Overexpression of human heme oxygenase-1 attenuates endothelial cell sloughing in experimental diabetes

Heme oxygenase (HO)-1 represents a key defense mechanism against oxidative injury. Hyperglycemia produces oxidative stress and various perturbations of cell physiology. The effect of streptozotocin (STZ)-induced diabetes on aortic HO activity, heme content, the number of circulating endothelial cells, and urinary 8-epi-isoprostane PGF2alpha (8-Epi) levels in control rats and rats overexpressing or underexpressing HO-1 was measured. HO activity was decreased in hyperglycemic rats. Hyperglycemia increased urinary 8-Epi, and this increase was augmented in rats underexpressing HO-1 and diminished in rats overexpressing HO-1. The number of detached endothelial cells and O2- formation increased in diabetic rats and in hyperglycemic animals underexpressing HO-1 and decreased in diabetic animals overexpressing HO-1 compared with controls. These data demonstrate that HO-1 gene transfer in hyperglycemic rats brings about a reduction in O2- production and a decrease in endothelial cell sloughing. Upregulation of HO-1 decreases oxidant production and endothelial cell damage and shedding and may attenuate vascular complications in diabetes.     

G protein-linked cell signaling and cardiovascular functions in diabetes/hyperglycemia

Vascular complications, including impaired contractility and increased cell proliferation, are the most common complications with diabetes. Chronic hyperglycemia seems to be an important contributing factor in this process. Various signaling pathways are implicated in diabetes/hyperglycemia-induced impaired vascular functions. Nonenzymatic glycation, enhanced production of diacylglycerol, increased activity of membranous protein kinase C (PKC), and increased oxidative stress have been proposed to explain the adverse effects of hyperglycemia on vascular smooth muscle cells. Hyperglycemia-induced stimulation of L-type Ca²⁺ channel via G protein-coupled adenylyl cyclase/cAMP and phospholipase C/PKC pathways also has been shown. In addition, hyperglycemia has been reported to decrease the availability of nitric oxide in humans, which may contribute to all the hemodynamic and physiological changes occuring in diabetes. G protein-adenylyl cyclase signaling that plays an important role in the regulation of cardiovascular functions also has been reported to be impaired in diabetes and under hyperglycemic conditions. In this review article, various G protein-linked cell signaling and functions in diabetes and hyperglycemia are discussed.     

Anti-oxidant effect of gold nanoparticles restrains hyperglycemic conditions in diabetic mice

Background  

Oxidative stress is imperative for its morbidity towards diabetic complications, where abnormal metabolic milieu as a result of hyperglycemia, leads to the onset of several complications. A biological antioxidant capable of inhibiting oxidative stress mediated diabetic progressions; during hyperglycemia is still the need of the era. The current study was performed to study the effect of biologically synthesized gold nanoparticles (AuNPs) to control the hyperglycemic conditions in streptozotocin induced diabetic mice.     

Hyperthermia-induced Hsp90·eNOS Preserves Mitochondrial Respiration in Hyperglycemic Endothelial Cells by Down-regulating Glut-1 and Up-regulating G6PD Activity

Uncoupling of NO production from NADPH oxidation by endothelial nitric-oxide synthase (eNOS) is enhanced in hyperglycemic endothelium, potentially due to dissociation of heat shock proteins 90 (Hsp90), and cellular glucose homeostasis is enhanced by a ROS-induced positive feed back mechanism. In this study we investigated how such an uncoupling impacts oxygen metabolism and how the oxidative phosphorylation can be preserved by heat shock (42 °C for 2 h, hyperthermia) in bovine aortic endothelial cells. Normal and heat-shocked bovine aortic endothelial cells were exposed to normoglycemia (NG, 5.0 mm) or hyperglycemia (30 mm). With hyperglycemia treatment, O₂ consumption rate was reduced (from V_O2max = 7.51 ± 0.54 to 2.35 ± 0.27 mm Hg/min/10⁶ cells), whereas in heat-shocked cells, O₂ consumption rate remained unaltered (8.19 ± 1.01 mm Hg/min/10 × 10⁶ cells). Heat shock was found to enhance Hsp90/endothelial NOS interactions and produce higher NO. Moreover, ROS generation in the hyperglycemic condition was also reduced in heat-shocked cells. Interestingly, glucose uptake was reduced in heat-shocked cells as a result of decrease in Glut-1 protein level. Glucose phosphate dehydrogenase activity that gives rise to NADPH generation was increased by hyperthermia, and mitochondrial oxidative metabolism was preserved. In conclusion, the present study provides a novel mechanism wherein the reduced oxidative stress in heat-shocked hyperglycemic cells down-regulates Glut-1 and glucose uptake, and fine-tuning of this pathway may be a potential approach to use for therapeutic benefit of diabetes mellitus.     

Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress

The potential of proline to suppress reactive oxygen species (ROS) and apoptosis in mammalian cells was tested by manipulating intracellular proline levels exogenously and endogenously by overexpression of proline metabolic enzymes. Proline was observed to protect cells against H(2)O(2), tert-butyl hydroperoxide, and a carcinogenic oxidative stress inducer but was not effective against superoxide generators such as menadione. Oxidative stress protection by proline requires the secondary amine of the pyrrolidine ring and involves preservation of the glutathione redox environment. Overexpression of proline dehydrogenase (PRODH), a mitochondrial flavoenzyme that oxidizes proline, resulted in 6-fold lower intracellular proline content and decreased cell survival relative to control cells. Cells overexpressing PRODH were rescued by pipecolate, an analog that mimics the antioxidant properties of proline, and by tetrahydro-2-furoic acid, a specific inhibitor of PRODH. In contrast, overexpression of the proline biosynthetic enzymes Delta(1)-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) resulted in 2-fold higher proline content, significantly lower ROS levels, and increased cell survival relative to control cells. In different mammalian cell lines exposed to physiological H(2)O(2) levels, increased endogenous P5CS and P5CR expression was observed, indicating that upregulation of proline biosynthesis is an oxidative stress response.     

Glucose-induced endoplasmic reticulum stress is independent of oxidative stress: A mechanistic explanation for the failure of antioxidant therapy in diabetes

Oxidative stress contributes to the pathogenesis of diabetes and its complications. However, a large number of interventional studies have failed to show any health benefits of antioxidants. The overwhelming failure of antioxidant therapy to prevent disease can be explained by inadequacy of the doses of antioxidants used, short duration of therapy, or poor timing of initiation of the supplementation. A more likely reason for failure of antioxidants to reduce diabetes-related complications is the multiplicity of mechanisms of glucotoxicity that are independent of oxidative stress. Recently, endoplasmic reticulum (ER) stress has emerged as an important contributor to diabetes-related complications. Multiple lines of experimental evidence indicate that ER stress in endothelial cells can be uncoupled from oxidative stress induced by hyperglycemia, and antioxidants can ameliorate the latter without altering the ER stress. These observations provide a novel mechanistic explanation for the failure of antioxidant therapy in interventional clinical trials.     

Polyol pathway-dependent osmotic and oxidative stresses in aldose reductase-mediated apoptosis in human lens epithelial cells: role of AOP2

Aldose reductase (AR) has been implicated as a major contributor to the pathogenesis of diabetic cataracts. AR activation generates osmotic and oxidative stresses via the polyol pathway and induces cell death signals. Antioxidant protein 2 (AOP2) protects cells from oxidative stress. We investigated the effect of AR overexpression on polyol accumulation and on hyperglycemic oxidative stress and osmotic stress, as well as the effects of these stresses on human lens epithelial cell (hLEC) survival. hLECs overexpressing the AR became apoptotic during hyperglycemia and showed elevated levels of intracellular polyols. Glutathione and AOP2 levels were significantly decreased in these cells. Interestingly, supply of AOP2 and/or the AR inhibitor fidarestat protected the cells against hyperglycemia-induced death. Overexpression of AR increased osmotic and oxidative stresses, resulting in increased apoptosis in hLECs. Because AOP2 protects hyperglycemia-induced hLEC apoptosis, this molecule may have the potential to prevent hyperglycemia-mediated complications in diabetes.     

Are diabetic neuropathy,retinopathy and nephropathy caused by hyperglycemic exclusion of dehydroascorbate uptake by glucose transporters?

