Cells In High Glucose Condition Research Articles

The Key Role of Liraglutide in Preventing Autophagy of Vascular Smooth Muscle Cells in High Glucose Conditions

The glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide (LIRA) is a potential hypoglycemic drug with anti-atherosclerosis (AS) effects. Autophagy in the vascular smooth muscle cells (VSMCs) facilitates AS. However, the role of autophagy in the anti-AS mechanism of LIRA remains unclear. To examine the role and mechanisms of autophagy in LIRA’s improvement of the biological characteristics of VSMCs in high glucose conditions. Experimental animal study. VSMCs isolated from the thoracic aorta of male SD rats were subjected to a high glucose (HG) condition (25 mM) in Dulbecco’s Modified Eagle’s Medium with or without LIRA, the GLP-1 receptor antagonist exendin9-39 (Exe9-39), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), and autophagy inhibitors (3-methyladenine [3-MA] and bafilomycin A1 [Baf A1]). Acridine orange staining, western blotting, transmission electron microscopy, and mCherry-GFP-LC3 transfection were performed to evaluate the autophagy flux. Additionally, VSMC migration, calcification, proliferation, and apoptosis in HG conditions were observed. Addition of LIRA alone or in combination with autophagy inhibitors significantly downregulated Beclin, increased the LC3-II/LC3-I ratio, and upregulated p62 in VSMCs in HG conditions. Furthermore, autophagolysosome formation was markedly curbed after treatment with LIRA and/or autophagy inhibitors. Inhibition of autophagy by LIRA and/or the autophagy inhibitors attenuated VSMC phenotype conversion, proliferation, migration, and calcification and promoted VSMC apoptosis in HG conditions. This protective role of LIRA was augmented by LY294002, but inhibited by Exe9-39. LIRA plays a significant role in the improvement of the biological features of VSMCs in HG conditions.

Lens epithelial cell-derived exosome inhibits angiogenesis in ocular pathological neovascularization through its delivery of miR-146a-5p.

Abnormal ocular neovascularization, a major pathology of eye diseases, leads to severe visual loss. The role of lens epithelial cell (LEC)-derived exosomes (Lec-exo) is largely unknown. Thus, we aimed to investigate whether Lec-exo can inhibit abnormal ocular neovascularization and explore the possible mechanisms. In our study, we proved the first evidence that exosomes derived from LECs attenuated angiogenesis in both oxygen-induced retinopathy and laser-induced choroidal neovascularization mice models. Further in vitro experiments proved that Lec-exo inhibited proliferation, migration, and tube formation capability of human umbilical vein endothelial cells in high glucose condition. Further high-throughput miRNAs sequencing analysis detected that miR-146a-5p was enriched in Lec-exo. Mechanistically, exosomal miR-146a-5p was delivered to endothelial cells and bound to the NRAS coding sequence, which subsequently inactivated AKT/ERK signaling pathway. We successfully elucidated the function of Lec-exo in inhibiting abnormal ocular neovascularization, which may offer a promising strategy for treatment of abnormal ocular neovascularization.

High glucose enhances the aggressiveness of lung adenocarcinoma via activating epidermal growth factor receptor/signal transducer and activator of transcription 3 pathways

Epidemiological studies revealed hyperglycemia as a poor prognostic factor for lung adenocarcinoma with unclear molecular mechanisms. The present study thus aimed to investigate the effects of high glucose on the progression of lung adenocarcinoma and its underlying mechanisms. Lung adenocarcinoma cell lines, A549 and RERF-LC-KJ, were cultured in 5.6 mM glucose (normal glucose; NG) or 25 mM glucose (high glucose; HG) resembling euglycemia and hyperglycemia. Cells were examined for proliferation by the MTT assay, and migration-invasion using Transwell. The expressions of signaling proteins in epidermal growth factor receptor (EGFR) pathways and their downstream targets were investigated using Western blots. The effects of diabetes mellitus (DM) and hyperglycemia on lung adenocarcinoma growth in vivo were studied in streptozotocin-induced diabetic BALB/cAJcl-Nu/Nu mice and their nondiabetic counterparts. High glucose significantly promoted proliferation, migration, and invasion of lung adenocarcinoma cells compared with those in normal glucose (P<.05). Western blot analyses showed the increased ratio of pEGFR/EGFR in cells cultured in high glucose and subsequently activated the signal transducer and activator of transcription 3 (STAT3). Epithelial-mesenchymal (EMT) markers were also altered in lung adenocarcinoma cells in high glucose conditions, corresponding with increased migration and invasion abilities. Erlotinib, an EGFR inhibitor, significantly reversed high glucose-induced aggressive phenotypes confirming high glucose-enhancing lung adenocarcinoma progression via the activation of EGFR. DM and hyperglycemia also promoted the growth of lung adenocarcinoma xenografts in vivo in which erlotinib significantly suppressed the growth of tumors (P<.05) suggesting EGFR inhibitor as an effective therapeutic agent for lung adenocarcinoma with DM.

