High Glucose-induced Reactive Oxygen Species Research Articles

Sodium butyrate ameliorates high glucose-suppressed neuronal mitophagy by restoring PRKN expression via inhibiting the RELA-HDAC8 complex

ABSTRACT Damaged mitochondria accumulation in diabetes is one of the main features that contribute to increased incidence of cognitive impairment by inducing apoptosis. Butyrate is a major metabolite produced by microbiota that has neuroprotective effects by regulating mitochondrial function. However, detailed mechanisms underlying how butyrate can regulate neuronal mitophagy remain unclear. Here, we examined the regulatory effects of sodium butyrate (NaB) on high glucose-induced mitophagy dysregulation, neuronal apoptosis, and cognitive impairment and its underlying mechanisms in human-induced pluripotent stem cell-derived neurons, SH-SY5Ys, and streptozotocin (STZ)-induced diabetic mice. In our results, diabetic mice showed gut-microbiota dysbiosis, especially a decreased number of butyrate-producing bacteria and reduced NaB plasma concentration. NaB ameliorated high glucose-induced neuronal mitochondrial dysfunction by recovering PRKN/Parkin-mediated mitophagy. High glucose-induced reactive oxygen species (ROS) and -inhibited PRKAA/AMPKα stimulated the RELA/p65-HDAC8 complex, which downregulated PRKN protein expression by binding to the PRKN promoter region. NaB restored PRKN expression by blocking RELA nuclear translocation and directly inhibiting HDAC8 in the nucleus. In addition, HDAC8 overexpression inhibited the positive effect of NaB on high glucose-induced mitophagy dysfunction and neuronal apoptosis. Oral administration of NaB improved cognitive impairment in diabetic mice by restoring mitophagy in the hippocampus. Taken together, NaB ameliorates neuronal mitophagy through PRKN restoration by inhibiting RELA-HDAC8 complexes, suggesting that NaB is an important substance for protecting neuronal apoptosis in diabetes-associated cognitive impairment.

Untargeted metabolomics reveals the regulatory effect of geniposidic acid on lipid accumulation in HepG2 cells and Caenorhabditis elegans and validation in hyperlipidemic hamsters

BackgroundGeniposidic acid (GPA) alleviates oxidative stress and inflammation in mice However, whether it can effectively regulate lipid accumulation and prevent hyperlipidemia requires further investigation. PurposeThis study combined the untargeted metabolomics of cells and a Caenorhabditis elegans model to evaluate the anti-hyperlipidemic potential of GPA by modulating oxidative stress and regulating lipid metabolism. A golden hamster model of hyperlipidemia was used to further validate the lipid-lowering effect and mechanism of action of GPA. MethodsChemical staining, immunofluorescence, and flow cytometry were performed to examine the effects of GPA on lipid accumulation and oxidative stress. Untargeted metabolomic analysis of cells and C. elegans was performed using ultra-performance liquid chromatography coupled with quadrupole electrostatic field Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap MS) to identify biomarkers altered by GPA action, analyze the affected metabolic pathways, and validate the mechanisms by which GPA regulates lipid metabolism and oxidative stress. A golden hamster model of hyperlipidemia was established to test the lipid-lowering effects of GPA. Body weight, biochemical markers, rate-limiting enzymes, and key proteins were assessed. Hematoxylin and eosin (H&E) and Oil Red O staining were performed. ResultsPhenotypic data showed that GPA decreased free fatty acid (FFA)-induced lipid buildup and high reactive oxygen species (ROS) levels, reversed the decrease in mitochondrial membrane potential (MMP), and increased the cellular reduced glutathione/oxidized glutathione disulfide (GSH/GSSG) ratio. GPA also reduces high glucose-induced lipid build-up and ROS production in C. elegans. Metabolomic analysis showed that GPA affected purine, lipid, and amino acid metabolism. Moreover, GPA inhibited xanthine oxidase (XOD), glutamate dehydrogenase (GLDH), fatty acid synthase (FAS), phosphorylation of P38 MAPK, and upregulated the expression of SIRT3 and CPT1A protein production to control lipid metabolism and produce antioxidant benefits in cells and golden hamsters. ConclusionCurrent evidence suggests that GPA can effectively regulate lipid metabolism and the oxidative stress response, and has the potential to prevent hyperlipidemia. This study also provided an effective method for evaluating the mechanism of action of GPA.