Vitamin C exists in two major forms. The charged form, ascorbic acid (AA), is taken up into cells via sodium-dependent facilitated transport. The uncharged form, dehydroascorbate (DHA), enters cells via glucose transporters (GLUT) and is then converted back to AA within these cells. Cell types such as certain endothelial and epithelial cells as well as neurons that are particularly prone to damage during diabetes tend to be those that appear to be dependent on GLUT transport of DHA rather than sodium-dependent AA uptake. We hypothesize that diabetic neuropathies, nephropathies and retinopathies develop in part by exclusion of DHA uptake by GLUT transporters when blood glucose levels rise above normal. AA plays a central role in the antioxidant defense system. Exclusion of DHA from cells by hyperglycemia would deprive the cells of the central antioxidant, worsening the hyperglycemia-induced oxidative stress level. Moreover, AA participates in many cellular oxidation-reduction reactions including hydroxylation of polypeptide lysine and proline residues and dopamine that are required for collagen production and metabolism and storage of catecholamines in neurons. Increase in the oxidative stress level and metabolic perturbations can be expected in any tissue or cell type that relies exclusively or mainly on GLUT for co-transport of glucose and DHA including neurons, epithelial cells, and vascular tissues. On the other hand, since DHA represents a significant proportion of total serum ascorbate, by increasing total plasma ascorbate concentrations during hyperglycemia, it should be possible to correct the increase in the oxidative stress level and metabolic perturbations, thereby sparing diabetic patients many of their complications.     

Reducing oxidative stress in patients with type 2 diabetes mellitus: A primary care call to action

Background: Oxidative stress is believed to be the primary cause of the microvascular and macrovascular complications of type 2 diabetes mellitus (DM).Objective: This paper examines the evidence linking oxidative stress with long-term complications of type 2 DM and explores methods to minimize its effect.Methods: A literature search was performed to identify relevant studies for this review. Articles published in English from 2000 to 2008 were identified through searches of PubMed, Diabetes Care, and Google using the search terms oxidative stress, postprandial hyperglycemia, ACCORD Trial, and endothelial cell dysfunction.Results: The literature search identified 423 articles. Although chronic hyperglycemia can be effectively monitored and targeted using glycosylated hemoglobin concentrations, postprandial glucose levels are also important. Postprandial glucose excursions are exhibited by almost all patients with type 2 DM and are independent risk factors for cardiovascular morbidity and mortality. Furthermore, glucose fluctuations during the postprandial period elicit more oxidative stress than chronic, sustained hyperglycemia and can lead to endothelial dysfunction, vascular inflammation, and microvascular complications. In turn, endothelial dysfunction has been implicated in the development of vascular pathologies such as atherosclerosis. Pharmacologic interventions (eg, rapid-acting insulin analogues that target post-prandial glucose excursions) reduce oxidative stress and vascular inflammation and improve endothelial dysfunction.Conclusions: Given the important role of oxidative stress in the development of complications of type 2 DM, physi-cians should consider methods to reduce oxidative stress that may occur during both acute (postprandial) and chronic hyperglycemia. One critical aspect is to reduce postprandial glucose levels to <180 mg/dL while lowering fasting glucose levels to <110 mg/dL. By coaching patients to reach these goals, physicians and other health care professionals can minimize the risk of long-term complications of type 2 DM.     

Metabolic memory: mechanisms and implications for diabetic vasculopathies

In the past decades, a persistent progression of diabetic vascular complications despite reversal of hyperglycemia has been observed in both experimental and clinical studies. This durable effect of prior hyperglycemia on the initiation and progression of diabetic vasculopathies was defined as “metabolic memory”. Subsequently, enhanced glycation of cellular proteins and lipids, sustained oxidative stress, and prolonged inflammation were demonstrated to mediate this phenomenon. Recently, emerging evidence strongly suggests that epigenetic modifications may account for the molecular and phenotypic changes associated with hyperglycemic memory. In this review, we presented an overview on the discovery of metabolic memory, the recent progress in its molecular mechanisms, and the future implications related to its fundamental research and clinical application.     

Analysis of the oxidative damage of DNA,RNA, and their metabolites induced by hyperglycemia and related nephropathy in Sprague Dawley rats

