HG Conditions Research Articles

Sestrin‑2 regulates podocyte mitochondrial dysfunctionand apoptosis under high‑glucose conditions via AMPK.

Diabetic kidney disease (DKD) is a severe form of microangiopathy among diabetic patients, of which podocyte injury is one of the more predominant features. There is increasing evidence to suggest that mitochondrial dysfunction is associated with podocyte injury, thus contributing to the progression of DKD. Initially identified as a p53 target protein, the endogenous antioxidant protein, sestrin-2 (sesn2), has recently attracted attention due to its potential function in various inflammatory diseases. However, the association between sesn2 and podocytes in DKD remains unclear. In the present study, to elucidate the role of sesn2 in podocyte mitochondrial dysfunction, the effects of sesn2 on the regulation of AMP-activated protein kinase (AMPK) were examined in vitro and in vivo. Abnormal mitochondria were found in rats with streptozotocin-induced diabetes, and hyperglycemia downregulated the expression of sesn2. The upregulation of sesn2 increased the level of AMPK phosphorylation, and thus ameliorated mitochondrial dysfunction under high glucose conditions (HG). On the whole, these results suggest that sesn2 is associated with mitochondrial dysfunction in podocytes under HG conditions. In addition, the decreased expression of sesn2 may be a therapeutic target for DKD.

Miniature 3D-printed integrated electrochemical cell for trace voltammetric Hg(II) determination

This work describes an integrated miniature all-3D-printed device for the determination of trace Hg(II) by anodic stripping voltammetry (ASV). The device is fabricated through a single-step printing process using a dual-extruder 3D printer and is composed of a mini vessel (printed from a polylactic acid (PLA) filament) and of 3 thermoplastic electrodes printed from a carbon-loaded PLA conductive filament and integrated at the bottom of the vessel. The working electrode surface is modified with a thin gold film electroplated in situ. The formation of the gold deposit was studied by linear sweep voltammetry and optical microscopy and the electrode materials as well as the chemical and instrumental conditions for Hg(II) determination were optimized. The limit of detection for Hg(II) is 0.52 μg L−1, the within-sensor repeatability (expressed as the coefficient of variation of 8 measurements of 20 μg L−1 Hg(II) at the same device) is 3.9 % and the between-cell reproducibility (expressed as the coefficient of variation 5 measurements of 20 μg L−1 Hg(II) conducted at different cells) is 8.9 %. The 3D-printed device was successfully applied to the determination of Hg(II) in bottled water and fish oil samples.

Preliminary Research of Main Components of Dll4/ Notch -VEGF Signaling Pathway Under High-Glucose Stimulation In Vitro

Objective: To set up high-glucose stressed cell model and investigate the expression of main components of Dll4/Notch-VEGF signaling pathway under high-glucose stimulation. Method: The cells (human umbilical vein endothelial cells, HUVECs) model under high-glucose condition were set up at first, and then the expression of Dll4, Notch1, Notch4 and VEGF in HG-stressed cells with or without Notch pathway blockage were analyzed through RT-PCR and western blot. To observe the cell migration, we also evaluated the Transwell assay. Results: HUVECs stimulated with 30mmol/L HG was chosen as cell model. Results from RT-PCR and western blot showed that HG stimulation induced Dll4, Notch1 and VEGF expression and down-regulated Notch4. The expressions were reversed after Notch pathway blockage, meanwhile, the blockage of Notch pathway inhibited cell migration under HG condition. Conclusions: Function of Notch4 is worth further researching under HG stimulation. Combination therapy by blocking Dll4/Notch and VEGF pathways might provide us a new way for anti-neovascularization. Funding Statement: This work was supported by the research program of Health and Family Planning Commission of 212 Sichuan Province, China (17PJ534). Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: This study performed in accordance with the Declaration of Helsinki and have been approved by ethics committee of Teaching Hospital of Chengdu University of Traditional Chinese Medicine.

Distinct downstream signaling and the roles of VEGF and PlGF in high glucose-mediated injuries of human retinal endothelial cells in culture

Vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) plays a crucial role in breakdown of the blood-retinal barrier due to hyperpermeability in diabetic retinopathy (DR). However, the distinct signaling driven by VEGF and PlGF in the pathogenesis of DR remains unclear. In this study, we investigated VEGF- and PlGF- related signaling pathways and their roles in cultured human microvascular retinal endothelial cells (hRECs) under high glucose conditions (HG; 25 mM). Apoptotic cell death was evaluated, and FITC conjugated bovine serum albumin across monolayer hRECs served as an index of permeability. Western blots were used to assess the protein levels of VEGF and PlGF, as well as the phosphorylation of p38MAPK, STAT1 and Erk1/2. Knockdown of VEGF and PlGF was performed by using siRNA. Following HG treatment, increases of VEGF and PlGF as well as PKC activity were detected in hRECs. Increased phosphorylations of p38MAPKThr180/Thr182, STAT1Ser727, and Erk1/2Tyr202/Tyr185 as well as VEGFR1Tyr1213 and VEGFR2Tyr1175 were also detected in HG-treated hRECs. Inhibition of PKC activity by Go 6976 prevented HG-induced increases of phosphor-Erk1/2 and nitric oxide synthase (NOS1) expressions as well as hyperpermeability, whereas inhibition of p38MAPK pathway by SB203580 selectively suppressed activation of STAT1 and decreased apoptotic cell death under HG conditions. Moreover, VEGF knockdown predominantly inhibited activation of VEGFR2, and phosphorylation of p38MAPK and STAT1, as well as apoptotic cell death in HG-treated hRECs. Nevertheless, PlGF knockdown mainly suppressed phosphorylation of VEGFR1, PKC, and Erk1/2, as well as NOS1 expressions and hyperpermeability. Taken together, we provide evidence demonstrating that HG-induced elevation of PlGF is responsible for hyperpermeability mainly through increasing activation of PKC-Erk1/2-NOS axis via VEGFR1, while HG-induced elevation of VEGF is associated with induction of apoptotic cell death mainly through increasing activation of p38MAPK/STAT1 signaling via VEGFR2.