Metformin suppresses Oxidative Stress induced by High Glucose via Activation of the Nrf2/HO-1 Signaling Pathway in Type 2 Diabetic Osteoporosis

BackgroundMetformin (MET) is widely used as a first-line hypoglycemic agent for the treatment of type 2 diabetes mellitus (T2DM) and was also confirmed to have a therapeutic effect on type 2 diabetic osteoporosis (T2DOP). However, the potential mechanisms of MET in the treatment of T2DOP are unclear. ObjectiveTo clarify the effect of MET in T2DOP and to explore the potential mechanism of MET in the treatment of T2DOP. MethodsIn vitro, we used MC3T3-E1 cells to study the effects of MET on osteogenic differentiation and anti-oxidative stress injury in a high glucose (Glucose 25 mM) environment. In vivo, we directly used db/db mice as a T2DOP model and assessed the osteoprotective effects of MET by Micro CT and histological analysis. ResultsIn vitro, we found that MET increased ALP activity in MC3T3-E1 cells in a high-glucose environment, promoted the formation of bone mineralized nodules, and upregulated the expression of the osteogenesis-related transcription factors RUNX2, Osterix, and COL1A1-related genes. In addition, MET was able to reduce high glucose-induced reactive oxygen species (ROS) production. In studies on the underlying mechanisms, we found that MET activated the Nrf2/HO-1 signaling pathway and alleviated high-glucose-induced oxidative stress injury. In vivo results showed that MET reduced bone loss and bone microarchitecture destruction in db/db mice. ConclusionOur results suggest that MET can activate the Nrf2/HO-1 signaling pathway to regulate the inhibition of osteogenic differentiation induced by high glucose thereby protecting T2DOP.

Cellular interorganelle crosstalk in health and disease states: A glimpse on nephrology-related conditions

Cellular interorganelle crosstalk in medical sciences is a new discussion of mechanisms and pathways of physiological functions and pathogenesis of diseases. The organelles (“mitochondria”, nucleus, lysosome, endoplasmic reticulum, Golgi apparatus) are members of such functional units that are needed to perform specific tasks. Mitochondria are essential metabolic organelles in cells, but they also contribute to iron and calcium homeostasis, as well as in the regulation of apoptosis, and they are increasingly recognized as key signaling platforms. In the kidney, crosstalk between mitochondria with endoplasmic reticulum is at mitochondria-associated endoplasmic reticulum membrane in regulating calcium homeostasis. Another crosstalk between these organelles is about autophagic mechanisms. Autophagy is triggered in the kidney in response to acute kidney injury and supports against kidney injury. High glucose-induced reactive oxygen species can be produced by both enzymatic and nonenzymatic pathways. The nucleotide-binding domain and leucine-rich repeat (NLR) family or nucleotide-binding and oligomerization domain (NOD)-like receptors family pyrin domain containing 3 (NLRP3) inflammasome plays a role in inducing infectious defenses via inflammatory cytokines. The NLRP3 inflammasome is activated by the mitochondria-associated endoplasmic reticulum membrane. It has a role in nephrocalcinosis-related chronic kidney disease. This review article is a summary of interorganelle crosstalk in health and disease states, especially in kidney and nephrology-related conditions.

Vascular hyperacetylation is associated with vascular smooth muscle dysfunction in a rat model of non-obese type 2 diabetes

BackgroundAdvanced type 2 diabetes mellitus (T2DM) accelerates vascular smooth muscle cell (VSMC) dysfunction which contributes to the development of vasculopathy, associated with the highest degree of morbidity of T2DM. Lysine acetylation, a post-translational modification (PTM), has been associated with metabolic diseases and its complications. Whether levels of global lysine acetylation are altered in vasculature from advanced T2DM remains undetermined. We hypothesized that VSMC undergoes dysregulation in advanced T2DM which is associated with vascular hyperacetylation.MethodsAged male Goto Kakizaki (GK) rats, a non-obese murine model of T2DM, and age-matched male Wistar rats (control group) were used in this study. Thoracic aortas were isolated and examined for measurement of global levels of lysine acetylation, and vascular reactivity studies were conducted using a wire myograph. Direct arterial blood pressure was assessed by carotid catheterization. Cultured human VSMCs were used to investigate whether lysine acetylation participates in high glucose-induced reactive oxygen species (ROS), a crucial factor triggering diabetic vascular dysfunction.ResultsThe GK rats exhibited marked glucose intolerance as well as insulin resistance. Cardiovascular complications in GK rats were confirmed by elevated arterial blood pressure and reduced VSMC-dependent vasorelaxation. These complications were correlated with high levels of vascular global lysine acetylation. Human VSMC cultures incubated under high glucose conditions displayed elevated ROS levels and increased global lysine acetylation. Inhibition of hyperacetylation by garcinol, a lysine acetyltransferase and p300/CBP association factor (PCAF) inhibitor, reduced high glucose-induced ROS production in VSMC.ConclusionThis study provides evidence that vascular hyperacetylation is associated with VSMC dysfunction in advanced T2DM. Understanding lysine acetylation regulation in blood vessels from diabetics may provide insight into the mechanisms of diabetic vascular dysfunction, and opportunities for novel therapeutic approaches to treat diabetic vascular complications.

