Metformin enhances METTL14-Mediated m6A methylation to alleviate NIT-1 cells apoptosis induced by hydrogen peroxide.

Injuries to pancreatic ß-cells are intricately linked to the onset of diabetes mellitus (DM). Metformin (Met), one of the most widely prescribed medications for diabetes and metabolic disorders, has been extensively studied for its antioxidant, anti-aging, anti-glycation, and hepatoprotective activities. N6-methyladenosine (m6A) plays a crucial role in the regulation of ß-cell growth and development, and its dysregulation is associated with metabolic disorders. This study aimed to elucidate the mechanistic basis of m6A involvement in the protective effects of Met against oxidative damage in pancreatic ß-cells. Hydrogen peroxide (H2O2) was employed to induce ß-cell damage. Remarkably, Met treatment effectively increased methylation levels and the expression of the methyltransferase METTL14, subsequently reducing H2O2-induced apoptosis. Knocking down METTL14 expression using siRNA significantly compromised cell viability. Conversely, targeted overexpression of METTL14 specifically in ß-cells substantially enhanced their capacity to withstand H2O2-induced stress. Molecular evidence suggests that the anti-apoptotic properties of Met may be mediated through Bcl-xL and Bim proteins. In conclusion, our findings indicate that Met induces METTL14-mediated alterations in m6A methylation levels, thereby shielding ß-cells from apoptosis and oxidative damage induced by oxidative stress.

Increased serum extrachromosomal circular DNA SORBS1circle level is associated with insulin resistance in patients with newly diagnosed type 2 diabetes mellitus.

BACKGROUND: Extrachromosomal circular DNAs (eccDNAs) exist in human blood and somatic cells, and are essential for oncogene plasticity and drug resistance. However, the presence and impact of eccDNAs in type 2 diabetes mellitus (T2DM) remains inadequately understood. METHODS: We purified and sequenced the serum eccDNAs obtained from newly diagnosed T2DM patients and normal control (NC) subjects using Circle-sequencing. We validated the level of a novel circulating eccDNA named sorbin and SH3-domain- containing-1circle97206791-97208025 (SORBS1circle) in 106 newly diagnosed T2DM patients. The relationship between eccDNA SORBS1circle and clinical data was analyzed. Furthermore, we explored the source and expression level of eccDNA SORBS1circle in the high glucose and palmitate (HG/PA)-induced hepatocyte (HepG2 cell) insulin resistance model. RESULTS: A total of 22,543 and 19,195 eccDNAs were found in serum samples obtained from newly diagnosed T2DM patients and NC subjects, respectively. The T2DM patients had a greater distribution of eccDNA on chromosomes 1, 14, 16, 17, 18, 19, 20 and X. Additionally, 598 serum eccDNAs were found to be upregulated, while 856 eccDNAs were downregulated in T2DM patients compared with NC subjects. KEGG analysis demonstrated that the genes carried by eccDNAs were mainly associated with insulin resistance. Moreover, it was validated that the eccDNA SORBS1circle was significantly increased in serum of newly diagnosed T2DM patients (106 T2DM patients vs. 40 NC subjects). The serum eccDNA SORBS1circle content was positively correlated with the levels of glycosylated hemoglobin A1C (HbA1C) and homeostasis model assessment of insulin resistance (HOMA-IR) in T2DM patients. Intracellular eccDNA SORBS1circle expression was significantly enhanced in the high glucose and palmitate (HG/PA)-induced hepatocyte (HepG2 cell) insulin resistance model. Moreover, the upregulation of eccDNA SORBS1circle in the HG/PA-treated HepG2 cells was dependent on generation of apoptotic DNA fragmentation. CONCLUSIONS: These results provide a preliminary understanding of the circulating eccDNA patterns at the early stage of T2DM and suggest that eccDNA SORBS1circle may be involved in the development of insulin resistance.

Protective effects of metformin on pancreatic ß-cell ferroptosis in type 2 diabetes in vivo.

Metformin (Met) is the recommended first-line therapeutic drug for type 2 diabetes mellitus (T2DM) and exerts protective effects on ß-cell damage. Ferroptosis, a new form of cell death, is associated with pancreatic islet injury in patients with T2DM. However, the protective effects of Met treatment against ß-cell damage through ferroptosis modulation remain under-reported. This study investigated the in vivo effects of Met treatment on pancreatic ß-cell ferroptosis using two different diabetic mouse models, namely, low-dose streptozotocin (STZ) and high-fat diet (HFD)-induced diabetic mice and db/db mice. Met treatment significantly restored insulin release, reduced cell mortality, and decreased the overproduction of lipid-related reactive oxygen species in the islets of both STZ/HFD-induced diabetic mice and db/db mice. Administration of the Ras-selective lethal 3 injection significantly attenuated the antiferroptosis effects of Met. Mechanistically, Met treatment alleviated ß-cell ferroptosis in T2DM, which was associated with the regulation of the GPX4/ACSL4 axis in the islets. In conclusion, our findings highlight the significance of ferroptosis in T2DM ß-cell damage and provide novel insights into the protective effects of Met against islet ß cells.