Sodium Tanshinone IIA Silate Alleviates High Glucose Induced Barrier Impairment of Human Retinal Pigment Epithelium through the Reduction of NF-κB Activation via the AMPK/p300 Pathway

ABSTRACTPurpose: The disruption of retinal pigment epithelium (RPE) barrier may perform a crucial role in the pathogenesis of diabetic retinopathy (DR). AMPK exerts several salutary effects on photoreceptors and the RPE function and improves retina abnormalities. The current study aimed to determine whether sodium tanshinone IIA silate (STS) has an inhibitory effect on ARPE-19 cell monolayer permeability under high glucose conditions, and establish the underlying mechanism.Methods: We used a model of high glucose (25 mmol glucose, HG) condition mimicking diabetes in ARPE-19 cells, to assess the protective effects of STS. The barrier function of RPE cells were measured by Transepithelial Electrical Resistance (TEER) and fluorescein isothiocyanate (FITC)–dextran permeability. The interaction of NF-κB p65 and p300 were tested using immunoblotting and immunoprecipitation and immunofluorescence assay. Protein levels were assayed using Western blot.Results: We found STS promoted the phosphorylation of AMP-activated protein kinase (AMPK) at T172 in RPE cells, and STS treatment thus inhibited ARPE-19 cell monolayer permeability under HG condition, similar to the permeability under normal glucose (5.5 mmol glucose, NG). Moreover, we found that STS obviously prevented the colocalization of NF-κB and p300, and significantly inhibited their binding, subsequent decreased ARPE-19 cell monolayer permeability. Notably, Compound C (CC), a specific inhibitor of AMPK, blocked STS-mediated inhibition of ARPE-19 cell monolayer permeability.Conclusions: STS inhibited HG-induced RPE permeability possibly through the reduction of NF-κB activation via the AMPK/p300 pathway. The protective effects of STS were attained through the suppression of p300-mediated NF-κB acetylation and STS might be utilized for treatment of DR, in terms of preventing inflammation.

The Cytotoxic Effects of Geranylgeranylacetone Are Attenuated in the High-Glucose Condition.

Geranylgeranylacetone (GGA) has been used as an antiulcer drug and also is known as inducer of heat shock protein 70 that has cytoprotective effects especially in hyperglycemic condition. In contrast, cytotoxicity of GGA has also been reported. Some studies have reported that GGA suppresses cell growth and induces apoptosis in cell models of human leukemia, ovarian carcinoma, and colon cancer in vitro. Therefore, the aim of this study was to determine whether GGA can have a cytotoxic effect on a human cervical cancer cell line (HeLa), human colorectal adenocarcinoma cells (Caco-2), and human embryonic kidney cells 293 (HEK) in normal-glucose and high-glucose environments (NG and HG, respectively). The results showed that 100 μM GGA inhibited proliferation of HeLa cells only in NG environment despite inhibiting proliferation of Caco-2 and HEK cells regardless of glucose concentration. Cell viability assay revealed that GGA decreased viability of HeLa, Caco-2, and HEK cells only in NG environment. Flow cytometric analyses revealed that the type of cell death was a combination of necrosis and apoptosis. Our study revealed that difference in cytotoxicity of GGA is influenced by glucose condition. The cytotoxic effects of GGA are attenuated in the HG condition. Since both cytotoxic and cytoprotective effects are reported about GGA, further research is needed about the mechanism of the cytotoxic effects.

P6309Impaired secretion of clusterin in pericardial fluid of diabetics, a deleterious outcome for the cardiac micro-vasculature