3-Hydroxybutyrate Ameliorates the Progression of Diabetic Nephropathy.

Studies report beneficial effects of 3-hydroxybutyrate (3-OHB) on the treatment of type 2 diabetes and obesity, but the effects of 3-OHB on diabetic nephropathy have not been elucidated. This study was designed to investigate the efficacy and mechanism of 3-OHB against progression of diabetic nephropathy (DN). Mice (db/db) were fed normal chow, high-fat, or ketogenic diets (KD) containing precursors of 3-OHB. Hyperglycemia was determined based on random glucose level (≥250 mg/dL). Fasting blood glucose and body weights were measured once a week. Twenty four-hour urine albumin to creatinine ratio was determined 5 weeks after the differential diet. Energy expenditure was measured 9 weeks after the differential diet. Body weights were significantly lower in the KD group than those in other groups, but no significant differences in fasting blood glucose levels among three groups were observed. Urine albumin to creatinine ratio and serum blood urea nitrogen (BUN) to creatinine ratio in the KD group were significantly lower than in other groups. Histologic and quantitative analysis of mesangial area suggested that KD delayed the progression of DN phenotype in db/db mice. Metabolic cage analysis also revealed that KD increased energy expenditure in db/db mice. In vitro studies with proximal tubular cells revealed that 3-OHB stimulated autophagic flux. 3-OHB increased LC3 I to LC3 II ratio, phosphorylation of AMPK, beclin, p62 degradation, and NRF2 expression. Moreover, we found that 3-OHB attenuated high glucose-induced reactive oxygen species (ROS) levels in proximal tubular cells. In vivo study also confirmed increased LC3 and decreased ROS levels in the kidney of KD mice. In summary, this study shows in both in vivo and in vitro models that 3-OHB delays the progression of DN by augmenting autophagy and inhibiting oxidative stress.

Pectin-Lyase-Modified Ginseng Extract and Ginsenoside Rd Inhibits High Glucose-Induced ROS Production in Mesangial Cells and Prevents Renal Dysfunction in db/db Mice.

Diabetes increases the incidence rate of chronic renal disease. Pectin-lyase-modified ginseng (GS-E3D), with enhanced ginsenoside Rd content, has been newly developed. In this study, renal protective roles of GS-E3D in type-2 diabetic db/db mice were investigated. The generation of reactive oxygen species (ROS) induced by high glucose (25 mM) was reduced by ES-E3D (75%) and ginsenoside Rd (60%). Diabetic db/db mice received 100 or 250 mg/kg/day of GS-E3D daily via oral gavage for 6 weeks. Albuminuria and urinary 8-hydroxy-2′-deoxyguanosine (8-OhdG, an oxidative stress marker) levels were increased in db/db mice and the levels recovered after GS-E3D treatment. In renal tissues, TUNEL-positive cells were decreased after GS-E3D treatment, and the increased apoptosis-related protein expressions were restored after GS-E3D treatment. Therefore, GS-E3D has a potent protective role in diabetes-induced renal dysfunction through antioxidative and antiapoptotic activities. These results may help patients to select a dietary supplement for diabetes when experiencing renal dysfunction.

566-P: Sulodexide Suppresses NLRP3 Inflammasome Activation via NOX4/ROS Signaling in the Retinal Microvasculature