Focal adhesion kinase–related pathways may be suppressed by metformin in vascular smooth muscle cells in high glucose conditions

IntroductionCardiovascular diseases are known as one of the important causes of death in patients with diabetes mellitus. Metformin is used as an oral medication for reducing blood sugar. In this study, the effects of metformin were investigated on the FAK gene expression levels, pFAK protein values, cell viability and migration rate of VSMCs in high glucose conditions.Materials and methodsThe FAK gene expression levels and pFAK protein values were evaluated in VSMCs treated with different doses of metformin (1, 5 and 7 mM), based on cell viability using RT‐qPCR, western blotting and MTT techniques. The cellular migration was evaluated by scratch assay.ResultsThe FAK gene expression levels reduced significantly in metformin‐treated VSMCs at 24 h and 48 h periods (p < .0008 and p < .0001, respectively). The pFAK protein values reduced significantly at 24 h (5 mM and 7 mM metformin doses) and 48 h periods (p < .001). In agreement with pFAK protein values, cellular migration reduced significantly at 24 h and 48 h periods (p < .001).ConclusionThe results showed that metformin may suppress the proliferation and migration of VSMCs via FAK‐related pathways and may retard the progression of vessel stenosis in diabetes.

The effect of metformin on the metabolism of human vascular smooth muscle cells in high glucose conditions.

Metformin is widely used in type 2 diabetic patients as an antihyperglycemic drug. The aim of this study was to investigate the effect of metformin on the metabolism of vascular smooth muscle cells in high glucose conditions. The vascular smooth muscle cells were cultured in DMEM F12 containing glucose as high as 25mM. The preconditioned cells were then treated with metformin in doses of 1, 5, and 7mM for 24h. MTT method was used to determine cell viability. Biochemical parameters including lactate, glucose, total protein, creatinine, and triglyceride were measured in the cell culture after the treatment with metformin. Oil Red O staining method was used to stain the lipids in the cells. Metformin reduced significantly (p<0.001) VSMC proliferation in a concentration-dependent manner. With the increase of glucose uptake by VSMCs, the cell lipid deposition was not changed. Other biochemical parameters such as lactate, triglyceride, total protein, and creatinine were significantly changed in the cell culture (p<0.05). Metformin increased the glucose uptake impacting metabolic pathways in VSMCs. It also increased the lactate efflux and protein metabolism without the change in cellular lipid deposition in high glucose conditions.

Antioxidant Polyphenols of Antirhea borbonica Medicinal Plant and Caffeic Acid Reduce Cerebrovascular, Inflammatory and Metabolic Disorders Aggravated by High-Fat Diet-Induced Obesity in a Mouse Model of Stroke.