Metformin alleviates glucolipotoxicity-induced pancreatic ß cell ferroptosis through regulation of the GPX4/ACSL4 axis.

Ferroptosis, a new type of cell death, is associated with pancreatic ß cell damage. However, the role of glucolipotoxicity in inducing ß cell ferroptosis remains unclear. Metformin (Met), exenatide (Exe), and saxagliptin (Sax) are frequently used anti-hyperglycaemic drugs. However, their protective effects on ß cells through ferroptosis modulation are not well-established. In this study, we observed significant ferroptosis in NIT-1 cells and primary mouse islets after exposure to high glucose and palmitate (HG/PA). Compared to Exe and Sax, Met significantly alleviated glucolipotoxicity-induced pancreatic ß cell ferroptosis. Blocking the activity of glutathione peroxidase 4 (GPX4) with Ras-selective lethal 3 or inhibiting its expression by small interfering RNA transfection significantly attenuated the anti-ferroptosis effects of Met. Mechanistically, Met alleviates HG/PA-induced ß cell ferroptosis by regulating the GPX4/ACSL4 axis. Collectively, our findings highlight the significance of ferroptosis in pancreatic ß cell glucolipotoxicity-induced injury and provide novel insights into the protective effects of Met on islet ß cells.

LncRNA HEM2ATM improves obesity-associated adipose tissues meta-inflammation and insulin resistance by interacting with heterogeneous nuclear ribonucleoprotein U.

Obesity is a complicated metabolic disease characterized by meta-inflammation in adipose tissues. In this study, we explored the roles of a new long non-coding RNA (lncRNA), HEM2ATM, which is highly expressed in adipose tissue M2 macrophages, in modulating obesity-associated meta-inflammation and insulin resistance. HEM2ATM expression decreased significantly in adipose tissue macrophages (ATMs) obtained from epididymal adipose tissues of high-fat diet (HFD)-induced obese mice. Overexpression of macrophage HEM2ATM improved meta-inflammation and insulin resistance in the adipose tissues of HFD-fed mice. Functionally, HEM2ATM negatively regulated the production of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in macrophages. Mechanistically, HEM2ATM bound to heterogeneous nuclear ribonucleoprotein U (hnRNP U), suppressed hnRNP U translocation from the nucleus to the cytoplasm, hindered the function of cytoplasmic hnRNP U on TNF-α and IL-6 mRNA stabilization, and decreased the secretion of TNF-α and IL-6. Collectively, HEM2ATM is a novel suppressor of obesity-associated meta-inflammation and insulin resistance.

Decreased Urine N6-methyladenosine level is closely associated with the presence of diabetic nephropathy in type 2 diabetes mellitus.

Background: To investigate the dynamic changes of urine N6-methyladenosine (m6A) levels in patients with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) and evaluate the clinical significance. Methods: First, the levels of urine m6A were examined and compared among 62 patients with T2DM, 70 patients with DN, and 52 age- and gender-matched normal glucose tolerant subjects (NGT) by using a MethyIFIashTM Urine m6A Quantification Kit. Subsequently, we compared the concentrations of urine m6A between different stages of DN. Moreover, statistical analysis was performed to evaluate the association of urine m6A with DN. Results: The levels of m6A were significantly decreased in patients with DN [(16.10 ± 6.48) ng/ml], compared with NGT [(23.12 ± 7.52) ng/ml, P < 0.0001] and patients with T2DM [(20.39 ± 7.16) ng/ml, P < 0.0001]. Moreover, the concentrations of urine m6A were obviously reduced with the deterioration of DN. Pearson rank correlation and regression analyses revealed that m6A was significantly associated with DN (P < 0.05). The areas under the receiver operator characteristics curve (AUC) were 0.783 (95% CI, 0.699 - 0.867, P < 0.0001) for the DN and NGT groups, and 0.737 (95% CI, 0.639 - 0.835, P < 0.0001) for the macroalbuminuria and normoalbuminuria groups, and the optimal cutoff value for m6A to distinguish the DN from NGT and the macroalbuminuria from normoalbuminuria cases was 0.4687 (diagnostic sensitivity, 71%; diagnostic specificity, 76%) and 0.4494 (diagnostic sensitivity, 79%; diagnostic specificity, 66%), respectively. Conclusions: The levels of urine m6A are significantly decreased in patients with DN and change with the deterioration of DN, which could serve as a prospective biomarker for the diagnosis of DN.