Aims: Sulodexide (SDX) protects endothelial cells via restoring endothelial glycocalyx, but the effect of SDX on endothelial inflammation remains unknown. We showed that SDX potently inhibited the activation of NLRP3 inflammasome in diabetic retinas, but the underlying mechanism was not clear. As reactive oxygen species (ROS) activates NLRP3 inflammasome, and NADPH oxidase 4 (NOX4) is a key generator of ROS in endothelial cells, this study thus investigated if NOX4/ROS signaling mediates the effect of SDX on NLRP3 inflammasome, hoping to provide novel insights into SDX in treating diabetic retinopathy (DR). Methods: Diabetes was induced in mice by high fat diet and streptozotocin, followed by i.p. injection of SDX (10mg/kg) every other day for 3 months. Human retinal microvascular endothelial cells were exposed to high glucose, accompanied with overexpression or knocking down of NOX4 and a serial concentration of SDX. The retinal levels of NOX4, NLRP3 inflammasome, inflammatory factors, junction components, vascular leakage and ROS levels were determined. Results: SDX treatment dramatically suppressed the hyperglycemia-induced overexpression of NOX4 and activation of NLRP3 inflammasome in retinas compared to controls. Moreover, SDX preserved the levels of ZO-1, VE-cadherin and ameliorated the retinal vascular permeability. In addition, SDX significantly ameliorated high glucose-induced NOX4 up-regulation, ROS production and NLRP3 inflammasome activation, and restored the reduction of ZO-1 and VE-cadherin in endothelial cells. Conversely, overexpression of NOX4 exacerbated endothelial inflammation through over production of ROS and subsequent activation of NLRP3 inflammasome, which was rescued by SDX treatment. Conclusion: Our study suggests that SDX protects the retinal microvasculature by inhibiting NOX4/ROS-induced NLRP3 inflammasome activation, thus provides novel evidence of utilizing SDX for the treatment of DR. Disclosure T. Li: None. X. He: None. R. Peng: None. X. Fu: None. G. Shi: None. Y. Chen: None. Funding Research and Development Plans in Key Areas of Guangdong Province (2019B020227003); National Natural Science Foundation of China (81770826); National Key Research and Development Program of China (2017YFA0105803)

484-P: Abnormal Ketone Body Metabolism Caused by Bdh1 Deficiency Promoted the Progression of Diabetic Kidney Disease by Activating ROS

Objective: This study was intended to reveal the mechanism of ketone body metabolism abnormality caused by bdh1 deficiency promoting the progression of diabetic kidney disease(DKD) by activating reactive oxygen species (ROS). Methods: Eight-week-old wild type(n=8) and db/db (n=8) mice were treated with normal diet and high-fat diet for three months, respectively. Then two groups of mice kidney samples were taken for RNA-Seq analysis to select the target gene. It was verified by qRT-PCR, Western blot, immunofluorescence, and immunohistochemistry in mice kidney. A high glucose-induced ROS activation model of HK-2 renal cells was established, and then siRNA was used to interfere with bdh1. Results: RNA-seq showed that the pathway of ketone body metabolism in DKD changed significantly, and further analysis showed that bdh1, the key protein of ketone body metabolism, decreased significantly. The sequencing result was further confirmed by qRT-PCR, Western blot, immunofluorescence, and immunohistochemistry, and we found that bdh1 was mainly expressed in tubular epithelial cells. In order to identify the molecular mechanism of bdh1 involved in DKD, we constructed the high glucose-induced ROS activation model of HK-2 cells in vitro. It was found that bdh1 was significantly down-regulated in high glucose treated HK-2 cells. The knockdown of bdh1 by siRNA could promote the production of ROS induced by high glucose. Further research found that bdh1 promoted the expression of nrf2 then inhibited ROS through the ketone body metabolism pathway, thus protected HK-2 cells in a high glucose environment. Conclusions: Bdh1 promoted the expression of nrf2 through the pathway of ketone body metabolism and inhibited the production of ROS to protect HK-2 cells in a high glucose environment. Abnormal ketone body metabolism caused by bdh1 deficiency participated in the progress of DKD. Disclosure S. Wan: None. F. Teng: None. Z. Jiang: None. Y. Xu: None.

High glucose inhibits vascular endothelial Keap1/Nrf2/ARE signal pathway via downregulation of monomethyltransferase SET8 expression.

Hyperglycemia-mediated reactive oxygen species (ROS) accumulation plays an important role in hyperglycemia-induced endothelial injury. Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway inhibition participates in hyperglycemia-induced ROS accumulation. Our previous study indicated that SET8 overexpression inhibits high glucose-mediated ROS accumulation in human umbilical vein endothelial cells (HUVECs). In the present study, we hypothesize that SET8 may play a major role in high glucose-induced ROS accumulation via modulation of Keap1/Nrf2/ARE pathway. Our data indicated that high glucose mediated cell viability reduction, ROS accumulation, and Nrf2/ARE signal pathway inhibition via upregulation of Keap1 expression in HUVECs. Moreover, high glucose inhibited the expressions of SET8 and H4K20me1 (a downstream target of SET8). SET8 overexpression improved high glucose-mediated Keap1/Nrf2/ARE pathway inhibition and endothelial oxidation. Consistently, the effects of sh-SET8 were similar to that of high glucose treatment and were reversed by si-Keap1. A mechanistic study found that H4K20me1 was enriched at the Keap1 promoter region. SET8 overexpression attenuated Keap1 promoter activity and its expression, while mutant SET8 R259G did not affect Keap1 promoter activity and expression. The results of this study demonstrated that SET8 negatively regulates Keap1 expression, thus participating in high glucose-mediated Nrf2/ARE signal pathway inhibition and oxidative injury in HUVECs.