Abstract Background Extracellular vesicles are vital mediators of cell-to-cell communications. We previously demonstrated that in the adult heart, exosomes released in the pericardial fluid (PF) have the functional capability to induce cell survival and angiogenesis in recipient endothelial cells. It has previously been shown that PF exosomes from patients with an existing myocardial infarction (MI) contain clusterin (CLU). Remarkably, secreted CLU is cardio-protective as evidenced by enhanced arteriogenesis and reduced apoptosis in animal models of MI. We hypothesize that type 2 Diabetes Mellitus (T2DM) along with ischemic heart disease (IHD) alters PF EV content impairing its reparative angiogenic potential. Aim To investigate the role of T2DM in modulating human PF exosomal content and its vascular action. Methods and results PF samples were collected from 3 different groups of patients (N=3): 1) Ischemic heart disease (IHD) with or 2) without T2DM, and 3) non-ischaemic, non-diabetic controls operated for mitral valve repair. The impact of PF exosomes on human coronary microvascular ECs (HCMECs) was evaluated by cell based functional assays for measuring apoptosis, proliferation and angiogenesis. Interestingly, unlike PF exosomes from control patients, PF exosomes from DM patients with IHD promoted EC apoptosis and impaired angiogenesis. To investigate the effect of T2DM on PF content, we performed high throughput proteomic and metabolomic analysis of whole PF and PF EVs. We identified proteins and metabolites that were differentially expressed under T2DM condition by using the R package Limma. Employing a network approach, protein and metabolic data were integrated by using our newly developed inhouse R package Metabosignal. This networks approach revealed an interesting interaction circuit involving protein CLU. The T2DM-associated reduction of CLU level in the PF, identified from the “omics” analyses was confirmed by ELISA (n=8 patients per group). To examine the effects of DM and ischemia on CLU intracellular expression and secretion, HCMECs were exposed to high D-glucose (HG, 25 mM) and hypoxia (1%O2). In line with our PF data, HCMECs exposed to HG and hypoxia showed lower levels of both CLU mRNA and secreted CLU (ELISA). Importantly, recombinant CLU (rCLU) treatment on HCMECs rescued their angiogenic potential, induced cell proliferation and protected them from apoptosis under both HG and hypoxia conditions. In addition, silencing of endogenous levels of CLU in HCMECs impaired angiogenesis, suggesting that CLU might be an inherent component of the angiogenesis machinery in ECs. Conclusions This study suggests that DM reduces the level of secreted CLU in the PF, depriving myocardial micro-vessels of this protective and regenerative mediator. Restoring diminished CLU levels in the diabetic heart could be a possible therapeutic approach for contrasting diabetic micro-angiopathy. Acknowledgement/Funding British Heart Foundation Program Grant

High Glucose Level Impairs Human Mature Bone Marrow Adipocyte Function Through Increased ROS Production.

Bone marrow adipocytes (BMAds) accumulate in aging, menopause, and metabolic diseases such as Type 2 diabetes. These osteoporotic conditions are associated with oxidative stress and hyperglycemia which are both considered as critical factors underlying bone fragility. Glucose excess and reactive oxygen species (ROS) are known to favor adipogenesis over osteoblastogenesis. In this study, we investigated whether high glucose exposure could determine dysfunction of mature BMAds, specifically through ROS production. The effects of low (LG, 5 mM) or high glucose (HG, 25 mM) concentrations were examined using human bone mesenchymal stromal cells (hBMSCs) in the time course of differentiation, and, up to 21 days once adipocytes were mature. HG did not alter the adipocyte differentiation process of hBMSCs. Yet, after 21 days under HG exposure, PPARG, CEBPA, and adiponectin mRNA expressions were decreased. These alterations were also observed following adipogenic inducer withdrawal as well as in adipocytes fully differentiated in LG then cultured in HG for the last 11 days. Without inducers, HG condition also led to decreased leptin mRNA level. Importantly, intracellular and extracellular ROS concentrations measured using Amplex Red were significantly raised by 50% under HG exposure. This rise was observed once adipocytes ended differentiation and was reproduced within the different cell culture settings without any cytotoxicity. Among genes involved in ROS metabolism, the mRNA level of the H2O2 generating enzyme NOX4 was found upregulated in the presence of HG. Following cell separation, mature BMAds were shown to overproduce ROS and to display the gene alterations in contrast to non-lipid-laden cells. Finally, a non-lethal treatment with a pro-oxidant agent under LG condition reduces the mRNA levels of PPARG, adiponectin, and leptin as the HG condition does in the absence of inducers, and amplifies the effect of glucose excess on gene expression. HG concentration drives mature BMAds toward altered expression of the main adipokines and transcriptional factors. These perturbations are associated with a rise in ROS generation likely mediated through enhanced expression of NOX4. Mature BMAds are thus responsive to changes in glucose and ROS concentrations, which is relevant regarding with their phenotype and function in age- or metabolic disease-related osteoporosis.

Podoplanin mediates the renoprotective effect of berberine on diabetic kidney disease in mice.

