Role of TRPV4 channels in high glucose-induced neurotoxicity in a neuronal-like cell model

BackgroundBabies born to mothers with pregestational diabetes have a high risk for congenital heart defects (CHD). Embryonic stem cells (ESCs) are excellent in vitro models for studying the effect of high glucose on cardiac lineage specification because ESCs can be differentiated into cardiomyocytes. ESC maintenance and differentiation are currently performed under high glucose conditions, whose adverse effects have never been clarified.MethodWe investigated the effect of high glucose on cardiomyocyte differentiation from a well-characterized ESC line, E14, derived from mouse blastocysts. E14 cells maintained under high glucose (25 mM) failed to generate any beating cardiomyocytes using the hanging-drop embryonic body method. We created a glucose-responsive E14 cell line (GR-E14) through a graduated low glucose adaptation. The expression of stem cell markers was similar in the parent E14 cells and the GR-E14 cells.ResultsGlucose transporter 2 gene was increased in GR-E14 cells. When GR-E14 cells were differentiated into cardiomyocytes under low (5 mM) or high (25 mM) glucose conditions, high glucose significantly delayed the appearance and reduced the number of TNNT2 (Troponin T Type 2)-positive contracting cardiomyocytes. High glucose suppressed the expression of precardiac mesoderm markers, cardiac transcription factors, mature cardiomyocyte markers, and potassium channel proteins. High glucose impaired the functionality of ESC-derived cardiomyocytes by suppressing the frequencies of Ca2+ wave and contraction.ConclusionsOur findings suggest that high glucose inhibits ESC cardiogenesis by suppressing key developmental genes essential for the cardiac program.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0446-5) contains supplementary material, which is available to authorized users.

The present studies investigated the role of high glucose inhibit cell proliferation in kidney tubular epithelial cells. In LLC-PK1 cells, p21 protein (not p27 or p15) increased after 24-to -48-h exposure to high glucose. The key role of p21 protein on cell cycle arrest was confirmed by the use of p21shRNA, effectively decreased p21 protein levels and reversed the effect of high glucose on inhibition cellular proliferation. Furthermore, high glucose treatment leads to an increase in p21 mRNA and promoter activity independent on osmotic pressure change. To further define the signaling pathway through which high glucose induces p21, we have performed a detailed functional analysis on the p21 promoter. Through both deletion and mutation analysis of the p21 promoter, these results indicate maximal p21 promoter activity requires both strong distal and proximal regions, suggesting synergistic interactions between transcription factors recognizing these sites. We focus on proximal regions of p21 promoter and define sp1-3 site has a pivotal role on the activation of high glucose. EMSA assays demonstrate that high glucose would increase sp1-3 element binding complex, two proteins of the complex are sp1 and Smads. To investigate the role of Smads involve the activation of p21 promoter, we found high glucose would increase Smad2/3 phosphorylation and binding activity by EMSA assays after 24 h exposure. Although TGFβ1 antibody and TGFβ type I receptor inhibitor (SB431542) could decrease high glucose induced p3TP-lux, they had no effect on the activation of p21 promoter by high glucose, suggesting independent on TGFβ1/ TGFβ type I receptor. From transient transfectioned dominant negative Smad3∆c plasmids, we got it can decrease hyperglycemia-induced p21 promoter activation and protein expression. We also treated with Smad3 antisense oligonucleotides for 10 days in STZ-induced diabetic mice and found this treatment could reduce p21 and TGFβRI protein expression and reduce the induction of kidney weight in vivo. Taken together, these results suggest that Smads activation plays a pivotal role in the transcriptional activation of the p21 gene by high glucose in vitro and in vivo. Because Smad3 protein involved high glucose regulate p21 protein expression but the regulation independent on TGFβ1/ TGFβ type I receptor, so we tried to find out how high glucose activate Smad2/3 protein. We found PI3K, PKC, p38 and ROS signaling pathway involve high glucose activate p21 promoter and protein expression, but except PI3K signaling pathway, there are many researches about how high glucose activate PKC, p38, ROS signaling and regulate gene expression. So we focus on the relationship between high glucose and phosphoinositide-3 kinase (PI3K). We analysed PI3K and Akt kinase activity under high glucose condition, and got high glucose can increase their activity, otherwise, PI3K inhibitor LY294002 and dominant negative PI3K subunit p85 plasmid could decrease hyperglycemia-induced p21 promoter activation and protein expression. Furthermore, PI3K inhibitor LY294002 and dominant negative PI3K subunit p85 plasmid could reverse high glucose induced cellular hypertrophy. LY294002 also decreased p21 binding with cyclin E and cdk2. In molecular mechanism, we also confirmed by EMSA assays : LY294002 could decrease hyperglycemia-induced sp1-3 element binding activity and Smad binding element (SBE) binding activity. In another way, we found antioxidant reagent (N-acetyl-L-cystein) and NADPH oxidase inhibitor (DPI) could reverse hyperglycemia-induced p21 protein expression, and LY294002 could decrease H2O2 - induced p21 protein expression, this result suggested reactive oxygen species (ROS) generated by high glucose, mainly H2O2 , stimulates PI3K signaling pathway to induce p21 expression. Recently, PI3K/mTOR signaling pathway has been demonsted involve protein translation and play an important role on pathologic hypertrophy. Because we find high glucose can activate PI3K and induce cellular hypertrophy, so we use mTOR specific inhibitor rapamycin to investigate its role under high glucose condition. We got the mTOR specific inhibitor rapamycin can reverse high glucose induced p21 promoter, mRNA and protein expression. Otherwise, rapamycin also could reverse high glucose inhibit cellular proliferation and cellular hypertrophy. These result indicated PI3K/mTOR signaling pathway indeed involved high glucose induce cellular hypertrophy by regulating p21 protein expression. Taken together, these results suggest that high glucose activated PI3K which related Smad3 and mTOR protein activation and played a pivotal role in the transcriptional activation of the p21 gene under high glucose condition, otherwise, PKC, p38 and ROS signaling pathway involve high glucose activate p21 promoter and protein expression.

