Glucose-treated Cells Research Articles

Diabetes compromises tight junction protein claudin 14 in the urinary bladder.

Infections are common in patients with diabetes. Moreover, increasing incidence of antibiotic resistance impedes the complete bacterial clearance and calls for alternative treatment strategies. Along with antibacterial resistance, compromised host conditions create a favorable condition for the disease progression. In particular, cell junction proteins are of major importance as they contribute to a tight cell barrier, protecting against invading pathogens. However, the impact of high glucose on cell junction proteins has received little attention in the urinary bladder but merits closer investigation. Here, we report that during diabetes the expression of cell junction protein, claudin 14 is compromised in the human urine exfoliated cells and in the urinary bladder of type 2 diabetic mouse. Further in vitro analysis confirmed a direct correlation of lower intracellular calcium levels with claudin 14 expression in high glucose-treated human uroepithelial cells. Moreover, external calcium supplementation in high glucose-treated cells significantly affected the cell migration and restored the claudin 14 expression through focal adhesion and β-1 integrins. Strengthening the epithelial barrier is essential, especially in individuals with diabetes where basal calcium levels could contribute.

SWATH-proteomics reveals Mathurameha, a traditional anti-diabetic herbal formula, attenuates high glucose-induced endothelial dysfunction through the EGF/NO/IL-1β regulatory axis

Mathurameha is a traditional Thai herbal formula with a clinically proven effect of blood sugar reduction in patients with diabetes mellitus, but its anti-diabetic complication potential is largely unknown. This study aimed to elucidate the effects of Mathurameha and its underlying mechanisms against high glucose-induced endothelial dysfunction in human endothelial EA.hy926 cells. After confirming no cytotoxic effects, the cells were treated with normal glucose (NG), high glucose (HG), or high glucose plus Mathurameha (HG + M) for 24 h. A quantitative label-free proteomic analysis using the sequential window acquisition of all theoretical mass spectra (SWATH-MS) approach identified 24 differentially altered proteins among the three groups: 7 between HG and NG, 9 between HG + M and NG, and 13 between HG + M and HG. Bioinformatic analyses suggested a potential anti-diabetic action through the epidermal growth factor (EGF) pathway. Subsequent functional validations demonstrated that Mathurameha reduced the EGF secretion and the intracellular reactive oxygen species (ROS) level in high glucose-treated cells. Mathurameha also exhibited a stimulatory effect on nitric oxide (NO) production while significantly reducing the secretion of endothelin-1 (ET-1) and interleukin-1β (IL-1β) in high glucose-treated cells. In conclusion, our findings demonstrated that Mathurameha attenuated high glucose-induced endothelial dysfunction through the EGF/NO/IL-1β regulatory axis. SignificanceThis study reveals the potential of Mathurameha, a traditional Thai herbal formula, in mitigating high glucose-induced endothelial dysfunction, a common complication in diabetes mellitus. Using proteomics and bioinformatic analyses followed by functional validations, the present study highlights the protective effects of Mathurameha through the EGF/NO/IL-1β regulatory axis. These findings support its potential use as a therapeutic intervention for diabetic vascular complications and provide valuable information for developing more effective anti-diabetic drugs.

Scopoletin alleviates high glucose-induced toxicity in human renal proximal tubular cells via inhibition of oxidative damage, epithelial-mesenchymal transition, and fibrogenesis.

Recent years of evidence suggest the crucial role of renal tubular cells in developing diabetic kidney disease. Scopoletin (SCOP) is a plant-based coumarin with numerous biological activities. This study aimed to determine the effect of SCOP on renal tubular cells in developing diabetic kidney disease and to elucidate mechanisms. In this study, SCOP was evaluated in vitro using renal proximal tubular (HK-2) cells under hyperglycemic conditions to understand its mechanism of action. In HK-2 cells, SCOP alleviated the high glucose-generated reactive oxygen species (ROS), restored the levels of reduced glutathione, and decreased lipid peroxidation. High glucose-induced alteration in the mitochondrial membrane potential was markedly restored in the SCOP-treated cells. Moreover, SCOP significantly reduced the high glucose-induced apoptotic cell population in the Annexin V-FITC flow cytometry study. Furthermore, high glucose markedly elevated the mRNA expression of fibrotic and extracellular matrix (ECM) components, namely, transforming growth factor (TGF)-β, alfa-smooth muscle actin (α-SMA), collagen I, and collagen III, in HK-2 cells compared to the untreated cells. SCOP treatment reduced these mRNA expressions compared to the high glucose-treated cells. Collagen I and TGF-β protein levels were also significantly reduced in the SCOP-treated cells. Further findings in HK-2 cells revealed that SCOP interfered with the epithelial-mesenchymal transition (EMT) in the high glucose-treated HK-2 cells by normalizing E-cadherin and downregulating the vimentin and α-SMA proteins. In conclusion, SCOP modulates the high glucose-generated renal tubular cell oxidative damage and accumulation of ECM components and may be a promising molecule against diabetic nephropathy.