Metabolic disorders related to obesity and type 2 diabetes are associated with aggravated cerebrovascular damages during stroke. In particular, hyperglycemia alters redox and inflammatory status, leading to cerebral endothelial cell dysfunction, blood–brain barrier (BBB) disruption and brain homeostasis loss. Polyphenols constitute the most abundant dietary antioxidants and exert anti-inflammatory effects that may improve cerebrovascular complications in stroke. This study evaluated the effects of the characterized polyphenol-rich extract of Antirhea borbonica medicinal plant and its major constituent caffeic acid on a high-fat diet (HFD)-induced obesity mouse model during ischemic stroke, and murine bEnd3 cerebral endothelial cells in high glucose condition. In vivo, polyphenols administered by oral gavage for 12 weeks attenuated insulin resistance, hyperglycemia, hyperinsulinemia and dyslipidemia caused by HFD-induced obesity. Polyphenols limited brain infarct, hemorrhagic transformation and BBB disruption aggravated by obesity during stroke. Polyphenols exhibited anti-inflammatory and antioxidant properties by reducing IL-1β, IL-6, MCP-1, TNF-α and Nrf2 overproduction as well as total SOD activity elevation at the cerebral or peripheral levels in obese mice. In vitro, polyphenols decreased MMP-2 activity that correlated with MCP-1 secretion and ROS intracellular levels in hyperglycemic condition. Protective effects of polyphenols were linked to their bioavailability with evidence for circulating metabolites including caffeic acid, quercetin and hippuric acid. Altogether, these findings show that antioxidant polyphenols reduced cerebrovascular, inflammatory and metabolic disorders aggravated by obesity in a mouse model of stroke. It will be relevant to assess polyphenol-based strategies to improve the clinical consequences of stroke in the context of obesity and diabetes.

MiR-146a-5p modulates cellular senescence and apoptosis in visceral adipose tissue of long-lived Ames dwarf mice and in cultured pre-adipocytes.

MicroRNAs (miRNAs) are potent regulators of multiple biological processes. Previous studies have demonstrated that miR-146a-5p increases in normal mice during aging, while long-living Ames dwarf (df/df) mice maintain youthful levels of this miRNA. The aim of this study was to elucidate the involvement of miR-146a-5p in modulating cellular senescence and apoptosis in visceral adipose tissue of df/df mice and cultured pre-adipocytes. To test the effects of miR-146a-5p overexpression on visceral adipose tissue, wild-type, and df/df mice, were treated with miRNA-negative control-base and df/df were transfected with 4 or 8µg/g of a miR-146a-5p mimetic, respectively. Effects of miR-146a-5p overexpression were also evaluated in 3T3-L1 cells cultured under high and normal glucose conditions. Treatment with miR-146a-5p mimetic increased cellular senescence and inflammation and decreased pro-apoptotic factors in visceral adipose tissue of df/df mice. The miR-146a-5p mimetic induced similar effects in 3T3-L1 cells cultivated at normal but not high glucose levels. Importantly, 3T3-L1 HG cells in high glucose conditions showed significantly higher expression of miR-146a-5p than 3T3-L1 grown in normal glucose conditions. These results indicate that miR-146a-5p can be a marker for cellular senescence. This miRNA represents one of the significant SASP factors that if not precisely regulated, can accentuate inflammatory responses and stimulate senescence in surrounding non-senescent cells. The role of miR-146a-5p is different in healthy versus stressed cells, suggesting potential effects of this miRNA depend on overall organismal health, aging, and metabolic state.

FKBPL and SIRT-1 Are Downregulated by Diabetes in Pregnancy Impacting on Angiogenesis and Endothelial Function.

Diabetes in pregnancy is associated with adverse pregnancy outcomes including preterm birth. Although the mechanisms leading to these pregnancy complications are still poorly understood, aberrant angiogenesis and endothelial dysfunction play a key role. FKBPL and SIRT-1 are critical regulators of angiogenesis, however, their roles in pregnancies affected by diabetes have not been examined before in detail. Hence, this study aimed to investigate the role of FKBPL and SIRT-1 in pre-gestational (type 1 diabetes mellitus, T1D) and gestational diabetes mellitus (GDM). Placental protein expression of important angiogenesis proteins, FKBPL, SIRT-1, PlGF and VEGF-R1, was determined from pregnant women with GDM or T1D, and in the first trimester trophoblast cells exposed to high glucose (25 mM) and varying oxygen concentrations [21%, 6.5%, 2.5% (ACH-3Ps)]. Endothelial cell function was assessed in high glucose conditions (30 mM) and following FKBPL overexpression. Placental FKBPL protein expression was downregulated in T1D (FKBPL; p<0.05) whereas PlGF/VEGF-R1 were upregulated (p<0.05); correlations adjusted for gestational age were also significant. In the presence of GDM, only SIRT-1 was significantly downregulated (p<0.05) even when adjusted for gestational age (r=-0.92, p=0.001). Both FKBPL and SIRT-1 protein expression was reduced in ACH-3P cells in high glucose conditions associated with 6.5%/2.5% oxygen concentrations compared to experimental normoxia (21%; p<0.05). FKBPL overexpression in endothelial cells (HUVECs) exacerbated reduction in tubule formation compared to empty vector control, in high glucose conditions (junctions; p<0.01, branches; p<0.05). In conclusion, FKBPL and/or SIRT-1 downregulation in response to diabetic pregnancies may have a key role in the development of vascular dysfunction and associated complications affected by impaired placental angiogenesis.