Effects of high glucose on the production of heparan sulfate proteoglycan by mesangial and epithelial cells

Diabetic patients are susceptible to severe inflammatory periodontitis manifesting as swollen gingiva with bleeding, but the underlying mechanism is not well understood. Our purpose was to determine the effect of a high glucose (HG) condition on the interleukin-6/soluble interleukin-6 receptor (IL-6/sIL-6R)-induced activation of signaling and vascular endothelial growth factor (VEGF) expression in human gingival fibroblasts (HGFs). In this study, HGFs were cultured for at least two passages under a normal glucose (NG; 5.5 mM) condition or high glucose (25 mM) condition. Importantly, the HG condition significantly induced expression of gp130 mRNA in HGFs compared with levels in control cells. Consistent with the expression of its mRNA, the HG condition also increased the expression of gp130 protein, and phosphorylation of the tyrosine residue by gp130 was enhanced significantly by IL-6/sIL-6R stimulation. Furthermore, the HG condition enhanced the IL-6/sIL-6R-induced phosphorylation of p44/42 MAPK and led to subsequent activation of CCAAT/enhancer binding protein in nuclei. In contrast, there was no significant difference in phosphorylation of JNK between the HG and NG condition. Interestingly, HGFs increased IL-6/sIL-6R-induced VEGF165 mRNA expression and VEGF165 secretion under the HG condition compared with levels under the NG condition. In contrast, the induction of VEGF165 secretion was partially inhibited by PD98059 (selective p44/42 MAPK inhibitor) under the HG condition. In addition, the VEGF165 secretion was completely inhibited by the combination of PD98059 and SP600125 (JNK inhibitor). Our findings suggest that the HG condition indirectly increases VEGF expression via activation of gp130-mediated p44/42 MAPK-CCAAT/enhancer binding protein signaling in HGFs. Thus, elevated VEGF secretion in HGFs under the HG condition may play a role in the development of the severe periodontitis observed in diabetic patients.