Hyperglycemia-caused podocyte injury plays a crucial role in the progress of diabetic kidney disease. Podoplanin, one of the podocyte-associated molecules, is closely related to the integrity of the glomerular filtration barrier. A number of studies demonstrate that berberine could ameliorate renal dysfunction in diabetic mice with nephropathy, but the molecular mechanisms have not been fully elucidated. In this study, we explored the relationship between the renoprotective effect of berberine and podoplanin expression in streptozotocin (STZ)-induced diabetic mice as well as mouse podocytes (MPC5 cells) cultured in high glucose (HG, 30 mM) medium. We found that the expression levels of podoplanin were significantly decreased both in the renal glomerulus of STZ-induced diabetic mice and HG-cultured MPC5 cells. We also demonstrated that NF-κB signaling pathway was activated in MPC5 cells under HG condition, which downregulated the expression level of podoplanin, thus leading to increased podocyte apoptosis. Administration of berberine (100, 200 mg/kg every day, ig, for 8 weeks) significantly improved hyperglycemia and the renal function of STZ-induced diabetic mice and restored the expression level of podoplanin in renal glomerulus. In high glucose-cultured MPC5 cells, treatment with berberine (30–120 μM) dose-dependently decreased the apoptosis rate, increased the expression of podoplanin, and inhibited the activation of NF-κB signaling pathway. When podoplanin expression was silenced with shRNA, berberine treatment still inhibited the NF-κB signaling pathway, but its antiapoptotic effect on podocytes almost disappeared. Our results suggest that berberine inhibits the activation of NF-κB signaling pathway, thus increasing the podoplanin expression to exert renoprotective effects.

Protective effects of pyrroloquinoline quinone against oxidative stress-induced cellular senescence and inflammation in human renal tubular epithelial cells via Keap1/Nrf2 signaling pathway

Oxidative stress-induced cellular senescence and inflammation are important biological events in diabetic nephropathy (DN). Our recent studies have found that pyrroloquinoline quinone (PQQ) has protective effects against HG-induced oxidative stress damage and apoptosis in HK-2 cells. Nevertheless, whether PQQ has the effect of anti-inflammation and anti-senescence in HK-2 cells remains unclear. Here, we showed that low-dose PQQ treatment (100 nM) downregulates the expression of P16, P21, IL-1β, TNF-α and NF-κB in HG cultured HK-2 cells. A low dose of PQQ also upregulated the protein expression of SOD2, CAT and inhibited the generation of ROS. We also indicated that PQQ affected the activity of Keap1/Nrf2 pathway, increased the nuclear accumulation of Nrf2 and the downstream pathway protein expression of Keap1/Nrf2 signaling pathway (HO-1, NQO-1, GST and GPx-3). When ML385 was added to inhibit the activity of Keap1/Nrf2 signaling pathway, the effects of PQQ on anti-oxidative stress, anti-inflammation and anti-senescence in HK-2 cells under HG condition were weakened. In conclusion, our results suggest that PQQ could modulate HG-induced inflammation and senescence in HK-2 cells via the inhibition of ROS generation and achieves the protective effects through Keap1/Nrf2 pathway and upregulating the expression of its target protein.

Hyperglycemia-Driven Inhibition of AMP-Activated Protein Kinase α2 Induces Diabetic Cardiomyopathy by Promoting Mitochondria-Associated Endoplasmic Reticulum Membranes In Vivo.

Fundc1 (FUN14 domain containing 1), an outer mitochondrial membrane protein, is important for mitophagy and mitochondria-associated endoplasmic reticulum membranes (MAMs). The roles of Fundc1 and MAMs in diabetic hearts remain unknown. The aims of this study, therefore, were to determine whether the diabetes mellitus-induced Fundc1 expression could increase MAM formation, and whether disruption of MAM formation improves diabetic cardiac function. Levels of FUNDC1 were examined in the hearts from diabetic patients and nondiabetic donors. Levels of Fundc1-induced MAMs and mitochondrial and heart function were examined in mouse neonatal cardiomyocytes exposed to high glucose (HG, 30 mmol/L d-glucose for 48 hours), and in streptozotocin-treated cardiac-specific Fundc1 knockout mice and cardiac-specific Fundc1 knockout diabetic Akita mice, as well. FUNDC1 levels were significantly elevated in cardiac tissues from diabetic patients in comparison with those from nondiabetic donors. In cultured mouse neonatal cardiomyocytes, HG conditions increased levels of Fundc1, the inositol 1,4,5-trisphosphate type 2 receptor (Ip3r2), and MAMs. Genetic downregulation of either Fundc1 or Ip3r2 inhibited MAM formation, reduced endoplasmic reticulum-mitochondrial Ca2+ flux, and improved mitochondrial function in HG-treated cardiomyocytes. Consistently, adenoviral overexpression of Fundc1 promoted MAM formation, mitochondrial Ca2+ increase, and mitochondrial dysfunction in cardiomyocytes exposed to normal glucose (5.5 mmol/L d-glucose). In comparison with nondiabetic controls, levels of Fundc1, Ip3r2, and MAMs were significantly increased in hearts from streptozotocin-treated mice and Akita mice. Furthermore, in comparison with control hearts, diabetes mellitus markedly increased coimmunoprecipitation of Fundc1 and Ip3r2. The binding of Fundc1 to Ip3r2 inhibits Ip3r2 ubiquitination and proteasome-mediated degradation. Cardiomyocyte-specific Fundc1 deletion ablated diabetes mellitus-induced MAM formation, prevented mitochondrial Ca2+ increase, mitochondrial fragmentation, and apoptosis with improved mitochondrial functional capacity and cardiac function. In mouse neonatal cardiomyocytes, HG suppressed AMP-activated protein kinase activity. Furthermore, in cardiomyocytes of Prkaa2 knockout mice, expression of Fundc1, MAM formation, and mitochondrial Ca2+ levels were significantly increased. Finally, adenoviral overexpression of a constitutively active mutant AMP-activated protein kinase ablated HG-induced MAM formation and mitochondrial dysfunction. We conclude that diabetes mellitus suppresses AMP-activated protein kinase, initiating Fundc1-mediated MAM formation, mitochondrial dysfunction, and cardiomyopathy, suggesting that AMP-activated protein kinase-induced Fundc1 suppression is a valid target to treat diabetic cardiomyopathy.