222-OR: High Glucose Level Increases Expression of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Receptors

Introduction: Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by a novel coronavirus, SARS-CoV-2. The SARS-CoV-2 virus infects the host human cells through its surface receptors human angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2). This study aims to investigate the effects of high glucose on the expression of ACE2 and TMPRSS2 receptors using macrophage (CD14+ve cells) as a biomarker in vivo and human endothelial cells in vitro. Methods: Mononuclear cells were isolated by Ficoll density centrifuge method from whole blood. CD14 cells were isolated from MNCs by MACS kit. The migratory capacity of CD14 cells was evaluated using the CytoSelect Migration Assay kit. CFU-Hill colony formation assay was also done on MNCs. In vitro studies using HUVEC were done by culturing the cells in normal glucose (NG, 5.5mmol/L) or high glucose (HG, 20 mmol/L), followed by mRNA assays at day 3 and 7. Results: All the COVID patients had high glucose levels (&amp;gt;300 mg/dl). The migratory function of CD14 cells show moderately increased migration compared to similar hematopoietic cells from nondiabetic COVID negative patients. In general, CD14 cells showed low expression of ACE2 and/or TMPRSS2 in all samples. For our in-vitro experiments using cultured HUVEC mRNA expression of ACE2 and/or TMPRSS2 receptors were higher in HG compared to NG at day 7. No expression was observed at day 3 either in NG or HG. An elevated gene expression of inflammatory marker, IL-6 and apoptosis marker, P53 were observed in HG at both days 3 and 7. Conclusions: A low expression of SARS-CoV-2 receptors were observed on CD14 macrophage cells from patients which may indicate poor recognition of the virus particle by CD14 cells. Higher expression of the SARS-CoV-2 receptors were noted when HUVEC cells were cultured in HG for 7 days. Conclusion: Longer exposure of human endothelial cells in high glucose condition increase the SARS-CoV-2 receptor expression. Disclosure S. Nandula: None. R. Janapala: None. S. Sen: None. Funding Clinical and Translational Science Institute at Children’s National

Molecular and Biochemical Pathways of Catalpol in Alleviating Diabetes Mellitus and Its Complications

Catalpol isolated from Rehmannia glutinosa is a potent antioxidant and investigated against many disorders. This review appraises the key molecular pathways of catalpol against diabetes mellitus and its complications. Multiple search engines including Google Scholar, PubMed, and Science Direct were used to retrieve publications containing the keywords “Catalpol”, “Type 1 diabetes mellitus”, “Type 2 diabetes mellitus”, and “diabetic complications”. Catalpol promotes IRS-1/PI3K/AKT/GLUT2 activity and suppresses Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose 6-phosphatase (G6Pase) expression in the liver. Catalpol induces myogenesis by increasing MyoD/MyoG/MHC expression and improves mitochondria function through the AMPK/PGC-1α/PPAR-γ and TFAM signaling in skeletal muscles. Catalpol downregulates the pro-inflammatory markers and upregulates the anti-inflammatory markers in adipose tissues. Catalpol exerts antioxidant properties through increasing superoxide dismutase (sod), catalase (cat), and glutathione peroxidase (gsh-px) activity in the pancreas and liver. Catalpol has been shown to have anti-oxidative, anti-inflammatory, anti-apoptosis, and anti-fibrosis properties that in turn bring beneficial effects in diabetic complications. Its nephroprotective effect is related to the modulation of the AGE/RAGE/NF-κB and TGF-β/smad2/3 pathways. Catalpol produces a neuroprotective effect by increasing the expression of protein Kinase-C (PKC) and Cav-1. Furthermore, catalpol exhibits a cardioprotective effect through the apelin/APJ and ROS/NF-κB/Neat1 pathway. Catalpol stimulates proliferation and differentiation of osteoblast cells in high glucose condition. Lastly, catalpol shows its potential in preventing neurodegeneration in the retina with NF-κB downregulation. Overall, catalpol exhibits numerous beneficial effects on diabetes mellitus and diabetic complications.