α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders.

Purpose Cysteinyl leukotriene receptor 1 (CysLTR1), originally described as a proinflammatory G protein-coupled receptor, has been shown to possess diverse nonimmunological properties. One of these functions is to modulate glucose-stimulated insulin secretion in β cells. Furthermore, the inhibition of CysLTR1 increases retinal cell survival in early diabetic retinopathy. Nevertheless, the potential of CysLTR1 to modulate glucose levels in retinal vascular cells, such as endothelial cells (ECs) and pericytes (PCs), is unknown. Therefore, we determined the intracellular glucose levels in retinal cells in vitro after the inhibition of CysLTR1 under standard and high-glucose culture conditions. Methods Primary human ECs, PCs, and the ARPE-19 cell line were cultured under standard (5.5 mmol/l glucose + 27.5 mmol/l mannitol) and high-glucose (33.0 mmol/l) conditions in the absence and presence of the specific CysLTR1 antagonists montelukast and zafirlukast for 1, 3, and 7 days. CysLTR1 expression was determined by immunofluorescence microscopy. CysLT secretion was measured by enzyme-linked immunosorbent assay. The effects of high glucose and CysLTR1 inhibition on cell viability and intracellular glucose levels were analyzed by luminescence-based assays. Furthermore, the transendothelial and transepithelial electrical resistance of the ECs and ARPE-19 monolayers was measured. Results CysLTR1 inhibition under standard glucose culture conditions increased the cellular glucose levels in retinal ECs, PCs, and ARPE-19 cells after 1 and 3 days of treatment. Under high-glucose culture conditions, CysLTR1 inhibition for 1 day reduced the intracellular glucose level in ARPE-19 cells. However, CysLTR1 inhibition for 3 days increased the level of intracellular glucose in ARPE-19 cells under high-glucose culture conditions. Furthermore, CysLTR1 inhibition reduced the tightness of the EC and ARPE-19 monolayers under standard culture conditions but increased the tightness of the ARPE-19 monolayers under high-glucose conditions. Conclusions CysLTR1 is considered a potential target for the treatment of type 2 diabetes and early diabetic retinopathy. Our data revealed that CysLTR1 activity directly regulates cellular glucose levels in retinal cells, supporting these hypotheses. Interestingly, the effect of CysLTR1 activity on glucose levels was reversed under acute metabolic stress. Thus, the activity of CysLTR1 appears to be more complex in terms of glucose metabolism and needs to be studied in more detail.

ABSTRACTBackgroundHigh blood glucose is a well‐established risk factor for poor outcomes in ischemic stroke. However, the underlying molecular mechanisms linking high blood glucose to worsened stroke outcomes remain unclear.ObjectivesPrevious studies have implicated the NLRP3 inflammasome, a key mediator of neuroinflammation, in cerebral ischemia/reperfusion (I/R) injury. Under high blood glucose conditions, NLRP3 activation is amplified, potentially driving a vicious cycle of inflammation and neuronal death. Yet, how high blood glucose specifically modulates NLRP3 activation and its downstream pathways remains unclear. This study aimed to investigate the specific mechanisms by which high glucose enhances NLRP3 inflammasome activity and contributes to worsened brain injury following cerebral I/R.MethodsWe employed a combination of in vitro and in vivo experimental approaches to explore the impact of high glucose on NLRP3 inflammasome activation and its consequences on ischemic stroke outcomes. In vitro experiments were conducted by culturing various immune cells in high‐glucose conditions to evaluate the activation of the NLRP3 inflammasome and the mitochondrial association of HK2. In vivo, mice with genetic knockouts of Nlrp3, Pycard (the gene encoding ASC), or microglial‐specific Hk2 were subjected to transient middle cerebral artery occlusion (tMCAO).ResultsOur findings revealed that the activation of the NLRP3 inflammasome was enhanced post cerebral I/R under high glucose and a N‐terminal truncation of NLRP3 (miniNLRP3) was induced. Overexpression of PKA could promote the generation of miniNLRP3, while inhibition of PKA decreased the generation of miniNLRP3. In addition, treatment with pan serine protease could block PKA and LPS mediated generation of miniNLRP3. Overexpression of the N‐terminal truncation of NLRP3 could potentiate the activation of the NLRP3 inflammasome under high glucose conditions by promoting the dissociation of Hexokinase 2 (HK2) from mitochondria. In addition, knockout of Nlrp3, Pycard, or microglial Hk2, could all attenuate cerebral I/R‐induced brain injury under high blood glucose in mice.ConclusionOur study elucidates PKA‐mediated generation of a 30 kD N‐terminal truncation of NLRP3 (miniNLRP3) in a serine protease‐dependent manner, which could potentiate the activation of the NLRP3 inflammasome under high glucose conditions via promoting the dissociation of HK2 from mitochondria. These findings add a new dimension to our understanding of NLRP3 regulation in the context of stroke injury, and suggest that the PKA‐miniNLRP3‐HK2‐NLRP3 pathway is a promising therapeutic strategy to improve stroke outcomes in patients with elevated blood glucose levels.

