn, suppresses nuclear factor erythroid 2-related factor 2 and its responsive antioxidant enzymes, resulting in diabetic embryopathy.

89 - Oxidative Stress-induced miR-27a Targets the Redox gene Nrf2 in Diabetic Embryopathy

Maternal diabetes induces neural tube defects (NTDs), and oxidative stress is a causal factor for maternal diabetes-induces NTDs. The redox gene Nrf2 (nuclear factor-erythroid 2-related factor 2) is the master regulator of the cellular antioxidant system. In the present study, we aimed to determine whether maternal diabetes inhibits Nrf2 expression and Nrf2-controlled antioxidant genes through the redox-sensitive miR-27a. We used a well-established type 1 diabetic embryopathy mouse model induced by streptozotocin for our in vivo studies. Embryos at E8.5 were harvested for analysis of Nrf2, Nrf2-controlled antioxidant genes and miR-27a expression. To determine if mitigating oxidative stress inhibits the increase of miR-27a and the decrease of Nrf2 expression, we induced diabetic embryopathy in SOD2 (mitochondrial-associated antioxidant gene)-overexpressing mice. This model exhibits reduced mitochondria reactive oxygen species even in the presence of hyperglycemia. To investigate the causal relationship between miR-27a and Nrf2 in vitro, we examined C17.2 neural stem cells under normal and high glucose conditions. We observed that the mRNA and protein levels of Nrf2 were significantly decreased in E8.5 embryos from diabetic dams compared to those from nondiabetic dams. High glucose also significantly decreased Nrf2 expression in a dose- and time- dependent manner in cultured neural stem cells. Our data revealed that miR-27a was up-regulated in E8.5 embryos exposed to diabetes, and that high glucose increased miR-27a levels in a dose- and time-dependent manner in cultured neural stem cells. In addition, we found that a miR-27a inhibitor abrogated the inhibitory effect of high glucose on Nrf2 expression, and a miR-27a mimic suppressed Nrf2 expression in cultured neural stem cells. Furthermore, our data indicated that the Nrf2-controlled antioxidant enzymes glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cyteine ligase modifier subunit (GLCM), and glutathione S-transferase A1 (GSTA1) were downregulated by maternal diabetes in E8.5 embryos and high glucose in cultured neural stem cells. Inhibiting miR-27a restored expression of GCLC, GLCM and GSTA1. Overexpressing SOD2 reversed the maternal diabetes-induced increase of miR-27a and suppression of Nrf2 and Nrf2-controlled antioxidant enzymes. Our study demonstrates that maternal diabetes-induced oxidative stress increases miR-27a, which, in turn, suppresses Nrf2 and its responsive antioxidant enzymes, resulting in diabetic embryopathy.

Effects of High Glucose on Cell Viability and Differentiation in Primary Cultured Schwann Cells: Potential Role of ERK Signaling Pathway.

Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes mellitus and hyperglycemia is considered to be the major factor in the development and progression of DPN. Because of the contribution of Schwann cells (SCs) to the pathology of DPN, we investigated the effects of high glucose on cell proliferation, apoptosis and differentiation in primary cultured SCs. Cell Counting Kit-8 (CCK-8) assay and Hoechst staining showed that high glucose inhibited SCs proliferation and increased apoptosis ratio in time and concentration dependent manner. Western blot and real-time quantitative PCR analysis revealed that the major myelin proteins and genes expressions including P0, MAG and Krox-20, were downregulated time dependently in SCs exposed to high glucose from 48 to 96h. To further elucidate the underlying pathogenic mechanisms, we also explored the role of ERK signaling pathway in high glucose induced SC injury, which has been proved to drive demyelination of peripheral nerves. The western blot analysis showed that compared with control group phosphorylation level of ERK was increased by 14.3% in SCs exposed to high glucose for 72h (P<0.01). Using immunocytochemistry analysis, we observed that the ERK specific inhibitor U0126 blocked the ERK activation induced by high glucose and reversed the inhibitory effect of high glucose on P0 expression. Taken together, these results suggest that high glucose can cause damage in primary cultured SCs and may exert the inhibitory effect on SC differentiation and myelination through ERK signaling activation.

Abstract 11691: Atorvastatin Reverses Impaired Voltage-gated K + Channels-mediated Coronary Vasodilation By Upregulating PPARγ In Diabetes (Best of Basic Science Abstract)