Sodium-Glucose Co-Transporter 2 Inhibitors Correct Metabolic Maladaptation of Proximal Tubular Epithelial Cells in High-Glucose Conditions.

Glucose filtered in the glomerulus is actively reabsorbed by sodium-glucose co-transporter 2 (SGLT2) in proximal tubular epithelial cells (PTEC) and passively returned to the blood via glucose transporter 2 (GLUT2). Healthy PTEC rely primarily on fatty acid beta-oxidation (FAO) for energy. In phase III trials, SGLT2 inhibitors improved outcomes in diabetic kidney disease (DKD). Tubulointerstitial renal fibrosis due to altered metabolic reprogramming of PTEC might be at the root of the pathogenesis of DKD. Here, we investigated the molecular mechanism of SGLT2 inhibitors’ renoprotective effect by examining transcriptional activity of Spp1, which encodes osteopontin, a key mediator of tubulointerstitial renal fibrosis. With primary cultured PTEC from Spp1-enhanced green fluorescent protein knock-in mice, we proved that in high-glucose conditions, increased SGLT2- and GLUT-mediated glucose uptake is causatively involved in aberrant activation of the glycolytic pathway in PTEC, thereby increasing mitochondrial reactive oxygen species (ROS) formation and transcriptional activation of Spp1. FAO activation did not play a direct role in these processes, but elevated expression of a tubular-specific enzyme, myo-inositol oxygenase, was at least partly involved. Notably, canagliflozin blocked overexpression of myo-inositol oxygenase. In conclusion, SGLT2 inhibitors exerted renoprotective effects by inhibiting aberrant glycolytic metabolism and mitochondrial ROS formation in PTEC in high-glucose conditions.

Proteomic study of in vitro osteogenic differentiation of mesenchymal stem cells in high glucose condition.

Patients with diabetes have been widely reported to be at an increased risk of secondary osteoporosis. Osteoporosis is caused by an imbalance in bone remodeling due to increased bone resorption and/or decreased osteoblast-dependent bone formation. In this study, mesenchymal stem cells (MSCs) were used as a disease model to determine the effects of high glucose levels on MSC-osteoblast development. The results indicated that under high glucose conditions, MSCs had reduced cell viability and increased number of β-galactosidase-positive cells. Furthermore, in vitro osteogenesis was shown to be reduced in MSCs cultured in osteogenic differentiation medium at 10, 25, and 40mM glucose as demonstrated by Alizarin red S staining and alkaline phosphatase activity assay. Moreover, a proteomic study was performed in MSCs cultured with 25 and 40mM glucose. The proteomic results demonstrated that 12 proteins were up- and downregulated in bone marrow-derived mesenchymal stem cells cultured with high glucose in a dose-dependent manner. The findings presented here contribute to our understanding of the mechanism of diabetes mellitus responsible for bone loss. However, the exact mechanism of action of hyperglycemia on bone deformability requires additional studies.

Grape seed proanthocyanidins improves mitochondrial function and reduces oxidative stress through an increase in sirtuin 3 expression in EA.hy926 cells in high glucose condition.

Proanthocyanidins are phenolic compounds abundant in the diet, commonly found in grapes and derivatives, foods known for their health-promoting benefits. There is previous evidence showing the antidiabetic activity of proanthocyanidins, however, their mechanisms of action have not been fully elucidated. This study evaluated the capacity of grape seed proanthocyanidins extract (GSPE) to modulate oxidative stress, nitric oxide levels, mitochondrial dysfunction, apoptosis, and sirtuin expression in endothelial cells EA.hy926 under high glucose condition. In addition, the possible toxic effects of GSPE was evaluated in a zebrafish embryos model. The results showed that GSPE was able to enhance cell viability and avoid the disturbance in redox metabolism induced by high glucose. Moreover, GSPE was able to avoid mitochondria dysfunction and the increased in p53 and poly-(ADP-ribose) polymerase expression induced by high glucose exposition. These effects were attributed to the increase in expression of sirtuin 3, a protein able to regulate mitochondrial function. GSPE in an effective concentration did not show toxic effects in zebrafish embryos model. Taken together, these data elucidate the key molecular target of GSPE for future pharmacological interventions in diabetic patients.