PurposeSeveral roles are attributed to the myometrium including sperm and embryo transport, menstrual discharge, control of uterine blood flow, and labor. Although being a target of diabetes complications, the influence of high glucose on this compartment has been poorly investigated. Both miRNAs and IGF1R are associated with diabetic complications in different tissues. Herein, we examined the effects of high glucose on the expression of miRNAs and IGF1R signaling pathway in the human myometrium.MethodsHuman myometrial explants were cultivated for 48 h under either high or low glucose conditions. Thereafter, the conditioned medium was collected for biochemical analyses and the myometrial samples were processed for histological examination as well as miRNA and mRNA expression profiling by qPCR.ResultsMyometrial structure and morphology were well preserved after 48 h of cultivation in both high and low glucose conditions. Levels of lactate, creatinine, LDH and estrogen in the supernatant were similar between groups. An explorative screening by qPCR arrays revealed that 6 out of 754 investigated miRNAs were differentially expressed in the high glucose group. Data validation by single qPCR assays confirmed diminished expression of miR-215-5p and miR-296-5p, and also revealed reduced miR-497-3p levels. Accordingly, mRNA levels of IGF1R and its downstream mediators FOXO3 and PDCD4, which are potentially targeted by miR-497-3p, were elevated under high glucose conditions. In contrast, mRNA expression of IGF1, PTEN, and GLUT1 was unchanged.ConclusionsThe human myometrium responds to short-term exposure (48 h) to high glucose concentrations by regulating the expression of miRNAs, IGF1R and its downstream targets.

Background Several types of cells participate in the formation of proliferative membrane in proliferative retinopathy (PVR), and the proliferation, migration and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells play an important role.Many studies have confirmed high blood glucose is the basic pathogenesis of diabetic retinopathy (DR).However, whether EMT could be induced in RPE cells under the high glucose condition has not been reported. Objective This study was to investigate the effects of high glucose on the migration and EMT of RPE cells in high glucose culture model in vitro. Methods Human RPE cell line D407 were cultured and passaged in DMEM/F12 medium with 10% fetal bovine serum, and 6-8 generations of cells were used in experiment.The cells were divided into 3 groups based on different glucose concentrations in medium.The glucose at the final concentration 5.5 mmol/L or 60.0 mmol/L was respectively used in the normal control group or high glucose group, and the DMEM with 5.5 mmol/L glucose and mannitol was used in the hypertonic control group.The migration rate of the cells were detected 0, 24, 48 and 72 hours after scratching by wound-scratch test.Real-time PCR was used to detect the relative expressions of zonula occludens-1 (ZO-1) and α-smooth muscle actin (α-SMA) in the cells. Results Cultured cells showed a polygon shape with the clear nucleolus and dense arrangement in the normal control group and the hypertonic control group, but the cells were larger and elongated with the lapse of culture time with the indistinct structure and loose arrangement.At 48 hours after scratching, migrating cells were seen in the scratching area, and the scratching area disappeared at 72 hours after scratching in the high glucose group, but the scratching area still was existed in the normal control group or hypertonic control group.The migrating rate of the cells was higher in the high glucose group than that in the normal control group or hypertonic control group, showing total differences among 3 groups and various time points (Fgroup =328.600, P=0.000;Ftime=773.270, P=0.000).Compared with the normal control group, the expression level of ZO-1 mRNA was significantly lower, and α-SMA mRNA level was higher 48 hours and 72 hours in the high glucose group than those in the normal control group (all at P<0.05). Conclusions High glucose induce the migration and EMT of RPE cells in vitro, which may be associated with the pathogenesis of proliferative diabetic retinopathy. Key words: Human; Retinal pigment epithelium cell; High glucose; Epithelial-mesenchymal transition; Diabetes mellitus/complication, retinopathy; Cell culture