Protective effects of glucagon-like peptide-1 on cardiac remodeling by inhibiting oxidative stress through mammalian target of rapamycin complex1/p70 ribosomal proteinS6 kinase pathway in diabetes mellitus.

Aims/IntroductionAlthough increased reactive oxygen species (ROS) generation is a major mechanism leading to cardiac remodeling in diabetes mellitus, research into the effects of anti‐oxidation on diabetic cardiac remodeling remains scarce and controversial. Glucagon‐like peptide‐1 (GLP‐1) shows potential anti‐oxidative effects besides lowering blood glucose. The objective of this research was to investigate the effects of GLP‐1 on cardiac remodeling and the molecular mechanism involved in diabetes mellitus.Materials and MethodsStreptozotocin‐induced diabetic rats received exenatide treatment for 3 months. Cardiac function, cardiac weight index and myocardial interstitial fibrosis were measured. Cardiomyocytes were cultured in high‐glucose medium with GLP‐1 treatment. The ROS production, apoptosis and the levels of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase protein expression in cardiomyocytes were analyzed.ResultsExperimental diabetes mellitus showed impaired cardiac diastolic function, increased brain natriuretic peptide expression and increased interstitial collagen deposition in the myocardium, which were ameliorated by exenatide treatment. Exenatide reduced myocardial ROS production and apoptosis in diabetes mellitus. Also, high glucose‐induced ROS generation and apoptosis in cardiomyocytes were inhibited by GLP‐1, as well as the levels of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase phosphorylation. Furthermore, GLP‐1 treatment upregulated adenosine monophosphate‐activated protein kinase activity in high‐glucose‐induced cardiomyocyte.ConclusionsGlucagon‐like peptide‐1 protects the cardiomyocytes from oxidative stress and apoptosis in diabetes mellitus, which might contribute to the improvement of cardiac remodeling. The cardiac protection of GLP‐1 might be dependent on inhibition of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase, through an adenosine monophosphate‐activated protein kinase‐mediated pathway.

Propofol attenuates high glucose-induced P66shc expression in human umbilical vein endothelial cells through Sirt1.

Perioperative hyperglycemia is a common metabolic disorder in clinic settings. Hyperglycemia leads to endothelial inflammation, endothelial cell apoptosis, and dysfunction, thus resulting in endothelial injury. Propofol (2,6-diisopropylphenol) is a widely used intravenous anesthetic in clinic settings. Our previous study indicated that propofol inhibits mitochondrial reactive oxygen species (ROS) production via down-regulation of phosphatase A2 (PP2A) expression, inhibition of Ser36-p66shc dephosphorylation and mitochondrial translocation, thus improving high glucose-induced endothelial injury. The expression of p66shc was inhibited by propofol in hyperglycemic human umbilical vein endothelial cells (HUVECs). However, the mechanism by which propofol inhibits p66shc expression in hyperglycemic HUVECs is still obscure. In the present study, we mainly examined how propofol inhibited high glucose-induced p66shc expression in HUVECs. Compared with 5 mM glucose treatment, high glucose increased p66shc expression and decreased sirt1 expression, which was inhibited by propofol treatment. Moreover, EX527 (a sirt1 inhibitor) reversed the effect of propofol against high glucose-induced p66shc expression. However, EX527 did not reverse the effects of propofol against high glucose-induced ROS accumulation, endothelial inflammation, and apoptosis. Furthermore, when cells were incubated with propofol, EX527, and FTY720 (a PP2A activator) simultaneously, the effects of propofol against high glucose-induced ROS accumulation, inflammation, and apoptosis were reversed. Our results suggested that propofol inhibited high glucose-induced p66shc expression via upregulation of sirt1 expression in hyperglycemic HUVECs. Moreover, propofol protects against high glucose-mediated ROS accumulation and endothelial injury via both inhibition of p66shc expression and dephosphorylation of Ser36-p66shc.

ROS induces epithelial-mesenchymal transition via the TGF-β1/PI3K/Akt/mTOR pathway in diabetic nephropathy.