Upregulation of Lysyl Oxidase Expression in Vitreous of Diabetic Subjects: Implications for Diabetic Retinopathy.

Animal studies have shown diabetes-induced lysyl oxidase (LOX) upregulation promotes blood-retinal-barrier breakdown and retinal vascular cell loss associated with diabetic retinopathy (DR). However, it is unclear whether changes in LOX expression contribute to the development and progression of DR. To determine if vitreous LOX levels are altered in patients with DR, 31 vitreous specimens from subjects with advanced proliferative DR (PDR), and 27 from non-diabetics were examined. The two groups were age- and gender-matched (57 ± 12 yrs vs. 53 ± 18 yrs; 19 males and 12 females vs. 17 males and 10 females). Vitreous samples obtained during vitrectomy were assessed for LOX levels using ELISA. LOX was detected in a larger number of PDR subjects (58%) than in non-diabetic subjects (15%). Additionally, ELISA measurements showed a significant increase in LOX levels in the diabetic subjects with PDR, compared to those of non-diabetic subjects (68.3 ± 112 ng/mL vs. 2.1 ± 8.2 ng/mL; p < 0.01). No gender difference in vitreous LOX levels was observed in either the diabetic or non-diabetic groups. Findings support previous reports of increased LOX levels in retinas of diabetic animals and in retinal vascular cells in high glucose condition, raising the prospect of targeting LOX overexpression as a potential target for PDR treatment.

Untargeted Metabolic Profiling Cell-Based Approach of Pulmonary Artery Smooth Muscle Cells in Response to High Glucose and the Effect of the Antioxidant Vitamins D and E.

Pulmonary arterial hypertension (PAH) is a multi-factorial disease characterized by the hyperproliferation of pulmonary artery smooth muscle cells (PASMCs). Excessive reactive oxygen species (ROS) formation resulted in alterations of the structure and function of pulmonary arterial walls, leading to right ventricular failure and death. Diabetes mellitus has not yet been implicated in pulmonary hypertension. However, recently, variable studies have shown that diabetes is correlated with pulmonary hypertension pathobiology, which could participate in the modification of pulmonary artery muscles. The metabolomic changes in PASMCs were studied in response to 25 mM of D-glucose (high glucose, or HG) in order to establish a diabetic-like condition in an in vitro setting, and compared to five mM of D-glucose (normal glucose, or LG). The effect of co-culturing these cells with an ideal blood serum concentration of cholecalciferol-D3 and tocopherol was also examined. The current study aimed to examine the role of hyperglycemia in pulmonary arterial hypertension by the quantification and detection of the metabolomic alteration of smooth muscle cells in high-glucose conditions. Untargeted metabolomics was carried out using hydrophilic interaction liquid chromatography and high-resolution mass spectrometry. Cell proliferation was assessed by cell viability and the [3H] thymidine incorporation assay, and the redox state within the cells was examined by measuring reactive oxygen species (ROS) generation. The results demonstrated that PASMCs in high glucose (HG) grew, proliferated faster, and generated higher levels of superoxide anion (O2·−) and hydrogen peroxide (H2O2). The metabolomics of cells cultured in HG showed that the carbohydrate pathway, especially that of the upper glycolytic pathway metabolites, was influenced by the activation of the oxidation pathway: the pentose phosphate pathway (PPP). The amount of amino acids such as aspartate and glutathione reduced via HG, while glutathione disulfide, N6-Acetyl-L-lysine, glutamate, and 5-aminopentanoate increased. Lipids either as fatty acids or glycerophospholipids were downregulated in most of the metabolites, with the exception of docosatetraenoic acid and PG (16:0/16:1(9Z)). Purine and pyrimidine were influenced by hyperglycaemia following PPP oxidation. The results in addition showed that cells exposed to 25 mM of glucose were oxidatively stressed comparing to those cultured in five mM of glucose. Cholecalciferol (D3, or vitamin D) and tocopherol (vitamin E) were shown to restore the redox status of many metabolic pathways.