Aims: Voltage-gated K + (K v ) channels in vascular smooth muscle cells (VSMCs) play an important role in the regulation of coronary microcirculation. Atorvastatin (ATV) has shown some beneficial effects on vascular function, but the effect of ATV on K v channels-mediated coronary vasodilation remains unknown. The present study was designed to investigate the role of ATV in improving K v channels-mediated coronary dilator function in diabetes and the underlying mechanisms. Methods: Isolated VSMCs were incubated in normal or high glucose medium plus a different dose of ATV for 24 h at 37 o C. Patch-clamp recording and molecular biological techniques were used to assess the function and expression of K v channels. Control or type 2 diabetic rats were treated with ATV (50 mg/kg daily) by oral gavage for 10 weeks. Vasodilation of isolated rat coronary arteries was measured using a pressurized myograph. GW9662, the peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, was used to determine whether the mechanism of ATV-improved K v channel function can be explained by upregulation of PPARγ pathway. Results: Patch-clamp analysis revealed that high glucose reduced K v current density by 58.7 ± 4.6%, which was accompanied by a downregulation of K v 1.2 and K v 1.5 expression. Treatment with ATV reversed the inhibitory effect of high glucose on K v current density in a dose-dependent manner (1 μmol/L, 15.0 ± 2.6%; 10 μmol/L, 49.1 ± 3.8%; 100 μmol/L, 69.9 ± 4.8%; P &lt; 0.05). In addition, ATV restored high glucose-induced downregulation of K v channel protein expression, and the difference was significant in both 10 μmol/L and 100 μmol/L groups. For in vivo study, K v channels-mediated coronary vasodilation was decreased in diabetic rats, compared with controls (9.1 ± 1.3 vs. 36.7 ± 1.4%, P &lt; 0.05), whereas this decrease was partly corrected by ATV (25.0 ± 2.8 vs. 9.1 ± 1.3%, P &lt; 0.05). Treatment with ATV prominently increased protein expression of PPARγ both in vitro and in vivo . The effect of ATV in regulating K v channels and K v channels-mediated vasodilation was markedly blunted by GW9662. Conclusions: In conclusion, treatment with ATV activates PPARγ pathway and preserves K v channel activity in VSMCs, thus providing improvement of coronary dilator function in diabetic rats.

Superoxide Dismutase 1 In Vivo Ameliorates Maternal Diabetes Mellitus-Induced Apoptosis and Heart Defects Through Restoration of Impaired Wnt Signaling.

Oxidative stress is manifested in embryos exposed to maternal diabetes mellitus, yet specific mechanisms for diabetes mellitus-induced heart defects are not defined. Gene deletion of intermediates of Wingless-related integration (Wnt) signaling causes heart defects similar to those observed in embryos from diabetic pregnancies. We tested the hypothesis that diabetes mellitus-induced oxidative stress impairs Wnt signaling, thereby causing heart defects, and that these defects can be rescued by transgenic overexpression of the reactive oxygen species scavenger superoxide dismutase 1 (SOD1). Wild-type (WT) and SOD1-overexpressing embryos from nondiabetic WT control dams and nondiabetic/diabetic WT female mice mated with SOD1 transgenic male mice were analyzed. No heart defects were observed in WT and SOD1 embryos under nondiabetic conditions. WT embryos of diabetic dams had a 26% incidence of cardiac outlet defects that were suppressed by SOD1 overexpression. Insulin treatment reduced blood glucose levels and heart defects. Diabetes mellitus increased superoxide production, canonical Wnt antagonist expression, caspase activation, and apoptosis and suppressed cell proliferation. Diabetes mellitus suppressed Wnt signaling intermediates and Wnt target gene expression in the embryonic heart, each of which were reversed by SOD1 overexpression. Hydrogen peroxide and peroxynitrite mimicked the inhibitory effect of high glucose on Wnt signaling, which was abolished by the SOD1 mimetic, tempol. The oxidative stress of diabetes mellitus impairs Wnt signaling and causes cardiac outlet defects that are rescued by SOD1 overexpression. This suggests that targeting of components of the Wnt5a signaling pathway may be a viable strategy for suppression of congenital heart defects in fetuses of diabetic pregnancies.

Hyperglycemia Promotes Human Gastric Carcinoma Progression via Aquaporin 3.

Hyperglycemia plays an important role in the development of gastric carcinoma (GC). Aquaporin 3 (AQP3) is overexpressed in GC and involved in carcinogenesis and progression of GC. Hyperglycemia promotes AQP3 expression in human peritoneal mesothelial cells. To investigate whether hyperglycemia promotes progression of GC via AQP3. We enrolled 978 patients with GC and evaluated the correlation between preoperative fasting plasma glucose and clinicopathological features. AQP3 was detected by immunohistochemistry in human GC specimens. Western blotting and real-time quantitative polymerase chain reaction evaluated changes in AQP3 expression in human GC MGC803 and SGC7901 cells after co-culture with high glucose. Transwell migration and Cell Counting Kit-8 assays were used to determine migration and proliferation of GC cells. Hyperglycemia (fasting plasma glucose ≥6.1mM) correlated with tumor size, location, and pTNM stage. AQP3 expression in tumor tissue was associated with fasting plasma glucose levels. High glucose concentration upregulated AQP3 expression in a dose- and time-dependent manner. High glucose concentration promoted GC cell migration markedly, and AQP3 knockdown with siRNA could abolish the increase in cell migration. However, high glucose concentration inhibited cell proliferation, and AQP3 knockdown significantly enhanced the inhibitory effect of high glucose. The ERK and PI3K/AKT signaling pathways were involved in high glucose regulation of AQP3 in human GC cells. Hyperglycemia promotes GC progress via AQP3. This improves our understanding of the mechanism of hyperglycemia-induced carcinogenesis and provides a potential therapeutic strategy for GC.