Oxidative stress has been reported to serve an important role in the development and progression of diabetic nephropathy (DN). Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells promotes renal fibrosis in DN, while the mechanism of reactive oxygen species (ROS)-mediated EMT is not fully understood. The aim of the present study was to investigate the effect of high glucose-induced ROS on the activation of the transforming growth factor (TGF)-β1/phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in a normal rat kidney tubular epithelial cell line (NRK-52E) and rats with type 1 diabetes. In vitro, high glucose-stimulated ROS production resulted in increased TGF-β1 expression as well as an increase in the Akt and mTOR phosphorylation ratio, resulting in EMT. When cells were pre-treated with ROS inhibitors, changes in TGF-β1, Akt and mTOR were significantly ameliorated. In vivo, diabetic rats experienced a significant decline in renal function and severe renal fibrosis compared with control rats at 8 weeks following streptozocin injection. Levels of malondialdehyde and TGF-β1/PI3K/Akt/mTOR pathway activation were increased in the renal cortex of rats with diabetes compared with the control rats. Furthermore, renal fibrosis was further aggravated in DN compared with the control rats. The results of the present study suggest that ROS serves an important role in mediating high glucose-induced EMT and inhibits activation of the TGF-β1/PI3K/Akt/mTOR pathway. ROS may therefore have potential as a treatment approach to prevent renal fibrosis in DN.

Effects of resveratrol on regulation on UCP2 and cardiac function in diabetic rats.

Mitochondrial dysfunction is essential in the development and prognosis of diabetic cardiomyopathy (DCM). Resveratrol (RES) is thought as a mitochondrial protector. In this study, we hypothesized that RES may ameliorate mitochondrial function and consequently improve cardiac function in diabetic rats, and uncoupling protein 2 (UCP2) was involved in the protective effects of RES on DCM. Thirty rats were divided into three groups: normal control, DCM, and DCM+RES groups. DCM was induced by high-fat diet and streptozotocin (STZ) intraperitoneal injection, the rats in DCM+RES group received RES gavage for 16weeks. RES improved the insulin resistance, and reduced the level of triglyceride, cholesterol, and low density lipoprotein cholesterol (LDLc) in DCM rats (all P < 0.05). Echocardiographic and hemodynamic studies revealed that RES treatment reversed the impaired diastolic and systolic cardiac function in DCM rats. Meanwhile, RES improved myocardial structural disorder and fibrosis, reserved mitochondrial membrane potential level (P < 0.05), and suppressed myocardial apoptosis in DCM rats (P < 0.05). Myocardial mitochondrial respiratory enzyme activities were improved by RES treatment in DCM rats (P < 0.05), accompanied with attenuated reactive oxygen species (ROS) generation (P < 0.05). The expression of UCP2 was further increased by RES treatment both in the myocardium of DCM rats (P < 0.05) and in the H9c2 cardiomyocytes incubated with high-glucose (P < 0.05). The protective effects of RES on high glucose-induced ROS generation, MPTP opening, Cyto c release, and cell apoptosis were all blunted by inhibiting the expression of UCP2 (all P < 0.05). In conclusion, RES treatment improved cardiac function and inhibited cardiomyocyte apoptosis, involving in ameliorating mitochondrial function in diabetic rats. UCP2 mediated the protective effects of RES on diabetic hearts.

Hypermethylated in cancer 1 (HIC1) mediates high glucose induced ROS accumulation in renal tubular epithelial cells by epigenetically repressing SIRT1 transcription

Reactive oxygen species (ROS) is a key regulator of an array of physiological and pathological processes. While essential for the host defense mechanism, excessive ROS generation and/or deficient clearance is blamed for the pathogenesis of human diseases. In the present study, we investigated the regulatory role of hypermethylated in cancer 1 (HIC1), a transcription factor, in high glucose-induced ROS accumulation in renal tubular epithelial cells (HK-2). Treatment with high glucose (HG) not only markedly up-regulated HIC1 expression but prompted its translocation into the nucleus. HG stimulation promoted HIC1 binding to the promoter of SIRT1, a known HIC1 target with anti-oxidative ability. The recruitment of HIC1 to the SIRT1 promoter was paralleled by the enrichment of trimethylated histone H3K27 and 5‑methyl cytosine, two well-characterized markers for trans-repression. HIC1 silencing with small interfering RNA abrogated SIRT1 repression by HG and at the same time weakened ROS accumulation in HK-2 cells. Knockdown or pharmaceutical inhibition of SIRT1 preempted the effect of HIC1 depletion by restoring ROS accumulation and down-regulating the expression of antioxidant genes. Mechanistically, HIC1 interacted with and recruited EZH2, an H3K27 trimethyltransferase, and DNA methyltransferase 1 (DNMT1) to repress SIRT1 transcription in response to HG stimulation. Depletion or inhibition of EZH2 or DNMT1 rescued SIRT1 expression and blocked ROS accumulation in HG-treated HK-2 cells. In conclusion, our data suggest that epigenetic repression of SIRT1 by HIC1 may contribute to HG-induced elevation of ROS levels in renal tubular epithelial cells.