The m6A Methyltransferase METTL3 Ameliorates Methylglyoxal-Induced Impairment of Insulin Secretion in Pancreatic ß Cells by Regulating MafA Expression.

Methylglyoxal, a major precursor of advanced glycation end products, is elevated in the plasma of patients with type 2 diabetes mellitus. Islet ß-cell function was recently shown to be regulated by N6-methyladenosine (m6A), an RNA modification consisting of methylation at the N6 position of adenosine. However, the role of m6A methylation modification in methylglyoxal-induced impairment of insulin secretion in pancreatic ß cells has not been clarified. In this study, we showed that treatment of two ß-cell lines, NIT-1 and ß-TC-6, with methylglyoxal reduced m6A RNA content and methyltransferase-like 3 (METTL3) expression levels. We also showed that silencing of METTL3 inhibited glucose-stimulated insulin secretion (GSIS) from NIT-1 cells, whereas upregulation of METTL3 significantly reversed the methylglyoxal-induced decrease in GSIS. The methylglyoxal-induced decreases in m6A RNA levels and METTL3 expression were not altered by knockdown of the receptor for the advanced glycation end product but were further decreased by silencing of glyoxalase 1. Mechanistic investigations revealed that silencing of METTL3 reduced m6A levels, mRNA stability, and the mRNA and protein expression levels of musculoaponeurotic fibrosarcoma oncogene family A (MafA). Overexpression of MafA greatly improved the decrease in GSIS induced by METTL3 silencing; silencing of MafA blocked the reversal of the MG-induced decrease in GSIS caused by METTL3 overexpression. The current study demonstrated that METTL3 ameliorates MG-induced impairment of insulin secretion in pancreatic ß cells by regulating MafA.

Exenatide ameliorates hydrogen peroxide-induced pancreatic ß-cell apoptosis through regulation of METTL3-mediated m6A methylation.

Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, is a commonly used hypoglycemic agent in clinical practice; it inhibits reactive oxygen species-induced pancreatic ß-cell apoptosis. N6-methyladenosine (m6A) is produced by the methylation of RNA N6 residues and has recently been shown to play a crucial role in the regulation of islet ß-cell growth and development. However, the involvement of m6A methylation in the ß-cell protective effects of exenatide has not been clarified. In this study, the m6A-methylated RNA content and methyltransferase-like 3 (METTL3) expression levels in NIT-1 cells and primary mouse islets were found to significantly decrease following treatment with hydrogen peroxide (H2O2). Treatment with exenatide induced an increase in m6A content and METTL3 expression in the H2O2-treated NIT-1 cells and islets. Moreover, METTL3 silencing resulted in NIT-1 cell apoptosis under normal culture conditions. METTL3 upregulation significantly ameliorated H2O2-induced apoptosis in NIT-1 cells and primary islets. Furthermore, the anti-apoptotic effects of exenatide were obviously reversed by METTL3 knockdown. In conclusion, these findings suggest that exenatide elicits its anti-apoptotic effects in pancreatic ß-cells by promoting m6A methylation through the upregulation METTL3 expression.

A SGLT2 Inhibitor Dapagliflozin Alleviates Diabetic Cardiomyopathy by Suppressing High Glucose-Induced Oxidative Stress in vivo and in vitro.

Diabetic cardiomyopathy (DCM) is a serious complication of diabetes mellitus (DM). One of the hallmarks of the DCM is enhanced oxidative stress in myocardium. The aim of this study was to research the underlying mechanisms involved in the effects of dapagliflozin (Dap) on myocardial oxidative stress both in streptozotocin-induced DCM rats and rat embryonic cardiac myoblasts H9C2 cells exposed to high glucose (33.0 mM). In in vivo studies, diabetic rats were given Dap (1 mg/ kg/ day) by gavage for eight weeks. Dap treatment obviously ameliorated cardiac dysfunction, and improved myocardial fibrosis, apoptosis and oxidase stress. In in vitro studies, Dap also attenuated the enhanced levels of reactive oxygen species and cell death in H9C2 cells incubated with high glucose. Mechanically, Dap administration remarkably reduced the expression of membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits gp91phox and p22phox, suppressed the p67phox subunit translocation to membrane, and decreased the compensatory elevated copper, zinc superoxide dismutase (Cu/Zn-SOD) protein expression and total SOD activity both in vivo and in vitro. Collectively, our results indicated that Dap protects cardiac myocytes from damage caused by hyperglycemia through suppressing NADPH oxidase-mediated oxidative stress.