Autophagy impairment aggravates the inhibitory effects of high glucose on osteoblast viability and function

Autophagy is a highly regulated homoeostatic process involved in the lysosomal degradation of damaged cell organelles and proteins. This process is considered an important pro-survival mechanism under diverse stress conditions. A diabetic milieu is known to hamper osteoblast viability and function. In the present study, we explored the putative protective role of autophagy in osteoblastic cells exposed to an HG (high glucose) medium. HG was found to increase protein oxidation and triggered autophagy by a mechanism dependent on reactive oxygen species overproduction in osteoblastic MC3T3-E1 cells. MC3T3-E1 cell survival was impaired by HG and worsened by chemical or genetic inhibition of autophagy. These findings were mimicked by H2O2-induced oxidative stress in these cells. Autophagy impairment led to both defective mitochondrial morphology and decreased bioenergetic machinery and inhibited further osteoblast differentiation in MC3T3-E1 cells upon exposure to HG. These novel findings indicate that autophagy is an essential mechanism to maintain osteoblast viability and function in an HG environment.

Substrate Oxidation Control of Respiratory Rates in Primary Hepatocytes

The goal of our studies is a bioenergetics profiling of primary rat hepatocytes using the Seahorse-XF analyzer in order to assess adaptation in response to metabolic stress or disease. Cellular oxygen consumption rates (JO2) were compared in enriched medium (DMEM) vs a balanced salt solution (HBSS) without added substrates. Hepatocytes exhibited higher basal JO2 in DMEM compared to HBSS and showed a proportional increase in oligomycin-insensitive JO2. The fractional increase in JO2 by uncoupler was higher in DMEM than in HBSS, presumably due to substrate supply by amino acids present in DMEM. These data suggests that substrate oxidative pathways exert significant control over basal and uncoupled respiration rates in primary rat hepatocytes. To further test this hypothesis, we assessed JO2 under different substrate conditions, in DMEM or HBSS medium. Addition of mono-methylsuccinate (MMS), a mitochondrial Complex III substrate, resulted in a large concentration- dependent stimulation of basal JO2 of hepatocytes in HBSS but a more limited stimulation in DMEM, likely reflecting availability of alternate substrates. In DMEM, physiological glucose concentrations (11mM) had little stimulatory effect, while higher concentrations (25mM) inhibited O2 uptake, thus exhibiting a “Crabtree-like” effect, which was not overcome by uncoupler treatment. This inhibitory effect of high glucose was not evident in HBSS, where basal JO2 increased with higher concentrations of glucose. Oligomycin-insensitive JO2, as a fraction of basal O2 uptake remained similar under all substrate conditions in DMEM and HBSS, apart from a small decrease at the highest MMS concentration. These results suggest a significant control exerted by substrate oxidative pathways over basal and uncoupler-stimulated respiration rates in primary rat hepatocytes. Electron supply may limit the rate of uncoupled respiration in hepatocytes, underestimating the reserve capacity in the electron transport chain. Supported by NIH grants AA018873 and AA017261.

High glucose impairs EDHF-mediated dilation of coronary arterioles via reduced cytochrome P450 activity

Endothelium-derived hyperpolarizing factor (EDHF) is an important vasodilator that regulates the vasomotor function. However, it remains unclear whether diabetes/hyperglycemia-induced vascular impairments extend to the EDHF. The present study aims to determine the effect of high glucose (HG) on EDHF-mediated arteriolar dilation and the underlying mechanism. Porcine coronary arterioles were isolated and pressurized for vasomotor study. Cultured porcine coronary artery endothelial cells (ECs) were used for molecular and biochemical analysis. Our results demonstrate that bradykinin (BK)-simulated arteriolar dilation is mediated by nitric oxide (NO) and EDHF pathways. Direct incubation of HG impaired vasodilation to BK but not to sodium nitroprusside (endothelium-independent vasodilator). In the presence of inhibitors of endothelial NO synthase (eNOS) and cyclooxygenase, the EDHF-mediated dilation was reduced by HG incubation. The inhibitory effect of HG was prevented by treating the vessels with superoxide scavenger Tempol. In cultured coronary endothelial cells, HG reduced endothelial epoxyeicosatrienoic acid (EET) production as well as cytochrome P450 epoxygenase (CYP) activity. Furthermore, the superoxide production was elevated in ECs after HG incubation. Pretreatment with Tempol before HG incubation prevented the increase of cellular superoxide and abolished the decrease of CYP activity. Collectively, our results suggest that, in addition to NO-mediated pathway, HG impairs the EET/EDHF-mediated vasodilation in coronary arterioles via the elevated level of superoxide leading to inhibition of CYP activity in coronary ECs.