Hypoxia-induced sensitization of transient receptor potential vanilloid 1 involves activation of hypoxia-inducible factor-1 alpha and PKC

Hyperglycemia is widely considered to be the causal link between diabetes mellitus (DM) and diabetic complications. The purpose of this study is to determine the effects of high glucose in the presence of lipopolysaccharide (LPS) purified from the periodontal pathogen Porphyromonas gingivalis on human macrophages. Macrophages (U937) were treated with various concentrations of P. gingivalis-LPS under normal (5.5 mM) or high (25 mM) glucose conditions. Mitochondrial dehydrogenase activity was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay. The levels of inflammatory mediators secreted were determined using the enzyme-linked immunosorbent assay and the competitive enzyme immunoassay. The intracellular calcium chelator was used to examine whether the intracellular calcium was involved. Statistical differences were assessed using a one-way analysis of variance and Tukey multiple-comparison intervals with α = 0.05. High glucose condition enhanced the mitochondrial dehydrogenase activity in macrophages. P. gingivalis-LPS induced the secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and prostaglandin E(2) (PGE(2)) in a dose-dependent manner both in normal and high glucose conditions. The stimulatory effects by P. gingivalis-LPS were more evident when cells were cultured under high glucose conditions. Changes of intracellular calcium concentration were involved not only in high glucose-induced mitochondrial dehydrogenase activity but also in P. gingivalis-LPS-induced production of IL-6, TNF-α, or PGE(2), especially under the high glucose conditions. High glucose appeared to enhance the inflammatory response induced by the periodontal pathogen. The information generated may help to delineate the possible mechanisms by which hyperglycemia compromises the periodontal health of patients with DM.

Endothelial dysfunction leading to cardiovascular disease risk involves a decrease in nitric oxide (NO) production. In physiological conditions shear stress is a potent stimulation of endothelium-derived NO production and flow mediated NO production is regulated by the activation of endothelial NO synthase (eNOS). In endothelial cells, eNOS, aldose reductase (AR), a rate limiting enzyme of polyol pathway, and glutathione reductase (GR) share a NADPH as an obligate cofactor. In diabetec condition intracellular polyol pathway is activated and this may decrease shear stress-induced endothelial NO production and increase intracellular oxidative stress via inhibition of eNOS and GR by NADPH consumption. Therefore we investigated whethter AR inhibitor epalrestat improved endothelial NO production under high glucose condition to elucidate the mechanism of endothelial dysfunction in diabetes. We incubated human umbilical vein endothelial cells (HUVECs) in normal (5mM) and high (30mM) glucose condition for 72 hours, with or without epralrestat, or 100U/ml superoxide dismutase (SOD), respectively. After exchange of medium for Krebs’ buffer, HUVECs were exposed to 12dyne/cm2 steady laminar fluid shear stress for 5 minutes. NO release from HUVECs was measured as NO2 using a NOx analyzing HPLC system by Griess reaction. Next we harvested the cells in lysis buffer and analyzed phosphorylation of Akt (shear induced intracellular signal transduction) and eNOS by western blotting, and measured intracellular 8-OHdG and ratio of NADPH/NADP. In high glucose condition NO2 was decreased and 8-OHdG increased compared to low glucose. NO2 was restored and 8-OHdG was reduced by epalrestat significantly (p&lt;0.01, p&lt;0.05, respectively, vs. high glucose condition). In SOD-treated HUVECs, NO2 was not restored (n.s. vs. high glucose condition) despite of complete reduction of 8-OHdG (p&lt;0.01). Both Akt and eNOS phosphorylation by shear stress was affected neither by high glucose, epalrestat nor SOD. Intracellular NADPH/NADP ratio was decreased in high glucose condition, but this reduction was restored by epalrestat. These results showed that polyol pathway activation plays a key role in endothelial NO production under high glucose condition via a cofactor NADPH.