Abstract

We used a sensitive and accurate method based on isotope dilution high-performance liquid chromatography–triple quadrupole mass spectrometry (ID-LC-MS/MS) to determine the levels of 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosin (8-oxo-Gsn) in various tissue specimens, plasma, and urine of hyperglycemic Sprague Dawley rats induced by streptozotocin (STZ). The oxidative DNA and RNA damages were observed in various organs and the amounts of 8-oxo-dGsn and 8-oxo-Gsn derived from DNA and RNA were increased with hyperglycemic status. In contrast to the results of the nucleic acid samples derived from tissues, the levels of 8-oxo-Gsn in urine and plasma were significantly higher compared with that of 8-oxo-dGsn, which most likely reflected the RNA damage that occurs more frequently compared with DNA damage. For the oxidative stress induced by hyperglycemia, 8-oxo-Gsn in urine may be a sensitive biomarker on the basis of the results in urine, plasma, and tissues. In addition, high levels of urinary 8-oxo-Gsn were observed before diabetic microvascular complications. Based on that the 8-oxo-dGsn was associated with diabetic nephropathy and RNA was more vulnerable to oxidative stress compared with DNA. We also propose that 8-oxo-Gsn is correlated with diabetic nephropathy and that 8-oxo-Gsn in urine could be a useful and sensitive marker of diabetic nephropathy.     

Signaling properties of 4-hydroxyalkenals formed by lipid peroxidation in diabetes

Peroxidation of polyunsaturated fatty acids is intensified in cells subjected to oxidative stress and results in the generation of various bioactive compounds, of which 4-hydroxyalkenals are prominent. During the progression of type 2 diabetes mellitus, the ensuing hyperglycemia promotes the generation of reactive oxygen species (ROS) that contribute to the development of diabetic complications. It has been suggested that ROS-induced lipid peroxidation and the resulting 4-hydroxyalkenals markedly contribute to the development and progression of these pathologies. Recent findings, however, also suggest that noncytotoxic levels of 4-hydroxyalkenals play important signaling functions in the early phase of diabetes and act as hormetic factors to induce adaptive and protective responses in cells, enabling them to function in the hyperglycemic milieu. Our studies and others′ have proposed such regulatory functions for 4-hydroxynonenal and 4-hydroxydodecadienal in insulin secreting β-cells and vascular endothelial cells, respectively. This review presents and discusses the mechanisms regulating the generation of 4-hydroxyalkenals under high glucose conditions and the molecular interactions underlying the reciprocal transition from hormetic to cytotoxic agents.     

Cytochrome P450 2E1 and hyperglycemia-induced liver injury

Cytochrome P450 2E1 (CYP2E1), a microsomal enzyme involved in xenobiotic metabolism and generation of oxidative stress, has been implicated in promoting liver injury. The review deals with the changes in various cellular pathways in liver linked with the changes in regulation of CYP2E1 under hyperglycemic conditions. Some of the hepatic abnormalities associated with hyperglycemia-mediated induction of CYP2E1 include increased oxidative stress, changes in mitochondrial structure and function, apoptosis, nitrosative stress, and increased ketone body accumulation. Thus, changes in regulation of CYP2E1 are associated with the injurious effects of hyperglycemia in liver.     

Inhibition of CYP2E1 leads to decreased advanced glycated end product formation in high glucose treated ADH and CYP2E1 over-expressing VL-17A cells

Background

In recent years, there has been a growing interest to explore the association between liver injury and diabetes. Advanced glycated end product (AGE) formation which characterizes diabetic complications is formed through hyperglycemia mediated oxidative stress and is itself a source for ROS. Further, in VL-17A cells over-expressing ADH and CYP2E1, greatly increased oxidative stress and decreased viability have been observed with high glucose exposure.

Methods

In VL-17A cells treated with high glucose and pretreated with the different inhibitors of ADH and CYP2E1, the changes in cell viability, oxidative stress parameters and formation of AGE, were studied.

Results

Inhibition of CYP2E1 with 10 μM diallyl sulfide most effectively led to decreases in the oxidative stress and toxicity as compared with ADH inhibition with 2 mM pyrazole or the combined inhibition of ADH and CYP2E1 with 5 mM 4-methyl pyrazole. AGE formation was decreased in VL-17A cells when compared with HepG2 cells devoid of the enzymes. Further, AGE formation was decreased to the greatest extent with the inhibitor for CYP2E1 suggesting that high glucose inducible CYP2E1 and the consequent ROS aid AGE formation.

Conclusions

Thus, CYP2E1 plays a pivotal role in the high glucose induced oxidative stress and toxicity in liver cells as observed through direct evidences obtained utilizing the different inhibitors for ADH and CYP2E1.

General significance

The study demonstrates the role of CYP2E1 mediated oxidative stress in aggravating hyperglycemic insult and suggests that CYP2E1 may be a vital component of hyperglycemia mediated oxidative injury in liver.     