An evaluation of photobiomodulation effects on human gingival fibroblast cells under hyperglycemic condition: an in vitro study.

An in vitro study was designed to evaluate the effects of photobiomodulation (PBM) with 915-nm diode laser on human gingival fibroblast (HGF) cells under hyperglycemic condition. The HGF cells were cultured in Dulbecco's modified eagle medium (DMEM) medium containing 30mM glucose concentration for 48h to mimic the hyperglycemic condition. Subsequently, the cells received three sessions of PBM (915nm, continuous emission mode, 200 mW, energy density values of 3.2, 6, and 9.2J/cm2). Twenty-four hours post-irradiation, cell proliferation, expression of interleukin 6 (IL-6), and vascular endothelial growth factor (VEGF) were assessed with MTT and real-time polymerase chain reaction (PCR) tests, respectively. Also, reactive oxygen species (ROS) production was measured using CM-H2DCFDA fluorimetry. No changes were detected in the cell proliferation rate between the high glucose control group and laser-treated cells, while VEGF and IL-6 gene expression levels increased significantly after PBM in the high glucose-treated cells group. ROS level was significantly decreased in the irradiated cells in high-glucose medium compared with the high glucose control group. Our study revealed the inductive role of 915-nm-mediated PBM on VEGF and the inflammatory response while concurrently reducing reactive oxygen species production in HGF cells in hyperglycemic conditions.

An in vitro Model to Study Placental Functions in Gestational Diabetes Mellitus

Background: Gestational diabetes mellitus (GDM) is a serious pregnancy complication that affects around 14 % of pregnancies worldwide. It not only causes short-term complications in the mother and child but also significantly increases the risks of several chronic diseases, including cardiovascular diseases and diabetes. An excess production of the soluble vascular endothelial growth factor (VEGF) receptor 1 in the placenta, which is a natural inhibitor of VEGF, has been linked to various pregnancy complications. However, placentas affected by GDM often exhibit increased vascularization, suggesting that the role of VEGF in GDM differs from its impact on typical pregnancy complications. Unfortunately, limited studies on placental functions in GDM have been conducted due to a lack of reliable culture models. Methods: In this study, we developed an in vitro model of GDM using the human choriocarcinoma trophoblast cell line BeWo and examined the effects of VEGF on glucose uptake. The BeWo cells were treated with a high glucose-containing (25 mM) syncytialization differentiation medium to produce insulin-resistant cells. Insulin resistance was assessed by a glucose uptake assay using the fluorescent glucose analog 2-NBDG and flow cytometry analysis. Results: The cells treated with high glucose exhibited a significant decrease in glucose uptake, suggesting the successful development of glucose resistance in these cells. However, VEGF treatment did not show a significant impact on glucose uptake in high glucose-treated cells. Conclusions: It is important to note that glucose homeostasis is a complex process involving multiple cell types, and further studies are necessary to fully understand the functions of VEGF on GDM placentas. Nevertheless, the in vitro model we have developed and validated will be a valuable tool for studying placental pathophysiology in GDM and evaluating the effectiveness of potential therapeutic agents. HIGHLIGHTS BeWo cells are the choriocarcinoma cell line which were cultured in the in vitro condition BeWo cells were differentiated using forskolin and 8-Br Camp Insulin resistant model of BeWo cells were prepared by high glucose (25 mM) treatment The effect of VEGF on glucose homeostasis in GDM model of BeWo cells was observed GRAPHICAL ABSTRACT

Effect of metformin on the long non-coding RNA expression levels in type 2 diabetes: an in vitro and clinical trial study.