Upregulation of long non-coding RNA MEG3 in type 2 diabetes mellitus complicated with vascular disease: a case-control study.

Previous studies have indicated that long non-coding RNAs (lncRNAs) were closely related to diabetes. In this study, we aimed to explore the possible role and mechanism of lncRNA MEG3 in the occurrence and development of type 2 diabetes mellitus (T2DM) and its vascular complications. A case-control study involving 115 subjects was conducted, including 53 T2DM patients (37 patients with vascular complication and 16 patients without vascular complications) and 62 healthy subjects. We performed real-time polymerase chain reaction (RT-PCR) analysis of the lncRNA MEG3 and miR-146a levels in peripheral blood mononuclear cells (PBMCs) in the 115 samples. We found that the expression of lncRNA MEG3 was upregulated in the T2DM patients with vascular complication (DC group) compared with T2DM patients without vascular complication (D group) (P < 0.05) and the control group (P < 0.01). miR-146a levels in DC group were significantly lower compared with control group. There was a significant positive correlation between the expression of lncRNA MEG3 and glucose (GLU) (r = 0.301, P = 0.0011) and hemoglobin A1C (HbA1c) (r = 0.477, P = 0.0006). Our study suggests MEG3 may play as an important role in progression of diabetes-related vascular complications, contributing to a novel understanding of pathogenesis and prognosis for diabetes and its complications.

LncRNA LEGLTBC Functions as a ceRNA to Antagonize the Effects of miR-34a on the Downregulation of SIRT1 in Glucolipotoxicity-Induced INS-1 Beta Cell Oxidative Stress and Apoptosis.

Type 2 diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose and/or high serum free fatty acids. Chronic hyperlipidemia causes the dysfunction of pancreatic beta cells, which is aggravated in the presence of hyperglycemia (glucolipotoxicity). Long noncoding RNAs (lncRNAs) have been suggested to play key roles in type 1 diabetes mellitus development. However, their roles in glucolipotoxicity-induced beta cell dysfunction are not fully understood. In the present study, we identified the differentially expressed lncRNAs in INS-1 cells exposed to high glucose and palmitate (HG/PA). Among the dysregulated lncRNAs, NONRATT003679.2 (low expression in glucolipotoxicity-treated beta cells (LEGLTBC)) was involved in glucolipotoxicity-evoked rat islet beta cell damage. LEGLTBC functioned as a molecular sponge of miR-34a in INS-1 cells. Additionally, SIRT1 was identified as a target of miR-34a and LEGLTBC promoted SIRT1 expression by sponging miR-34a. The upregulation of LEGLTBC attenuated HG/PA-induced INS-1 cell injury through the promotion of SIRT1-mediated suppression of ROS accumulation and apoptosis. This is the first study to comprehensively identify the lncRNA expression profiling of HG/PA-treated INS-1 beta cells and to demonstrate that LEGLTBC functions as a competing endogenous RNA and regulates miR-34a/SIRT1-mediated oxidative stress and apoptosis in INS-1 cells undergoing glucolipotoxicity.

Tumour necrosis factor-related apoptosis-inducing ligand expression in patients with diabetic nephropathy.

OBJECTIVE: The objective of this study was to evaluate the expression profile of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in patients with diabetic nephropathy (DN). METHODS: A total of 126 Chinese subjects were enrolled in this study, including 42 patients with diabetes mellitus (DM), 42 patients with DN and 42 healthy controls. Real-time polymerase chain reaction was performed to analyze levels of TRAIL mRNA in peripheral blood mononuclear cells (PBMCs). Serum levels of soluble TRAIL (sTRAIL) and various cytokines were detected with a commercially available enzyme-linked immunosorbent assay kit. RESULTS: Compared with the control group, the levels of TRAIL mRNA in PBMCs and sTRAIL in sera were both significantly decreased in the DM and DN patients ( P < 0.05). Conversely, levels of interleukin (IL)-1, IL-6, tumour necrosis factor-α and monocyte chemotactic protein-1 were higher in the DN group than in the control group. Serum levels of TRAIL positively correlated with TRAIL mRNA levels in all of the subjects examined ( P < 0.05). CONCLUSIONS: These results provide support and a theoretical basis for further research of TRAIL in regard to the pathogenesis of DN.