Inhibition of NADPH oxidase-related oxidative stress-triggered signaling by honokiol suppresses high glucose-induced human endothelial cell apoptosis

Angiopathy is a major complication of diabetes. Abnormally high blood glucose is a crucial risk factor for endothelial cell damage. Nuclear factor-kappaB (NF-kappaB) has been demonstrated as a mediated signaling in hyperglycemia or oxidative stress-triggered apoptosis of endothelial cells. Here we explored the efficacy of honokiol, a small molecular weight natural product, on NADPH oxidase-related oxidative stress-mediated NF-kappaB-regulated signaling and apoptosis in human umbilical vein endothelial cells (HUVECs) under hyperglycemic conditions. The methods of morphological Hoechst staining and annexin V/propidium iodide staining were used to detect apoptosis. Submicromolar concentrations of honokiol suppressed the increases of NADPH oxidase activity, Rac-1 phosphorylation, p22(phox) protein expression, and reactive oxygen species production in high glucose (HG)-stimulated HUVECs. The degradation of IkappaBalpha and increase of NF-kappaB activity were inhibited by honokiol in HG-treated HUVECs. Moreover, honokiol (0.125-1 microM) also suppressed HG-induced cyclooxygenase (COX)-2 upregulation and prostaglandin E(2) production in HUVECs. Honokiol could reduce increased caspase-3 activity and the subsequent apoptosis and cell death triggered by HG. These results imply that inhibition of NADPH oxidase-related oxidative stress by honokiol suppresses the HG-induced NF-kappaB-regulated COX-2 upregulation, apoptosis, and cell death in HUVECs, which has the potential to be developed as a therapeutic agent to prevent hyperglycemia-induced endothelial damage.     

Endothelial cells' biophysical,biochemical, and chromosomal aberrancies in high‐glucose condition within the diabetic range

To date, many studies have been conducted to find out the underlying mechanisms of hyperglycemia‐induced complications in diabetes mellitus, attributed to the cellular pathologies of different cells—especially endothelial cells. However, there are still many ambiguities and unresolved issues to be clarified. Here, we investigated the alteration in biophysical and biochemical properties in human umbilical vein endothelial cells exposed to a high‐glucose concentration (30mM), comparable to glucose content in type 2 diabetes mellitus, over a course of 120 hours. In addition to a reduction in the rate of cell viability and induction of oxidative stress orchestrated by the high‐glucose condition, the dynamic of the fatty acid profile—including polyunsaturated, monounsaturated, and saturated fatty acids—was also altered in favor of saturated fatty acids. Genetic imbalances were also detected at chromosomal level in the cells exposed to the abnormal concentration of glucose after 120 hours. Moreover, the number of tip cells (CD31⁺/CD34⁺) and in vitro tubulogenesis capability negatively diminished in comparison to parallel control groups. We found that diabetic hyperglycemia was associated with a decrease in the cell‐cell tight junction and upregulation in vascular endothelial cadherin and zonula occludens (ZO)‐1 molecules after 72 and 120 hours of exposure to the abnormal glucose concentration, which resulted in a profound reduction in transendothelial electrical resistance. The surface plasmon resonance analysis of the human umbilical vein endothelial cells immobilized on gold‐coated sensor chips confirmed the loosening of the cell to cell intercellular junction as well as stable attachment of each cell to the basal surface. Our findings highlighted the disturbing effects of a diabetic hyperglycemia on either biochemical or biophysical properties of endothelial cells.     

Oleanolic Acid: A Novel Cardioprotective Agent That Blunts Hyperglycemia-Induced Contractile Dysfunction