It has been suggested that the anti-hyperglycemic effect of metformin could be associated with its impact on long non-coding RNA (lncRNA) expression levels. Accordingly, in the current study, we evaluated the effect of metformin on the expression of H19, MEG3, MALAT1, and GAS5 in in vitro and in vivo situations. The effect of hyperglycemia and metformin treatment on the lncRNAs expression level was evaluated in HepG2 cells. A total of 179 age- and sex-matched subjects, including 88 newly diagnosed patients with type 2 diabetes (T2D) and 91 healthy volunteers, were included in the case-control phase of the study. Moreover, 40 newly diagnosed patients participated in the study's open-labeled non-controlled clinical trial phase. The expression levels of lncRNA in HepG2 cells and whole blood samples were determined using QRT-PCR. In vitro results showed that hyperglycemia induced H19 and MALAT1 and decreased GAS5 expression levels. Moreover, metformin decreased H19 and increased GAS5 expression in high glucose-treated cells. Case-control study findings revealed that the circulating levels of H19, MALAT1, and MEG3 were significantlyelevated in T2D patients compared to the control subjects. Finally, results showed that the level of circulating H19 levels decreased while GAS5 increased in T2D patients after taking metformin for 2months. The results of the current study provided evidence that metformin could exert its effect in the treatment of T2D by altering the expression levels of H19 and GAS5.

Abstract P238: High Glucose Induces Interleukin-1β Synthesis In Renal Tubular Cells By Suppressing Autophagy

We showed that IL-1β derived from renal tubular epithelial cells initiates the inflammatory response that promotes salt sensitivity during diabetes. Yet, the intracellular mechanism that mediates IL-1β synthesis by tubular cells is unknown. We hypothesize that high glucose inhibits autophagy in tubular cells resulting in the accumulation of mitochondrial reactive oxygen spices (mtROS) that activate the NLRP3 inflammasome to produce IL-1β. Thirty-week-old db/db mice, a model of type 2 diabetes, and db/+ non-diabetic controls (n=6) were exposed to high salt diet (4% NaCl) for 4 weeks. At the end, mean arterial pressure was higher in db/db vs. db/+ mice (125±7 vs. 104±4 mmHg, P<0.05). db/db mice also displayed higher levels of IL-1β in tubular cells (E-cadherin+) compared to db/+ mice (22±4% vs. 7±2% of IL-1β + E-cadherin + cells, P<0.01 by flow cytometry). We measured two critical components of the autophagy machinery by Western blot. In db/db, we observed reduced LC3B-II expression (49% reduction, P<0.01) and increased p62 (2.7±0.3-fold increase, P<0.001) compared to db/+ mice indicating reduced autophagy. To investigate whether autophagy regulates IL-1β synthesis, we used a primary culture of tubular epithelial cells (TECs) (n=6). TECs exposed to high glucose (15mM) secreted higher levels of IL-1β (58±23 vs. 13±6 pg/ml, P>0.001) and displayed increased caspase-1 and NLRP3 expression compared to TECs exposed to low glucose (5mM). Autophagy analysis revealed low LCB3-II and high p62 suggesting that high glucose suppressed autophagy in tubular cells. This was associated with mtROS accumulation (1.9±0.2-fold increase, P<0.001 by MitoSox Red). We also analyzed mTOR, an inhibitor of autophagy. TECs exposed to high glucose showed higher levels of mTOR phosphorylated at Ser 2448 (a marker of activation) compared to low glucose-treated cells. Blocking mTOR with rapamycin (1μM) restored autophagy in high glucose-treated TECs, reduced caspase-1, NLRP3, and mitochondrial ROS and resulted in low IL-1β secretion. Thus, autophagy inhibition is a critical event behind the high glucose-mediated IL-1β secretion in tubular cells. Restoring autophagy might be a therapeutic target to blunt the inflammatory response that causes salt sensitivity during diabetes.

Folate derivatives, 5-methyltetrahydrofolate and 10-formyltetrahydrofolate, protect BEAS-2B cells from high glucose-induced oxidative stress and inflammation.