The Role of the Inflammatory Mediators TNF‐α and IFN‐γ in the Hyperglycemia‐induced PECAM‐1 Ubiquitination and Degradation in Diabetic Retinopathy

ObjectiveThe aim of this study was to investigate the role of TNF‐α and IFN‐γ in PECAM‐1 ubiquitination and subsequent degradation under hyperglycemic conditions in the diabetic retina.IntroductionInflammation is a major contributor to the development of diabetic retinopathy (DR). Two of the key mediators of inflammation, tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ), have been shown to contribute to the pathophysiological changes leading to the progression of DR. However, their role in the hyperglycemia‐induced loss of retinal PECAM‐1 is not known. Platelet endothelial cell adhesion molecule‐1 (PECAM‐1) is a cell surface protein that is heavily expressed on vascular endothelial cells, and has an important role in the maintenance of the endothelium and its functions. We have previously found a significant decrease in PECAM‐1 levels in the diabetic retina and cultured rat retinal microvascular endothelial cells (RRMECs) grown under high glucose conditions. Thus, an investigation into the possible mechanism/s of PECAM‐1 loss may help in understanding the molecular processes leading to DR, and provide a possible therapeutic target.Materials and MethodsFor our in vitro studies, primary RRMECs were grown in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for six days, with TNF‐α and/or IFN‐γ (20 ng/ml and 30 ng/ml, respectively) added 24 hrs prior to harvesting cells for analysis. For our animal studies, age‐matched male Wistar rats were injected with either vehicle (control), or Streptozotocin (STZ) (diabetic, 30 mg/kg/day) for three consecutive days in a model of type I diabetes. Retinas and plasma were collected eight weeks post‐STZ injection for analysis. PECAM‐1, TNF‐α, IFN‐γ, and ubiquitin levels were assessed using western blotting, ELISA, Immunofluorescence, and immunoprecipitation assays.ResultsTotal ubiquitination levels were significantly increased in the diabetic retina and in RRMECs grown under HG conditions (35% and 40% increase, respectively, p<0.05). Moreover, PECAM‐1 ubiquitination levels were significantly increased in RRMECs grown under hyperglycemic conditions (3‐fold, p<0.05). TNF‐α levels were significantly increased in diabetic STZ plasma and RRMECs grown under HG conditions (31%, 74.4% increase, respectively, p<0.05). Additionally, IFN‐γ levels were significantly increased in the diabetic retina (35% increase, p<0.05). PECAM‐1 levels were significantly decreased in RRMECs when treated with TNF‐α and/or IFN‐γ (60–75% decrease, p<0.05), with a significant increase in total ubiquitination levels (20–28% increase, p<0.05).ConclusionThese results indicate a possible role for TNF‐α and IFN‐γ in the hyperglycemia‐induced loss of PECAM‐1 in retinal endothelial cells, possibly by upregulating PECAM‐1 ubiquitination and subsequent degradation.Support or Funding InformationThis work was supported by funding from the National Institutes of Health (NIH) EY025632This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hyperglycemia‐Driven Inhibition of AMP‐Activated Protein Kinase a2 Induces Diabetic Cardiomyopathy by Promoting Mitochondria‐Associated Endoplasmic Reticulum Membranes in Vivo