Diabetes constitutes a major health challenge. Since cardiovascular complications are common in diabetic patients this will further increase the overall burden of disease. Furthermore, stress-induced hyperglycemia in non-diabetic patients with acute myocardial infarction is associated with higher in-hospital mortality. Previous studies implicate oxidative stress, excessive flux through the hexosamine biosynthetic pathway (HBP) and a dysfunctional ubiquitin-proteasome system (UPS) as potential mediators of this process. Since oleanolic acid (OA; a clove extract) possesses antioxidant properties, we hypothesized that it attenuates acute and chronic hyperglycemia-mediated pathophysiologic molecular events (oxidative stress, apoptosis, HBP, UPS) and thereby improves contractile function in response to ischemia-reperfusion. We employed several experimental systems: 1) H9c2 cardiac myoblasts were exposed to 33 mM glucose for 48 hr vs. controls (5 mM glucose); and subsequently treated with two OA doses (20 and 50 µM) for 6 and 24 hr, respectively; 2) Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min, followed by 20 min global ischemia and 60 min reperfusion ± OA treatment; 3) In vivo coronary ligations were performed on streptozotocin treated rats ± OA administration during reperfusion; and 4) Effects of long-term OA treatment (2 weeks) on heart function was assessed in streptozotocin-treated rats. Our data demonstrate that OA treatment blunted high glucose-induced oxidative stress and apoptosis in heart cells. OA therapy also resulted in cardioprotection, i.e. for ex vivo and in vivo rat hearts exposed to ischemia-reperfusion under hyperglycemic conditions. In parallel, we found decreased oxidative stress, apoptosis, HBP flux and proteasomal activity following ischemia-reperfusion. Long-term OA treatment also improved heart function in streptozotocin-diabetic rats. These findings are promising since it may eventually result in novel therapeutic interventions to treat acute hyperglycemia (in non-diabetic patients) and diabetic patients with associated cardiovascular complications.     

Therapeutic effects of stem cell on hyperglycemia,hyperlipidemia, and oxidative stress in alloxan-treated rats

Diabetes mellitus is the most common endocrine disorder that affects more than 285 million people worldwide. The purpose of this study was to investigate the effect of mesenchymal stem cells (MSCs) from the bone marrow of albino rats, on hyperglycemia, hyperlipidemia, and oxidative stress induced by intraperitoneal injection (i.p.) of alloxan at a dose of 150 mg/kg in rats. Injection of alloxan into rats resulted in a significant increase in serum glucose, total cholesterol, triglyceride, low density lipoprotein cholesterol, and sialic acid level and a significant decrease in serum insulin, high density lipoprotein-cholesterol, vitamin E, and liver glycogen as compared to their corresponding controls. Also, oxidative stress was noticed in pancreatic tissue as evidenced by a significant decrease in glutathione level, superoxide dismutase, glutathione-S-transferase activities, also a significant increase in malondialdehyde and nitric oxide levels when compared to control group. Treatment of diabetic rats with MSCs stem cells significantly prevented these alterations and attenuated alloxan-induced oxidative stress. In conclusion, rat bone marrow harbors cells that have the capacity to differentiate into functional insulin-producing cells capable of controlling hyperglycemia, hyperlipidemia, and oxidative stress in diabetic rats. This may be helpful in the prevention of diabetic complications associated with oxidative stress.     

Hyperinsulinemia-induced vascular smooth muscle cell (VSMC) migration and proliferation is mediated by converging mechanisms of mitochondrial dysfunction and oxidative stress

Atherosclerosis is one of the major complications of diabetes and involves endothelial dysfunction, matrix alteration, and most importantly migration and proliferation of vascular smooth muscle cells (VSMCs). Although hyperglycemia and hyperinsulinemia are known to contribute to atherosclerosis, little is known about the specific cellular signaling pathways that mediate the detrimental hyperinsulinemic effects in VSMCs. Therefore, we investigated the cellular mechanisms of hyperinsulinemia-induced migration and proliferation of VSMCs. VSMCs were treated with insulin (100 nM) for 6 days and subjected to various physiological and molecular investigations. VSMCs subjected to hyperinsulinemia exhibited increased migration and proliferation, and this is paralleled by oxidative stress [increased NADPH oxidase activity, NADPH oxidase 1 mRNA expression, and reactive oxygen species (ROS) generation], alterations in mitochondrial physiology (membrane depolarization, decreased mitochondrial mass, and increased mitochondrial ROS), changes in mitochondrial biogenesis-related genes (mitofusin 1, mitofusin 2, dynamin-related protein 1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, peroxisome proliferator-activated receptor gamma coactivator 1-beta, nuclear respiratory factor 1, and uncoupling protein 2), and increased Akt phosphorylation. Diphenyleneiodonium, a known NADPH oxidase inhibitor significantly inhibited migration and proliferation of VSMCs and normalized all the above physiological and molecular perturbations. This study suggests a plausible crosstalk between mitochondrial dysfunction and oxidative stress under hyperinsulinemia and emphasizes counteracting mitochondrial dysfunction and oxidative stress as a novel therapeutic strategy for atherosclerosis.