Folate (vitamin B9) and its biologically active derivatives are well-known antioxidant molecules protecting cells from oxidative degradation. The presence of high glucose, often found in diabetic patients, causes oxidative stress resulting in cellular stress and inflammatory injury. Cells in organs such as the lung are highly prone to inflammation, and various protective mechanisms exist to prevent the progressive disorders arising from inflammation. In the present study, the synthetic form of folate, i.e. folic acid, and active forms of folate, i.e. 5-methyltetrahydrofolate and 10-formyltetrahydrofolate, were evaluated for their antioxidant and antiinflammatory potential against high glucose (50 mM)–mediated oxidative stress and inflammation in BEAS-2B cells, an immortalised bronchial epithelial cell line. High glucose treatment showed a 67% reduction in the viability of BEAS-2B cells, which was restored to the viability levels seen in control cultures by the addition of active folate derivatives to the culture media. The DCFH-DA fluorometric assay was performed for oxidative stress detection. The high glucose–treated cells showed a significantly higher fluorescence intensity (1.81- and 3.8-fold for microplate assay and microscopic observation, respectively), which was normalised to control levels on supplementation with active folate derivatives. The proinflammatory NF-κB p50 protein expression in the active folate derivative–supplemented high glucose–treated cells was significantly lower compared to the folic acid treatment. In support of these findings, in silico microarray GENVESTIGATOR database analysis showed that in bronchiolar small airway epithelial cells exposed to inflammatory condition, folate utilization pathway genes are largely downregulated. However, the folate-binding protein gene, which encodes to the folate receptor 1 (FOLR1), is significantly upregulated, suggesting a high demand for folate by these cells in inflammatory situations. Supplementation of the active folate derivatives 5-methyltetrahydrofolate and 10-formyltetrahydrofolate resulted in significantly higher protection over the folic acid from high glucose–induced oxidative stress and inflammation. Therefore, the biologically active folate derivatives could be a suitable alternative over the folic acid for alleviating inflammatory injury-causing oxidative stress.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11626-022-00691-w.

Effect of dexmedetomidine on AKT/ERK signaling pathway and EMT-related proteins in high glucose-induced apoptosis in human renal tubular epithelial cells


 
 
 
 Purpose: To study the effect of dexmedetomidine (Dex) on AKTERK signaling pathway and EMT- related proteins in high glucose-induced apoptosis in human kidney tubular epithelial cells.
 Methods: HK-2 cells were assigned to control, high-glucose and Dex groups. Levels of ROS were determined using live cell station. Flow cytometry was used to measure cell apoptosis and cell cycle while Western blot was applied to assay levels of PI3K, Akt, p-Akt, ERK and p-ERK.
 Results: The ROS concentrations were markedly reduced in Dex group, relative to high glucose group (p < 0.05). Apoptosis was reduced in Dex group, relative to high glucose group, while P13K protein levels were significantly lower in high glucose and Dex groups than their corresponding control levels. In the high glucose-treated cells, AKT protein expression was downregulated, relative to control group, and p-AKT expression was markedly reduced in Dex group (p < 0.05). Protein expressions of ERK and p-ERK in high glucose group were lower than control values, but were significantly accentuated in Dex group, relative to high glucose group (p < 0.05).
 Conclusion: Dex mitigates high glucose-induced apoptosis of HK-2 cells, increases the proportion of cells in G1 phase, and reduces their EMT via a mechanism related to regulation of AKTERK signaling pathway-associated proteins. AKTERK signaling pathway-associated proteins provide insights into the development of drugs for the treatment of diabetic nephropathy.
 
 
 

Etoposide-induced protein 2.4 ameliorates high glucose-induced epithelial-mesenchymal transition by activating adenosine monophosphate-activated protein kinase pathway in renal tubular cells.

Epithelial-mesenchymal transition (EMT), known as the transition of tubular epithelial cells into fibroblasts, is one of the potential mechanisms of renal fibrosis, which promotes the development of diabetic kidney disease (DKD). Etoposide-induced protein 2.4 (EI24) is known as an endoplasmic reticulum (ER)-localized Bcl-2-binding transmembrane protein with various functions that can affect autophagy, apoptosis and differentiation. However, whether EI24 is involved in EMT of renal tubular epithelial cells and the exact mechanism is still not known. In this study, we first reported that EI24 expression was significantly downregulated in the kidneys of diabetic mice and in high glucose-stimulated HK2 cells. Knockdown of EI24 led to EMT of HK2 cells, as indicated by decreased E-cadherin and increased α-smooth muscle actin (α-SMA). Meanwhile, overexpression of EI24 ameliorated high glucose-induced EMT of HK2 cells via activation of the adenosine monophosphate-activated protein kinase (AMPK) pathway. Then, DNA methyltransferase (DNMT) inhibitor 5-Aza-2'-deoxycytidine (5-Aza) treatment enhanced EI24 expression and alleviated EMT in high glucose-treated HK2 cells and the kidneys of diabetic mice. Furthermore, DNMT1 and DNMT3a upregulation were found to be involved in the decrease of EI24 in high glucose-stimulated HK2 cells. Silencing of DNMT1 and DNMT3a effectively reversed high glucose-induced downregulation of EI24 and aggravation of EMT. Our findings demonstrate that the DNA methyltransferase-regulated EI24 affects EMT of renal tubular cells via AMPK signaling pathway. It is suggested that EI24 may be a potential therapeutic target for diabetic renal injury.