BackgroundFUN14 domain containing 1 (Fundc1), an outer mitochondrial membrane protein, is important for mitophagy and mitochondria‐associated endoplasmic reticulum (ER) membranes (MAMs). The roles of Fundc1 and MAMs in diabetic hearts remain unknown. The aims of this study therefore, were to determine if the diabetes‐induced Fundc1 expression could increase MAM formation, and whether disruption of MAM formation improves diabetic cardiac function.MethodsLevels of FUNDC1 were examined in the hearts from diabetic patients and non‐diabetic donors. Levels of Fundc1‐induced MAMs, and mitochondrial and heart function were examined in mouse neonatal cardiomyocytes exposed to high glucose (HG, 30 mmol/L D‐glucose for 48 h), as well as in streptozotocin (STZ)‐treated cardiac‐specific Fundc1 knockout (KO) mice and cardiac‐specific Fundc1 KO diabetic Akita mice.ResultsFUNDC1 levels were significantly elevated in cardiac tissues from diabetic patients compared to those in non‐diabetic donors. In cultured mouse neonatal cardiomyocytes, HG conditions increased levels of Fundc1, the inositol 1,4,5‐trisphosphate type 2 receptor (Ip3r2), and MAMs. Genetic downregulation of either Fundc1 or Ip3r2 inhibited MAM formation, reduced ER‐mitochondrial Ca2+ flux, and improved mitochondrial function in HG‐treated cardiomyocytes. Consistently, adenoviral overexpression of Fundc1 promoted MAM formation, mitochondrial Ca2+ overload, and mitochondrial dysfunction in cardiomyocytes exposed to normal glucose (5.5 mmol/L D‐glucose). Compared with non‐diabetic controls, levels of Fundc1, Ip3r2, and MAMs were significantly increased in hearts from STZ‐treated mice and Akita mice. Further, compared with control hearts, diabetes markedly increased co‐immunoprecipitation of Fundc1 and Ip3r2. The binding of Fundc1 to Ip3r2 inhibits Ip3r2 ubiquitination and proteasome‐mediated degradation. Cardiomyocyte‐specific Fundc1 deletion ablated diabetes‐induced MAM formation, prevented mitochondrial Ca2+ overload, mitochondrial fragmentation, and apoptosis with improved mitochondrial functional capacity and cardiac function. In mouse neonatal cardiomyocytes, HG suppressed AMP‐activated protein kinase (Ampk) activity. Furthermore, in cardiomyocytes of Prkaa2 KO mice, expression of Fundc1, MAM formation, and mitochondrial Ca2+ levels were significantly increased. Finally, adenoviral overexpression of a constitutively active mutant Ampk ablated HG‐induced MAM formation, mitochondrial Ca2+ overload, and mitochondrial dysfunction.ConclusionsWe conclude that HG conditions in diabetes suppress Ampk, initiating Fundc1‐mediated MAM formation, mitochondrial dysfunction, and cardiomyopathy, suggesting that Ampk‐induced Fundc1 suppression is a valid target to treat diabetic cardiomyopathy.Support or Funding InformationThis study was supported by funding from the following agencies: NHLBI (HL079584, HL080499, HL089920, HL110488, HL128014, HL132500, HL137371, HL142287, and HL140954), NCI (CA213022), NIA (AG047776), and AHA (16GRANT29590003). Dr. Zou is the Eminent Scholar in Molecular and Translational Medicine of Georgia Research Alliance.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Integrative Glycoproteomic and Phosphoproteomic Analyses to reveal Vascular Alterations in Response to a Hyperglycemic Microenvironment

Hyperglycemia‐induced vascular complications are preeminent factors for a decreased life expectancy in patients with diabetes. At a molecular level, increased concentrations of blood glucose is known to accelerate endothelial dysfunction through several negative intracellular signaling events. We hypothesize that these changes are not independent of spontaneous cell surface glycosylation. The cytosolic phosphoproteome and cell surface glycoproteome (both N‐and O‐glycoproteome) were isolated from rat microvascular endothelial cells (RMVECs) cultured under both normal (NG; 4.5 mM) and high glucose (HG; 25 mM) conditions for two weeks. This was followed by tandem mass spectrometry (MS/MS) and integrative bioinformatics analysis. Functional assays, namely tube formation, ROS production, and proliferation assays were performed on RMVECs in the presence or absence of HG conditions. The deleterious impact of hyperglycemic conditions on RMVEC function was confirmed by their inability to form robust tubes in vitro (n=3, p<0.05), and a diminished response to acetylcholine ex vivo (n=3, p<0.05). Hyperglycemic states resulted in differential glycosylation of several receptors, ion channels and cell surface antigens that are critical for endothelial functioning. Tandem MS/MS analysis revealed 87 proteins significantly increased in N‐glycosylation (65 unique) and 143 proteins significantly increased in O‐glycosylation (78 unique) in HG treated versus NG RMVECs (p<0.05). Targets included AT1R (N‐glycosylation: Unique HG, p<0.05), JAM‐A (O‐glycosylation: 2.05 fold, p<0.05) and CD59 (N‐glycosylation: 2.02 fold, p<0.05 and O‐glycosylation: Unique HG, p<0.05). Phosphopeptide pathway enrichment analysis revealed a 25% decrease in phosphorylation status, when filtered for scans (≥ 5) and p‐value (<0.05), in HG treated versus NG RMVECs. Pronounced alterations were observed in several components of insulin (INSR: 1.1 fold, p<0.05 and PKCβ: 2.3 fold, p<0.05), angiogenic (S1PR2: unique HG, p<0.05), and apoptotic (BCL2: −3.75 fold, p<0.05) signaling pathways. Furthermore, elimination of glucose moieties by the treating HG RMVECs with glycosidases, resulted in a normalization of several endothelial functional phenotypes. This study identified a multitude of changes in the glycosylation and phosphorylation status of several surface receptors and cytosolic proteins that are integral to vascular homeostasis. Since glycosidases reversed several of the impairments brought about by a hyperglycemic insult on the endothelium, reversion of dysfunctional endothelial cells to a healthy phenotype by negating a build‐up of glucose moieties on cell surface proteins may be a potential new avenue for the treatment of progressive cardiovascular complications in diabetes.Support or Funding InformationSupport for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University, Milwaukee, WI, USA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RETRACTED: Protective effect of DMDD, isolated from the root of Averrhoa carambola L., on high glucose induced EMT in HK-2 cells by inhibiting the TLR4-BAMBI-Smad2/3 signaling pathway