Activation of NLRP3 Inflammasome by Advanced Glycation End Products Promotes Pancreatic Islet Damage.

Accumulation of advanced glycation end products (AGEs) contributes to ageing and age-related diseases, especially type 2 diabetes. The NLRP3 inflammasome, as a vital component of the innate immune system, is implicated in the pathogenesis of type 2 diabetes. However, the role of the NLRP3 inflammasome in AGE-induced pancreatic islet damage remains largely unclear. Results showed that administration of AGEs (120 mg/kg for 6 weeks) in C57BL/6J mice induced an abnormal response to glucose (as measured by glucose tolerance and insulin release), pancreatic ß-cell ultrastructural lesion, and cell death. These effects were associated with an excessive superoxide anion level, significant increased protein expression levels for NADPH oxidase 2 (NOX2), thioredoxin-interacting protein (TXNIP), NLRP3, and cleaved IL-1ß, enhanced caspase-1 activity, and a significant increase in the levels of TXNIP-NLRP3 protein interaction. Ablation of the NLRP3 inflammasome or treatment with antioxidant N-acetyl-cysteine (NAC) clearly ameliorated these effects. In conclusion, our results reveal a possible mechanism for AGE-induced pancreatic islet damage upon NLRP3 inflammasome activation.

Metformin alleviates high glucose-mediated oxidative stress in rat glomerular mesangial cells by modulation of p38 mitogen-activated protein kinase expression in vitro.

The aim of the current study was to investigate the effects and mechanism of metformin in oxidative stress and p38 mitogen-activated protein kinase (p38MAPK) expression in rat glomerular mesangial cells (MCs) cultured in a high glucose medium. Rat glomerular MCs (HBZY-1) were cultured in complete medium and divided into the following five groups: Normal control (NC), high glucose (HG), metformin-treated, SB203580-treated (SB) and N-acetylcysteine-treated (NAC). The production of intracellular reactive oxygen species (ROS) in rat glomerular MCs was measured using flow cytometry. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the supernatant was detected using colorimetric analysis and an ELISA, respectively. p22phox mRNA levels in rat glomerular MCs were determined using reverse transcription-quantitative polymerase chain reaction. The levels of p22phox protein and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK) protein in rat glomerular MCs were determined by western blot analysis. Compared with the NC group, the activity of SOD in the supernatant was significantly reduced, whereas the levels of MDA in the supernatant, intracellular p22phox mRNA and protein, p-p38MAPK protein in addition to ROS production in rat glomerular MCs were significantly increased in the HG group (P<0.05). When metformin was added to the high glucose medium, the activity of SOD in supernatant fluid was increased significantly, whereas a significant reduction (P<0.05) was observed in the levels of MDA in the supernatant, intracellular p22phox mRNA and protein, p-p38MAPK protein in addition to ROS production in rat glomerular MCs. These results were similar to those obtained when SB203580 or N-acetylcysteine was added to the high glucose medium (P<0.05). In conclusion, metformin was suggested to alleviate high glucose-induced oxidative stress and p-p38MAPK protein expression in rat glomerular MCs, which may contribute to its reno­protective abilities in diabetes.

Rosiglitazone protects diabetic rats against kidney injury through the suppression of renal matrix metalloproteinase-9 expression.

In this study, the effects of rosiglitazone on renal matrix metalloproteinase-9 (MMP-9) expression and its possible renoprotective mechanisms were investigated in streptozotocin-induced diabetic rats. We examined the urinary excretion rates of albumin (ALB), retinal-binding protein (RBP) and MMP-9 in control healthy rats (group C, n = 8), untreated diabetic rats (group D, n = 8) and diabetic rats treated with rosiglitazone (5mg/kg/day) (group R, n = 8) at eighth week. The renal tissue of diabetic rats was obtained for observing renal pathological changes by electron microscope and examining the expression of renal MMP-9 mRNA by RT-PCR. Our results showed that urinary excretion rates of MMP-9. ALB and RBP were significantly increased concurrently with the expression of renal MMP-9mRNA in group D compared with those of group C. Rosiglitazone significantly reduced urinary excretion rates of ALB, RBP and MMP-9 as well as the expression of renal MMP-9 mRNA. In addition, urinary excretion rate of MMP-9 showed positive relationship with urinary excretion rates of ALB and RBP. In conclusion, rosiglitazone definitely protects diabetic rats against renal injury, which may be partly associated with decreasing expression of renal MMP-9 mRNA and urinary MMP-9 production.