2-Dodecyl-6-Methoxycyclohexa-2,5-Diene-1,4-Dione Ameliorates Diabetic Cognitive Impairment Through Inhibiting Hif3α and Apoptosis.

Diabetes mellitus (DM) is an independent risk factor for cognitive impairment. Although the etiology of diabetic cognitive impairment is complex and multifactorial, the hippocampus neuronal apoptosis is recognized as a main cause of diabetes-induced cognitive impairment. 2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) was purified from the roots of Averrhoa carambola L. Previous research demonstrated that DMDD was safe and effective in delaying some diabetic complications. However, the efficacy of DMDD to ameliorate diabetic cognitive impairment in type 2 diabetes mice has not been reported. In the present study, the behavioral evaluation was performed by Y maze and novel object recognition in db/db mice. Gene expression profiles were detected using mouse lncRNA microarray analysis in the hippocampi of db/db mice. Changes in the neurodegeneration-associated proteins and the apoptosis-related proteins were determined in both db/db mice and high glucose-treated HT22 cells by Western blotting. We observed that DMDD treatment significantly ameliorated the spatial working memory and object recognition memory impairment in db/db mice. Further study showed that neurodegeneration-associated protein tau was decreased after DMDD treatment in the hippocampi of db/db mice. Eleven lncRNAs and four mRNAs including pro-apoptotic gene Hif3a were significantly differently expressed after DMDD treatment in the hippocampi of db/db mice. The expression of Hif3a, cleaved parp, and caspase 3 proteins was significantly increased in the hippocampi of diabetic db/db mice compared with db/m control mice and then decreased after DMDD treatment. Similar beneficial effects of DMDD were observed in HG-treated HT22 cells. These data indicate that DMDD can alleviate cognitive impairment by inhibiting neuronal apoptosis through decreasing the expression of pro-apoptotic protein Hif3a. In conclusion, our study suggests that DMDD has great potential to be a new preventive and therapeutic compound for diabetic cognitive impairment.

Evaluation of Gallic Acid-Coated Gold Nanoparticles as an Anti-Aging Ingredient.

Hyperglycemic environment-induced oxidative stress-mediated matrix metalloproteinase-1 (MMP-1) plays a crucial role in the degradation of the extracellular matrix (ECM), which might contribute to premature skin aging. Synthesized, environmentally friendly gallic acid-coated gold nanoparticles (GA–AuNPs) have been evaluated as an anti-aging antioxidant. Their microstructure was characterized by transmission electron microscopy (TEM), which showed that GA–AuNPs are spherical when prepared at pH 11. Dynamic light scattering (DLS) analysis revealed that the average hydrodynamic diameter of a GA–AuNP is approximately 40 nm and with a zeta potential of −49.63 ± 2.11 mV. Additionally, the present data showed that GA–AuNPs have a superior ability to inhibit high glucose-mediated MMP-1-elicited type I collagen degradation in dermal fibroblast cells. Collectively, our data indicated that high-glucose-mediated ROS production was reduced upon cell treatment with GA–AuNPs, which blocked p38 MAPK/ERK-mediated c-Jun, c-Fos, ATF-2 phosphorylation, and the phosphorylation of NFκB, leading to the down-regulation of MMP-1 mRNA and protein expression in high glucose-treated cells. Our findings suggest that GA-AuNPs have a superior ability to inhibit high-glucose-mediated MMP-1-elicited ECM degradation, which highlights its potential as an anti-aging ingredient.