Hyperglycemia stimulated epithelial-mesenchymal transition (EMT) plays a critical role in initiating and progressing renal fibrosis in diabetic kidney disease (DKD). It is crucial to explore novel renal protective drugs for the treatment of DKD. The present study is to confirm our hypothesis and to accumulate the information for the application of DMDD (2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione) as a novel therapeutic agent to potentially inhibit renal fibrogenesis and EMT in the DKD. High glucose induced renal proximal tubular epithelial cell line (HK-2 cells) was cultured and treated with DMDD. The cell viability and DMDD cytotoxicity were assessed by CCK8. Immunofluorescence was used for detection of TLR4 and downstream protein in normal and high glucose induced HK-2 cells. HK-2 cells were transfected with lentivirus codifying for BAMBI (BMP and activin membrane bound inhibitor) and interfering RNA for determination of the effect of BAMBI over-expression and silencing, respectively. TLR4-BAMBI-Smad2/3 pathway was analyzed by means of RT-PCR and western blot. A high concentration (60mM) of glucose induced significant EMT process and TLR4 expression was increased obviously in this circumstance. DMDD inhibited high expressions of TLR4 and Smad2/3 in HG induced cells and decreased the expression of BAMBI. In addition, the effects of decreased BAMBI expression and increased Smad2/3 expression in HG cultured cells were reversed in the cells of TAK-242 (TLR4 signaling inhibitor) intervention. BAMBI gene silencing dramatically increased EMT process and the over-expression of BAMBI was opposite in HK-2 cells with HG condition. These observations of EMT were ameliorated when the HK-2 cells were pre-treated with DMDD. Our study demonstrates that DMDD treatment improves EMT in the HG induced HK-2 cells. In addition, DMDD significantly inhibits EMT by TLR4-BAMBI-Smad2/3 pathway, which hints that DMDD may be an alternative approach in diabetic renal injury.

The Mitochondria-Targeted Antioxidant MitoQ Modulates Mitochondrial Function and Endoplasmic Reticulum Stress in Pancreatic β Cells Exposed to Hyperglycaemia.

Mitochondria-targeted antioxidants such as mitoquinone (MitoQ) have demonstrated protective effects against oxidative damage in several diseases. The increase in reactive oxygen species (ROS) production during glucose metabolism in β cells can be exacerbated under hyperglycaemic conditions such as type 2 diabetes (T2D), thus contributing to β cell function impairment. In the present work, we aimed to evaluate the effect of MitoQ on insulin secretion, oxidative stress, endoplasmic reticulum (ER) stress and nuclear factor kappa B (NFκB) signalling in a pancreatic β cell line under normoglycaemic (NG, 11.1 mM glucose), hyperglycaemic (HG, 25 mM glucose) and lipidic (palmitic acid (PA), 0.5mM) conditions. We incubated the pancreatic β cell line INS-1E with or without MitoQ (0.5µM) under NG, HG and PA conditions. We then assessed the following parameters: glucose-induced insulin secretion, O₂ consumption (with a Clark-type electrode); mitochondrial function, oxidative stress parameters and calcium levels (by fluorescence microscopy); ER stress markers and NFκB-p65 protein levels (by western blotting). MitoQ increased insulin secretion and prevented the enhancement of ROS production and O₂ consumption and decrease in GSH levels that are characteristic under HG conditions. MitoQ also reduced protein levels of ER stress markers (GRP78 and P-eIF2α) and the proinflammatory nuclear transcription factor NFκB-p65, both of which increased under HG. MitoQ did not significantly alter ER stress markers under lipidic conditions. Our findings suggest that treatment with MitoQ modulates mitochondrial function, which in turn ameliorates endoplasmic reticulum stress and NFκB activation, thereby representing potential benefits for pancreatic β cell function.

Lipopolysaccharide (LPS) Aggravates High Glucose- and Hypoxia/Reoxygenation-Induced Injury through Activating ROS-Dependent NLRP3 Inflammasome-Mediated Pyroptosis in H9C2 Cardiomyocytes.

Diabetes aggravates myocardial ischemia-reperfusion (I/R) injury because of the combination effects of changes in glucose and lipid energy metabolism, oxidative stress, and systemic inflammatory response. Studies have indicated that myocardial I/R may coincide and interact with sepsis and inflammation. However, the role of LPS in hypoxia/reoxygenation (H/R) injury in cardiomyocytes under high glucose conditions is still unclear. Our objective was to examine whether lipopolysaccharide (LPS) could aggravate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by upregulating ROS production to activate NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes. H9C2 cardiomyocytes were exposed to HG (30 mM) condition with or without LPS, along with caspase-1 inhibitor (Ac-YVAD-CMK), inflammasome inhibitor (BAY11-7082), ROS scavenger N-acetylcysteine (NAC), or not for 24 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). The cell viability, lactate dehydrogenase (LDH) release, caspase-1 activity, and intracellular ROS production were detected by using assay kits. The incidence of pyroptosis was detected by calcein-AM/propidium iodide (PI) double staining kit. The concentrations of IL-1β and IL-18 in the supernatants were assessed by ELISA. The mRNA levels of NLRP3, ASC, and caspase-1 were detected by qRT-PCR. The protein levels of NF-κB p65, NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18 were detected by western blot. The results indicated that pretreatment LPS with 1 μg/ml not 0.1 μg/ml could efficiently aggravate HG and H/R injury by activating NLRP3 inflammasome to mediate pyroptosis in H9C2 cells, as evidenced by increased LDH release and decreased cell viability in the cells, and increased expression of NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18. Meanwhile, Ac-YVAD-CMK, BAY11-7082, or NAC attenuated HG- and H/R-induced H9C2 cell injury with LPS stimulated by reversing the activation of NLRP3 inflammasome-mediated pyroptosis. In conclusion, LPS could increase the sensitivity of H9C2 cells to HG and H/R and aggravated HG- and H/R-induced H9C2 cell injury by promoting ROS production to induce NLRP3 inflammasome-mediated pyroptosis.