Diabetes mellitus is associated with sensory abnormalities, including exacerbated responses to painful (hyperalgesia) or non-painful (allodynia) stimuli. These abnormalities are symptoms of diabetic peripheral neuropathy (DPN), which is the most common complication that affects approximately 50% of diabetic patients. Yet, the underlying mechanisms linking hyperglycemia and symptoms of DPN remain poorly understood. The transient receptor potential vanilloid 1 (TRPV1) channel plays a central role in such sensory abnormalities and shows elevated expression levels in animal models of diabetes. Here, we investigated the function of TRPV1 channels in sensory neurons cultured from the dorsal root ganglion (DRG) of neonatal mice, under control (5mM) and high glucose (25mM) conditions. After maintaining DRG neurons in high glucose for 1 week, we observed a significant increase in capsaicin (CAP)-evoked currents and CAP-evoked depolarizations, independent of TRPV1 channel expression. These functional changes were largely dependent on the expression of the receptor for Advanced Glycation End-products (RAGE), calcium influx, cytoplasmic ROS accumulation, PKC, and Src kinase activity. Like cultured neurons from neonates, mature neurons from adult mice also displayed a similar potentiation of CAP-evoked currents in the high glucose condition. Taken together, our data demonstrate that under the diabetic condition, DRG neurons are directly affected by elevated levels of glucose, independent of vascular or glial signals, and dependent on RAGE expression. These early cellular and molecular changes to sensory neurons in vitro are potential mechanisms that might contribute to sensory abnormalities that can occur in the very early stages of diabetes.

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Peritoneal fibrosis (PF) is caused by epithelial-mesenchymal transdifferentiation (EMT) in the peritoneum under high glucose (HG) conditions. The study aimed to explored the role of Insulin-like growth factor 1 receptor (IGF-1R) in the regulation of EMT in human peritoneal mesothelial cells (HPMCs). We used HG peritoneal dialysis fluid (PDF) to induce in vivo PF in mice, and treated HPMCs with HG in vitro to stimulate EMT. In the mice, the higher the glucose concentration in the dialysate, the more obvious the peritoneal tissue thickening and the more that collagen was deposited. The in vitro study indicated that the expression of IGF-1R, α-SMA, vimentin was upregulated, while the expression of occludin, ZO-1, and E-cadherin was downregulated in HPMCs under HG and IGF-1R overexpression conditions. Conversely, the expression of IGF-1R, α-SMA, and vimentin was downregulated, while the expression of occludin, ZO-1, and E-cadherin was upregulated in IGF-1R-underexpressed HPMCs under HG conditions. The cell migration abilities were increased, while the cell adhesion abilities were reduced in HPMCs under HG and IGF-1R overexpression conditions. In contrast, cell migration abilities were reduced, while cell adhesion abilities were increased in IGF-1Runderexpressed HPMCs under HG conditions. Targeting at IGF-1R may provide novel insights into the prevention and treatment of PF.