Exendin-4 inhibits high glucose-induced oxidative stress in retinal pigment epithelial cells by modulating the expression and activation of p66Shc

Purpose Activation of p66Sch, an adaptor protein, is associated with oxidative stress and apoptosis and has been implicated in the pathogenesis of diabetes-induced retinal pigment epithelial cell damage and diabetic retinopathy. Exendin-4 is a glucagon-like protein that protects against diabetic retinopathy, but the mechanism of action is not well understood. This study aimed to investigate whether Exendin-4 could protect against high glucose-induced oxidative stress and apoptosis in the adult human retinal pigment epithelial-19 cell line by modulating levels and activation of p66Shc and to study the underlying mechanisms. Materials and Methods Adult human retinal pigment epithelial-19 cells were cultured under low (5 µM) or high glucose (100 µM) conditions in the presence or absence of Exendin-4 and with or without pre-incubation with Exendin-9-39, a glucagon-like peptide-1 receptor antagonist. Results In a dose-dependent manner, Exendin-4 inhibited high glucose-induced cell death and decreased levels of reactive oxygen species, lactate dehydrogenase release, and single single-stranded DNA. At the most effective concentration (100 µM), Exendin-4 reduced mitochondrial levels of phospho-p66Shc (Ser36), cytoplasmic levels of cleaved caspase-3 and cytochrome-c, and NADPH oxidase levels in high glucose-treated cells. It also increased levels of glutathione and magnesium superoxide dismutase and protein levels of magnesium superoxide dismutase but downregulated total protein levels of protein kinase-β and p66Shc and inhibited c-Jun N-terminal kinase phosphorylation in both low- and high glucose-treated cells. All these Exendin-4 effects, however, were inhibited by Exendin-9-39. Conclusions Exendin-4 protects against high glucose-induced adult human retinal pigment epithelial-19 cell damage by increasing antioxidants, downregulating NADPH, and inhibiting mitochondria-mediated apoptosis, effects that are associated with the inhibition of c-Jun N-terminal kinase and downregulation of protein kinase-β and p66Shc.

Identification of sphingosine 1-phosphate level and MAPK/ERK signaling in pancreatic β cells.

Purpose Sphingosine kinase is a lipid kinase that phosphorylates sphingosine to generate sphingosine 1-phosphate (S1P). S1P regulates pancreatic islet β-cell endoplasmic reticulum stress and proliferation. Type 1 and type 2 diabetes share some key pathogenic processes. In this study, we investigated whether secretion of insulin and production of S1P is altered in alloxan and glucose-treated cells from the rat pancreatic β-cell line RIN-5F.Methods RIN-5F cells were treated with 2 mM alloxan and 20 mM glucose for 6 hours or 24 hours before being evaluated by enzyme linked immunosorbent assay (ELISA) and Western blotting.Results Insulin secretion and expression was higher in RIN-5F cells treated with glucose compared to control cells. In contrast, alloxan treatment did not affect insulin secretion and expression in RIN-5F cells. Interestingly, compared with normal control levels, S1P/EDG-5 was increased in both alloxan and glucose-treated pancreatic β cell than normal control. Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) inhibition strongly decreased the expression of insulin and S1P in glucose- or alloxan-treated RIN-5F cells.Conclusions We observe that production of S1P is increased in both diabetic cell models. In addition, MAPK/ERK signaling regulates secretion of insulin and S1P expression in pancreatic β-cells. Based on the literature and our findings, S1P may be a promising agent for the treatment of insulin-related disorders.

Casein Kinase 2-Interacting Protein-1 Alleviates High Glucose-Reduced Autophagy, Oxidative Stress, and Apoptosis in Retinal Pigment Epithelial Cells via Activating the p62/KEAP1/NRF2 Signaling Pathway.

Background Casein kinase 2-interacting protein-1 (CKIP-1) has been proved to be associated with complications of diabetes. Diabetic retinopathy is a main diabetic complication which usually leads to blindness. The current study aims to investigate the role of CKIP-1 in high glucose-treated retinal pigment epithelial (RPE) cells which is a component of blood-retinal barriers. Methods The RPE cells, ARPE-19, are treated with high glucose to mimic the diabetic stimulation. CKIP-1 was overexpressed in ARPE-19 cells to evaluate its effects on autophagy, oxidative stress, and apoptosis induced by high glucose treatment, using Western blot, immunofluorescence, and flow cytometry assays, respectively. Results CKIP-1 was expressed at a lower level in high glucose-treated cells than in normal glucose cells. Overexpression of CKIP-1 enhanced the Nrf2 translocation to the nucleus. Furthermore, high glucose-induced autophagy, oxidative stress, and apoptosis were inhibited after overexpression of CKIP-1. Also, CKIP-1 regulates the p62/Keap1/Nrf2 signaling, which might be the potential mechanism in this model. Conclusion In conclusion, CKIP-1 may be a potential therapeutic target that protects RPE cells from injury and subsequent diabetic retinopathy induced by high glucose.