Integrated on-chip sensor with sputtered Ag-Au-Au electrodes for the voltammetric determination of trace Hg(II)

This work describes a planar sensor chip with three integrated electrodes for the determination of Hg(II) by anodic stripping voltammetry (ASV). The sensor features Au-film working and auxiliary electrodes and a Ag-film reference electrode; all three metal-film electrodes are deposited on a silicon substrate by sputtering. Definition of the geometrical pattern of the sensor chip involves appropriate application of simple plastic masks during deposition of the metal electrodes making this approach simpler, faster and less laborious than photolithography. The Au-film working electrode is characterized by optical techniques and cyclic voltammetry and the chemical and instrumental conditions for Hg(II) determination by ASV are studied in detail. The limit of detection of Hg(II) is 0.6 μg L−1, the within-sensor repeatability (expressed as the coefficient of variation of 20 measurements at the 20 μg L−1 Hg(II) level) is 3.7% and the between-sensor reproducibility (expressed as the coefficient of variation of 6 measurements at different sensors at the 20 μg L−1 Hg(II) level) is 11.8%. The sensor chip is successfully applied to the determination of Hg(II) in a fish oil sample. The proposed fabrication approach offers scope for the mass-production of robust integrated sensors at low cost which are applicable to on-site analysis.

Mercury and arsenic in the surface peat soils of the Changbai Mountains, northeastern China: distribution, environmental controls, sources, and ecological risk assessment.

The potential toxic risk of mercury (Hg) and arsenic (As) in the soils of mining regions and other artificially disturbed lands receives considerable research attention. However, limited investigation has been conducted into the surface soils of natural globally distributed ecosystems, for example peatlands. In this study, we examine the distribution, controlling factors, sources, and potential ecological risks of Hg and As in 96 samples from 42 peatlands in the Changbai Mountains of northeastern China. The results showed that average concentrations (dry weight) of Hg and As at the samples sites were 169.1 ± 0.1 µg kg-1and 13.0 ± 7.7mgkg-1, respectively. The distribution of Hg is largely determined by latitude and altitude, while As is controlled more by pH, total organic carbon (TOC), and ratio of TOC and nitrogen (C/N) at the regional scale. Variations in TOC, C/N ratio, and redox conditions contribute to determining the distribution of Hg, while TOC and redox conditions mainly affected the distribution of Arsenic at the local scale. Mercury mostly comes from regional atmospheric wet deposition, whereas elevated concentrations of As are related to local anthropogenic activities. Overall, Hg and As in the peatlands of the Changbai Mountains pose a moderate level of potential risk to ecological health.

Cyclosporin A aggravates the Calcification of Vascular Smooth Muscle Cells under High Glucose conditions with a Calcifying Medium

Background Vascular calcification (VC) progresses substantially even in the case of stable kidney transplantation, and is a predictor of morbidity and mortality. However, the effect of cyclosporin A (CsA) on VC has not been reported in diabetic kidney transplant patients. In this study, we evaluated the effect of CsA on the VC of mouse vascular smooth muscle cells (VSMCs), in the diabetic milieu. Methods CsA of a concentration of 1.0 μmol/L was selected by the MTT assay for this study. To demonstrate the effect of CsA and high glucose (HG, 30mM) in the induction of the VC of the VSMCs, we determined alkaline phosphatase (ALP) activity, microscopic morphology of calcification, the expressions of the calcification and inflammation-related genes, and the intracellular calcium concentrations in VSMCs. Results Calcification was observed 14 days after exposure to a calcifying medium (sodium phosphate monobasic and dibasic mixture). On microscopic morphology, CsA alone did not induce calcification under HG conditions, but clearly increased calcification under HG with a calcifying medium. ALP activity increased under HG with CsA or a calcifying medium compared to HG conditions alone. CsA increased ALP activity under low glucose (LG, 5.5mM) with a calcifying medium, but markedly increased under HG with a calcifying medium. CsA significantly increased the mRNA expressions of the calcification markers (core binding factor-alpha 1, bone morphologic proteins 2) as well as those of the inflammatory marker (interleukin 6), under HG with a calcifying medium. Intracellular calcium concentrations was unchanged in CsA alone but significantly increased with the presence of a calcifying medium under both LG and HG conditions, Conclusion Considering the effect of CsA on VC, the vascular side effects of CsA need to be verified in diabetic transplant patients, in the future.