Curcumin Modulates DNA Methyltransferase Functions in a Cellular Model of Diabetic Retinopathy.

Hyperglycaemia-induced oxidative stress appears to be involved in the aetiology of diabetic retinopathy (DR), a major public health issue, via altering DNA methylation process. We investigated the effect of hyperglycaemia on retinal DNA methyltransferase (DNMT) expression in diabetic mice, using Gene Expression Omnibus datasets. We also evaluated the effect of curcumin both on high glucose-induced reactive oxygen species (ROS) production and altered DNMT functions, in a cellular model of DR. We observed that three months of hyperglycaemia, in insulin-deficient Ins2Akita mice, decrease DNMT1 and DNMT3a expression levels. In retinal pigment epithelium (RPE) cells, we also demonstrated that high glucose-induced ROS production precedes upregulation of DNMT expression and activity, suggesting that changes in DNMT function could be mediated by oxidative stress via a potential dual effect. The early effect results in decreased DNMT activity, accompanied by the highest ROS production, while long-term oxidative stress increases DNMT activity and DNMT1 expression. Interestingly, treatment with 25 μM curcumin for 6 hours restores ROS production, as well as DNMT functions, altered by the exposure of RPE to acute and chronic high glucose concentration. Our study suggests that curcumin may represent an effective antioxidant compound against DR, via restoring oxidative stress and DNMT functions, though further studies are recommended.

SET8 is involved in the regulation of hyperglycemic memory in human umbilical endothelial cells.

Hyperglycemic memory occurs in diabetic cardiovascular complications, but the underlying mechanism remains to be elucidated. Although the depletion of SET8 leads to increased mitochondrial oxidative stress via increasing cellular reactive oxygen species (ROS) production, the role of SET8 in hyperglycemic memory-induced mitochondrial dysfunction is not well understood. Here, we investigated the role of SET8 in this setting. Our results showed that high glucose-induced vascular inflammation, ROS production and apoptosis remained at high levels even when glucose returned to normal level. Elevated glucose reduced SET8 expression, which also remained at low level after returning to normoglycemia. SET8 overexpression protected cells from elevated glucose and hyperglycemic memory-induced endothelial injury by blocking ROS accumulation, attenuating vascular inflammation, and restoring nitric oxide production. Thus, our results suggest that SET8 may be a key mediator in hyperglycemic memory.

Hesperidin Prevents High Glucose-Induced Damage of Retinal Pigment Epithelial Cells.

The present study aimed to determine whether hesperidin, a plant-based active flavanone found in citrus fruits, can prevent high glucose-induced retinal pigment epithelial (RPE) cell impairment. Cultured human RPE cells (ARPE-19) were exposed to a normal glucose concentration (5.5 mM) for 4 d and then soaked in either normal (5.5 mM) or high (33.3 mM) concentrations of D-glucose with or without different concentrations of hesperidin (10, 20, or 40 µM) for another 48 h. The survival rates of the cells were measured using a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay. With the help of a fluorescent probe, the intracellular production of reactive oxygen species (ROS) was evaluated. Colorimetric assay kits were used to assess the antioxidant enzyme activities, and western blotting was used to measure the expression of apoptosis-related protein. Hesperidin was effective in inhibiting high glucose-induced ROS production, preventing loss of cell viability, and promoting the endogenous antioxidant defense components, including glutathione peroxidase, superoxide dismutase, catalase, and glutathione, in a concentration-dependent manner. Furthermore, high glucose triggered cell apoptosis via the upregulation of caspase-9/3, enhancement of cytochrome c release into the cytosol, and subsequent interruption of the Bax/Bcl-2 balance. These detrimental effects were ameliorated by hesperidin in a concentration-dependent manner. We conclude that through the scavenging of ROS and modulation of the mitochondria-mediated apoptotic pathway, hesperidin may protect RPE cells from high glucose-induced injury and thus may be a candidate in preventing the visual impairment caused by diabetic retinopathy.

Role of Family with Sequence Similarity 3A in High Glucose-induced Oxidative Damage of Human Umbilical Vein Endothelial Cells.