Wnt5a promotes renal tubular inflammation in diabetic nephropathy by binding to CD146 through noncanonical Wnt signaling

Immune and inflammatory factors have emerged as key pathophysiological mechanisms in the progression of diabetic renal injury. Noncanonical Wnt5a signaling plays an essential role in obesity- or diabetes-induced metabolic dysfunction and inflammation, but its explicit molecular mechanisms and biological function in diabetic nephropathy (DN) remain unknown. In this study, we found that the expression of Wnt5a and CD146 in the kidney and the level of soluble form of CD146 (sCD146) in serum and urine samples were upregulated in DN patients compared to controls, and this alteration was correlated with the inflammatory process and progression of renal impairment. Blocking the activation of Wnt5a signaling with the Wnt5a antagonist Box5 prevented JNK phosphorylation and high glucose-induced inflammatory responses in db/db mice and high glucose-treated HK-2 cells. Similar effects were observed by silencing Wnt5a with small-interfering RNA (siRNA) in cultured HK-2 cells. Knockdown of CD146 blocked Wnt5a-induced expression of proinflammatory cytokines and activation of JNK, which suggests that CD146 is essential for the activation of the Wnt5a pathway. Finally, we confirmed that Wnt5a directly interacted with CD146 to activate noncanonical Wnt signaling in HK-2 cells. Taken together, our findings suggest that by directly binding to CD146, Wnt5a-induced noncanonical signaling is a contributing mechanism for renal tubular inflammation in diabetic nephropathy. The concentration of sCD146 in serum and urine could be a potential biomarker to predict renal outcomes in DN patients.

NIPA2 regulates osteoblast function by modulating mitophagy in type 2 diabetes osteoporosis

The highly selective magnesium transporter non-imprinted in Prader-Willi/Angelman syndrome region protein 2 (NIPA2) has recently been associated with the development and progression of type 2 diabetes osteoporosis, but the mechanisms involved are still poorly understood. Because mitophagy is involved in the pathology of type 2 diabetes osteoporosis, the present study aimed to explore the relationship among NIPA2, mitophagy and osteoblast osteogenic capacity. NIPA2 expression was reduced in C57BKS background db/db mice and in vitro models of type 2 diabetes osteoporosis, and the activation of mitophagy in primary culture osteoblast-derived from db/db mice and in high glucose-treated human fetal osteoblastic cells (hFOB1.19) was observed. Knockdown, overexpression of NIPA2 and pharmacological inhibition of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) showed that NIPA2 increased osteoblast function, which was likely regulated by PTEN induced kinase 1 (PINK1)/E3 ubiquitin ligase PARK2 (Parkin)-mediated mitophagy via the PGC-1α/forkhead box O3a(FoxO3a)/mitochondrial membrane potential (MMP) pathway. Furthermore, the negative effect of mitophagy on osteoblast function was confirmed by pharmacological regulation of mitophagy and knockdown of Parkin. Taken together, these results suggest that NIPA2 positively regulates the osteogenic capacity of osteoblasts via the mitophagy pathway in type 2 diabetes.

High glucose up-regulates MMP-1 expression in endothelial cells via mTORC2-PKM2 pathway.

To investigate the role of pyruvate kinase isozyme type M2 (PKM2) in regulating the expression of matrix metalloproteinase-1 (MMP-1) in human umbilical vein endothelial cells (HUVECs) under high glucose exposure and the underlying mechanisms. HUVECs were exposed to concentration gradient (5.5, 15.0, 30.0 mmol/L) of D-glucose for 72 h. Western blotting was used to detect the expression of PKM2, p-PKM2 Y105 as well as its upstream and downstream proteins. The formation of PKM2 tetramer/dimer was examined. Immunofluorescence was used to detect the translocation of PKM2. The specific siRNAs, TEPP-46, and rapamycin were used to analyze the regulatory relations among PKM2, mammalian target of rapamycin (mTOR) complex, and MMP-1. Co-immunoprecipitation was used to detect the interaction of mTOR complex and PKM2. High glucose exposure upregulated the level of p-PKM2 Y105, and promoted PKM2 dimer formation and nuclear translocation as well as the expression of MMP-1 and Rictor in HUVECs. Down-regulation of PKM2 expression or the treatment of TEPP-46 reversed the high glucose induced-upregulation of MMP-1. Inhibition of mTOR singnal pathway by rapacymin reversed the phosphorylation of PKM2 on tyrosine 105 and the expression of MMP-1 in high glucose-treated cells. Knockdown the expression of Rictor decreased the phosphorylation of PKM2 on tyrosine 105, while knockdown the expression of Raptor upregulated the phosphorylation of PKM2 on tyrosine 105 under high glucose condition. Co-immunoprecipitation indicated the direct interaction between Rictor and PKM2. The phosphorylation of PKM2 promotes high glucose-induced upregulation of MMP-1, and mTORC2 participates in the upregulation of PKM2 phosphorylation under high glucose condition.