Leptin attenuates oxidative stress and neuronal apoptosis in hyperglycemic condition.

One of the main pathological mechanisms of neurotoxicity in diabetic situation is oxidative stress promoted by hyperglycemia. It has been shown that leptin has neuroprotective effects and may provide neuronal survival signals. This study was designed to reveal the possible neuroprotective effects of leptin in hyperglycemic conditions. Pheochromocytoma (PC12) cell viability was assessed via the MTT test. Cellular reactive oxygen species (ROS) generation was determined by DCFH-DA analysis. The malondialdehyde (MDA) and glutathione (GSH) levels were measured in high-glucose-treated PC12 cells with and without leptin cotreatment. Western blotting was performed to measure apoptosis markers (Cleaved caspase-3 and Bax/Bcl2 ratio). Elevation of glucose levels (100mmol/L) consecutively increased intracellular ROS and MDA level, and apoptosis in PC12 cells after 24h leptin administration (12 and 24nmol/L) decreased the high-glucose-induced cell toxicity, caspase-3 activation, and Bax/Bcl-2 ratio. Also, cotreatment with leptin (12 and 24nmol/L) significantly reduced oxidative damage to PC12 cells in high-glucose condition, as reflected by the diminution in MDA and ROS levels and the increase in GSH content. Our finding demonstrates that leptin has protective effects against hyperglycemia-induced neural damage. This could be related to the attenuation of oxidative stress and neural apoptosis.

The protective effects of metformin in an in vitro model of aging 3T3 fibroblast under the high glucose conditions.

Metformin is the most widely used anti-diabetic drug in the world. It reduces advanced glycation end product (AGEs)-induced ROS generation in high glucose condition. Protein glycation contributes to skin aging as it deteriorates the existing collagen by crosslinking. The progressive increase of AGE during aging not only causes oxidative damage to cellular macromolecules but also modulates the activation of transcription factors nuclear factor kappa-B(NF-kB). However, it is still unclear whether metformin can change collagen production and NF-kB activity induced by high glucose conditions in 3T3 fibroblast. The effects of metformin on proliferation, apoptosis, and collagen levels and NF-kB activity of in vitro cell aging model of 3T3 fibroblast cells in high glucose conditions. At first, we investigated the effects of 50mM high glucose concentration, with or without metformin, on 3T3 fibroblast proliferation, by BrdU immunostaining for cell proliferation. Apoptotic levels were analyzed by flow cytometric assay. NF-kB(p65) activity was measured by transcription factor assay kit and collagen I and III levels by Collagen Estimation Assay through ELISA. We observed that metformin exposure leads to decreased apoptosis levels and increased proliferation of 3T3 fibroblast in high glucose media. We also determined that metformin exposure leads to increased production of collagen I-III and decreased activation of NF-kB(p65) activity. The data are consistent with the observation that metformin has a protective effect in this in vitro model of aging 3T3 fibroblasts under high glucose conditions inducing cell proliferation, collagen I and III production, protection from apoptosis, and reducing NF-kB(p65) activity.

NLRC5 deficiency ameliorates diabetic nephropathy through alleviating inflammation.

NOD-like receptor family caspase recruitment domain family domain containing 5 (NLRC5) has important roles in inflammation and innate immunity. NLRC5 was highly expressed in kidney from streptozotocin-induced diabetic mice, db/ db mice and patients with diabetes. Based on that evidence, the present study was designed to explore the roles of NLRC5 in the progression of diabetic nephropathy (DN). We examined kidney injury, including inflammation and fibrosis in Nlrc5 gene knockout ( Nlrc5-/-) and wild-type (WT) diabetic mice. We found that Nlrc5-/- mice developed less-severe diabetic kidney injury compared with WT mice, exhibiting lower albuminuria, less fibronectin and collagen IV expression, and reduced macrophage infiltration but greater levels of podocin and nephrin in the diabetic kidney. The underlying mechanisms were further investigated in vitro with peritoneal macrophages and mesangial cells treated with high glucose. Reduced proinflammatory effect was observed in peritoneal macrophages from Nlrc5-/- mice, associated with NF-κB pathway suppression. Knocking down of NLRC5 in mesangial cells in high-glucose conditions was also associated with reduced NF-κB and TGF-β/Smad signaling. Taken together, NLRC5 promotes inflammation and fibrosis during DN progression partly through the effects on NF-κB and TGF-β/Smad pathways. NLRC5 may, therefore, be a promising therapeutic target for DN treatment.-Luan, P., Zhuang, J., Zou, J., Li, H., Shuai, P., Xu, X., Zhao, Y., Kou, W., Ji, S., Peng, A., Xu, Y., Su, Q., Jian, W., Peng, W. NLRC5 deficiency ameliorates diabetic nephropathy through alleviating inflammation.