Objective To investigate the role of family with sequence similarity 3A(Fam3A) in high glucose-induced damage of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were divided into control group and high glucose group, which were cultured in endothelial cell medium (ECM) containing 5.5 mmol/L of glucose and ECM containing 33.3 mmol/L of glucose, respectively. Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of Fam3A, whereas the protein expression of Fam3A was detected by enzyme-linked immunosorbent assay. HUVECs in control group and high glucose group were transfected with siNT and siFam3A, respectively, and the levels of reactive oxygen species(ROS), ATP, mitochondrial oxygen consumption rate(OCR), and P-p38 protein were detected.Results After HUVECs had been cultured for 24h, the relative mRNA expression of Fam3A between high glucose group and control group was 2.52±0.19 (t=13.296,P=0.000). The Fam3A protein level was (173.82±33.28)pg/ml in the high glucose group, which was significantly higher than that [(39.45±33.78)pg/ml] in the control group (t=4.907,P=0.006). The intracellular ROS content in siNT-high glucose group was (8217±794)RFU, which was significantly higher than that [(3982±398)RFU] in siNT control group (t=15.109,P=0.002). The intracellular ROS content of siFam3A high glucose group was (11 910±1 001)RFU, significantly higher than that [(4171±402)RFU] of siFam3A control group (t=9.705,P=0.010) and than that of siNT high glucose group (t=4.026,P=0.048). The relative amounts of ATP synthesis in siNT high glucose group, siFam3A control group and siFam3A high glucose group were (61.2±5.6)%, (94.6±8.4)%, and (29.7±2.7)% of the siNT control group respectively; thus, it was significantly lower in siNT high glucose group than in siNT control group (t=12.001,P=0.007) and was also significantly lower in siFam3A high glucose group than in siFam3A control group (t=20.742,P=0.002) and in siNT high glucose group(t=18.814,P=0.003). The mitochondrial OCR was (0.57±0.05)pMO2/(μg protein·min) in siNT high glucose group, significantly lower than that [(1.12±0.09)pMO2/(μg protein·min)] of siNT control group (t=6.804,P=0.021). The mitochondrial OCR of siFam3A high glucose group was (0.31±0.03)pMO2/(μg protein·min), significantly lower than that [(1.01±0.09)pMO2/(μg protein·min)] of siFam3A control group (t=19.876,P=0.003), which was significantly lower than that of siNT high glucose group (t=21.444,P=0.002). The relative expression of P-p38 in siNT high glucose group, siFam3A control group, and siFam3A high glucose group was 2.239±0.353, 0.816±0.120, and 1.160±0.185, respectively; thus, it was significantly higher in the siNT high glucose group than in siNT control group (t=6.075,P=0.026); in addition, it was significantly higher in the siFam3A high glucose group than in siFam3A control group (t=6.242,P=0.024) and significantly lower than in siNT high glucose group (t=9.686,P=0.010). Conclusions High glucose can induce high expression of Fam3A in HUVECs. Knockdown of Fam3A gene expression can exacerbate the decrease of ATP synthesis and mitochondrial OCR caused by high glucose and promote the generation of ROS in high glucose. Fam3A may regulate high glucose-induced ROS production in HUVECs via the p38 MAPK signaling pathway.

Lutein suppresses hyperglycemia-induced premature senescence of retinal pigment epithelial cells by upregulating SIRT1

Hyperglycemia contributes to the pathogenesis of diabetic complications, such as diabetic retinopathy, by affecting cellular redox state. In this study, we investigated the effects of lutein on hyperglycemia-induced premature senescence in retinal pigment epithelium (RPE) cells. In ARPE-19 cells, a spontaneously immortalized cell line derived from human RPE, lutein treatment significantly inhibited high glucose-triggered premature senescence and production of reactive oxygen species (ROS). Notably, lutein treatment increased SIRT1 mRNA and protein levels, suggesting that lutein exerted its beneficial effects in these cells by upregulating SIRT1 expression. Resveratrol, an activator of SIRT1, mimicked the inhibitory effects of lutein on high glucose-induced premature senescence and ROS generation, whereas sirtinol, a specific inhibitor of SIRT1, blocked these effects of lutein. Collectively, these observations indicate that lutein interferes with hyperglycemia-induced RPE senescence by modulating SIRT1 signaling. Practical applications Lutein, a xanthophyll carotenoid present in green leafy vegetables and eggs, has been consumed as a dietary supplement to improve eye health. Lutein exhibited anti-senescent properties by suppressing ROS generation through SIRT1 upregulation in RPE cells exposed to hyperglycemia mimicking diabetic conditions. Thus, we propose that lutein could be applied to therapeutic interventions aimed at treating hyperglycemia-induced retinal disorders.