Quercetin attenuates high glucose-induced injury in human retinal pigment epithelial cell line ARPE-19 by up-regulation of miR-29b.

Quercetin is a kind of distinctive bioactive flavonoid that has potent anti-oxidant, anti-inflammatory and anti-diabetic properties. The present article was designed to check the effect of quercetin on diabetic retinopathy. Adult retinal pigment epithelial cell line (ARPE)-19 cells were pre-treated with quercetin and then stimulated by high glucose. Cell damage was evaluated by CCK-8 assay, flow cytometer, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay, 2,7-dichlorofluorescein diacetate probe and western blot. The association between quercetin and miR-29b expression as well as the downstream pathways was studied by qRT-PCR and western blot. Pre-treating ARPE-19 cells with quercetin clearly attenuated high glucose-induced viability loss, apoptosis, MCP-1 and IL-6 overproduction and reactive oxygen species (ROS) generation. Quercetin down-regulated p53, Bax and cleaved-caspase-3 expression, while up-regulated CyclinD1, CDK4 and Bcl-2. miR-29b was low expressed in high glucose-treated cell, but quercetin elevated its expression. Moreover, the protective action of quercetin towards ARPE-19 cells was attenuated when miR-29b was suppressed. Also, quercetin promoted PTEN/AKT pathway, while inhibited NF-κB pathway via a miR-29b-dependent way. These data illustrated quercetin possibly possess the anti-diabetic retinopathy potential, as quercetin clearly attenuated high glucose-evoked damage in ARPE-19 cells. The protective action of quercetin may due to its regulation on miR-29b expression as well as PTEN/AKT and NF-κB pathways.

Long noncoding RNA KCNQ1OT1 induces pyroptosis in diabetic corneal endothelial keratopathy.

Diabetic corneal endothelial keratopathy is an intractable ocular complication characterized by corneal edema and endothelial decompensation, which seriously threaten vision. It has been suggested that diabetes is associated with pyroptosis, a type of programmed cell death via the activation of inflammation. Long noncoding RNA KCNQ1OT1 is commonly associated with various pathophysiological mechanisms of diabetic complications, including diabetic cardiomyopathy and diabetic retinopathy. However, whether KCNQ1OT1 is capable of regulating pyroptosis and participates in the pathogenesis of diabetic corneal endothelial keratopathy remains unknown. The aim of this study was to investigate the mechanisms of KCNQ1OT1 in diabetic corneal endothelial keratopathy. Here, we reveal that KCNQ1OT1 and pyroptosis can be triggered in diabetic human and rat corneal endothelium, along with the high glucose-treated corneal endothelial cells. However, miR-214 expression was substantially decreased in vivo and in experiments with cultured cells. LDH assay was also used to verify the existence of pyroptosis in high glucose-treated cells. Bioinformatics prediction and luciferase assays showed that KCNQ1OT1 may function as a competing endogenous RNA binding miR-214 to regulate the expression of caspase-1. To further analyze the KCNQ1OT1-mediated mechanism, miR-214 mimic and inhibitor were introduced into the high glucose-treated corneal endothelial cells. The results showed that upregulation of miR-214 attenuated pyroptosis; conversely, knockdown of miR-214 promoted it. In addition, KCNQ1OT1 knockdown by a small interfering RNA decreased pyroptosis factors expressions but enhanced miR-214 expression in corneal endothelial cells. To understand the signaling mechanisms underlying the prepyroptotic properties of KCNQ1OT1, si-KCNQ1OT1 was cotransfected with or without miR-214 inhibitor. The results showed that pyroptosis was repressed after silencing KCNQ1OT1 but was reversed by cotransfection with miR-214 inhibitor, suggesting that KCNQ1OT1 mediated pyroptosis induced by high glucose via targeting miR-214. Therefore, the KCNQ1OT1/miR-214/caspase-1 signaling pathway represents a new mechanism of diabetic corneal endothelial keratopathy progression, and KCNQ1OT1 could potentially be a novel therapeutic target.