Gac fruit extracts ameliorate proliferation and modulate angiogenic markers of human retinal pigment epithelial cells under high glucose conditions

Objective: To investigate the impact of the extracts of Gac fruit parts (peel, pulp, seed, and aril) on the cell viability and angiogenesis markers of human retinal pigment epithelial (ARPE- 19) cells under high glucose conditions. Methods: The effect of the extracts of Gac fruit peel, pulp, seed and aril on the ARPE-19 cells was determined using MTT viability assay, Trypan blue dye and morphological changes were observed using light microscopy. Enzyme-linked immunosorbent-based assay was performed to evaluate the effect of Gac fruit parts on the reactive oxygen species (ROS), vascular endothelial growth factor (VEGF) and pigmented epithelium-derived factor (PEDF) secretions. Results: High glucose (HG) at 30 mmol/L increased ARPE-19 cell viability and ROS and VEGF secretions. While, the exposure of ARPE- 19 cells in high glucose condition to Gac fruit extracts led to inhibition of cell viability, induced morphological changes, decreased ROS and VEGF secretions, and increased PEDF level. Gac pulp, seed, and aril at 1 000 μg/mL showed significant inhibition activities [(7.5 ± 5.1)%, (2.7 ± 0.5)%, (3.2 ± 1.1)%, respectively] against HG-induced ARPE-19 cell viability. The findings also demonstrated that Gac aril at 250 μg/mL significantly decreased ROS and VEGF levels [(40.6 ± 3.3) pg/mL, (107.4 ± 48.3) pg/mL, respectively] compared to ROS [(71.7 ± 2.9) pg/ mL] and VEGF [(606.9 ± 81.1) pg/mL] in HG untreated cells. Moreover, 250 μg/mL of Gac peel dramatically increased PEDF level [(18.2 ± 0.3) ng/mL] compared to that in HG untreated cells [(0.48 ± 0.39) ng/mL]. Conclusions: This study indicates that the extracts of Gac peel, pulp, seed and aril reduced cell viability, minimized ROS generations and showed angiogenic activities. Therefore, our findings open new insights into the potentiality of Gac fruit against HG-related diabetic retinopathy disease.

Effect of metformin and celecoxib on cytotoxicity and release of GDF-15 from human mesenchymal stem cells in high glucose condition.

Mesenchymal stem cells (MSCs) have therapeutic potential for treatment of diabetes. However, in vitro behavior of MSCs in high glucose condition as well as presence of glucose lowering agents is not fully understood. Because MSCs have an important role in tissue repair, we examined the effects of metformin and celecoxib on viability of MSCs in different glucose conditions. MSCs, from umbilical cord blood, were cultured in normoglycemic (glucose 5.5mM), midglycemic (glucose 10mM), and hyperglycemic (glucose 25mM) conditions, and the cell viability was evaluated by MTT assay. The cytotoxicity and secretion of GDF-15 were further tested in MSCs treated with metformin and celecoxib in various glucose concentrations. Our results showed that high glucose condition lowered viability of MSCs. Metformin treatment also inhibited proliferation of MSCs, but its toxicity was not changed in high glucose condition. Celecoxib induced cytotoxicity in MSCs, and the toxicity was increased in high glucose condition. Metformin and celecoxib induced release from MSCs; however, high glucose inhibited the metformin-induced GDF-15 release. These findings suggested that metformin did not increase the cytotoxicity of high glucose condition in MSCs. Moreover, celecoxib treatment in diabetic condition can reduce the viability of MSCs to proliferate and regenerate perhaps via change in release of GDF-15.