Nox2 Mediates Skeletal Muscle Insulin Resistance Induced by a High Fat Diet

Inflammation and oxidative stress through the production of reactive oxygen species (ROS) are consistently associated with metabolic syndrome/type 2 diabetes. Although the role of Nox2, a major ROS-generating enzyme, is well described in host defense and inflammation, little is known about its potential role in insulin resistance in skeletal muscle. Insulin resistance induced by a high fat diet was mitigated in Nox2-null mice compared with wild-type mice after 3 or 9 months on the diet. High fat feeding increased Nox2 expression, superoxide production, and impaired insulin signaling in skeletal muscle tissue of wild-type mice but not in Nox2-null mice. Exposure of C2C12 cultured myotubes to either high glucose concentration, palmitate, or H₂O₂ decreases insulin-induced Akt phosphorylation and glucose uptake. Pretreatment with catalase abrogated these effects, indicating a key role for H₂O₂ in mediating insulin resistance. Down-regulation of Nox2 in C2C12 cells by shRNA prevented insulin resistance induced by high glucose or palmitate but not H₂O₂. These data indicate that increased production of ROS in insulin resistance induced by high glucose in skeletal muscle cells is a consequence of Nox2 activation. This is the first report to show that Nox2 is a key mediator of insulin resistance in skeletal muscle.     

NEFA‐induced ROS impaired insulin signalling through the JNK and p38MAPK pathways in non‐alcoholic steatohepatitis

The aim of this study was to investigate the changes in hepatic oxidative phosphorylation (OXPHOS) complexes (COs) in patients and cows with non‐alcoholic steatohepatitis (NASH) and to investigate the mechanism that links mitochondrial dysfunction and hepatic insulin resistance induced by non‐esterified fatty acids (NEFAs). Patients and cows with NASH displayed high blood NEFAs, TNF‐α and IL‐6 concentrations, mitochondrial dysfunction and insulin resistance. The protein levels of peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α), mitofusin‐2 (Mfn‐2) and OXPHOS complexes (human: COI and COIII; cow: COI‐IV) were significantly decreased in patients and cows with NASH. NEFA treatment significantly impaired mitochondrial function and, increased reactive oxygen species (ROS) production, and excessive ROS overactivated the JNK and p38MAPK pathways and induced insulin resistance in cow hepatocytes. PGC‐1α and Mfn‐2 overexpression significantly decreased the NEFA‐induced ROS production and TNF‐α and IL‐6 mRNA expressions, reversed the inhibitory effect of NEFAs on mitochondrial function and attenuated the overactivation of the ROS‐JNK/p38MAPK pathway, alleviated insulin resistance induced by NEFAs in cow hepatocytes and HepG2 cells. These findings indicate that NEFAs induce mitochondrial dysfunction and insulin resistance mediated by the ROS‐JNK/p38MAPK pathway. PGC‐1α or Mfn‐2 overexpression reversed the lipotoxicity of NEFAs on mitochondrial dysfunction and insulin resistance. Our study clarified the mechanism that links hepatic mitochondrial dysfunction and insulin resistance in NASH.     

Insulin increases H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced pancreatic beta cell death

Insulin resistance results, in part, from impaired insulin signaling in insulin target tissues. Consequently, increased levels of insulin are necessary to control plasma glucose levels. The effects of elevated insulin levels on pancreatic beta (β) cell function, however, are unclear. In this study, we investigated the possibility that insulin may influence survival of pancreatic β cells. Studies were conducted on RINm, RINm5F and Min-6 pancreatic β-cells. Cell death was induced by treatment with H₂O₂, and was estimated by measurements of LDH levels, viability assay (Cell-Titer Blue), propidium iodide staining and FACS analysis, and mitochondrial membrane potential (JC-1). In addition, levels of cleaved caspase-3 and caspase activity were determined. Treatment with H₂O₂ increased cell death; this effect was increased by simultaneous treatment of cells with insulin. Insulin treatment alone caused a slight increase in cell death. Inhibition of caspase-3 reduced the effect of insulin to increase H₂O₂-induced cell death. Insulin increased ROS production by pancreatic β cells and increased the effect of H₂O₂. These effects were increased by inhibition of IR signaling, indicative of an effect independent of the IR cascade. We conclude that elevated levels of insulin may act to exacerbate cell death induced by H₂O₂ and, perhaps, other inducers of apoptosis.     

β-oxidation enzymes and the carnitine-dependent oxidation of palmitate and palmitoyl CoA in mitochondria from avocado

Two sites for the β-oxidation of fatty acids in avocado (Persea americana L.) mesocarp exist. One site is the microbody, the other the mitochondrion. It is apparent that the mitochondrial membrane barrier, which remains intact after sucrose density gradient centrifugation, prevents rapid access of acyl CoA substrates to matrix β-oxidation sites. Thus, intact mitochondria showed little β-oxidation enzyme activity. Rupturing of the mitochondrial membrane allowed rapid access of the acyl CoA substrates to matrix sites. Consequently, in ruptured mitochondria, high O₂-oxidation enzyme activities were measured. O₂ uptake studies further distinguished the two organellar sites of β-oxidation. During palmitoyl CoA oxidation, O₂ uptake was reduced by catalase and increased by KCN in the microbodies, whilst mitochondrial O₂ uptake was unaffected by catalase and reduced by KCN. This reflected the differing fates of FADH₂, produced during the first β-oxidation step, in the two organelles. In addition, only the mitochondrial β-oxidation of fatty acids was carnitine-dependent.     

Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210

Abstract

Background. Insulin protects cardiomyocytes from reactive oxygen species (ROS)-induced apoptosis after ischemic/reperfusion injury, but the mechanism is not clear. This study investigated the protective mechanism of insulin in preventing cardiomyocyte apoptosis from ROS injury. Methods. Rat cardiomyoblast H9c2 cells were treated with hydrogen peroxide (H₂O₂) or insulin at various concentrations for various periods of time, or with insulin and H₂O₂ for various periods of time. Cell viability was measured by the methylthiazolydiphenyl-tetrazolium bromide method. Cellular miR-210 levels were quantified using real-time RT-PCR. MiR-210 expression was also manipulated through lentivirus-mediated transfection. LY294002 was used to investigate involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Results. The percentage of viable cells was significantly and inversely associated with H₂O₂ concentration, an effect that was seemingly attenuated by insulin pretreatment. Treatments with H₂O₂ or insulin were associated with a significant increase in miR-210 levels. Manipulation of miR-210 expression by gene transfection showed that miR-210 could attenuate H₂O₂-induced cellular injury. Inhibition of the PI3K/Akt pathway by the Akt inhibitor LY294002 was associated with a decrease in miR-210 expression. Conclusion. Insulin stimulated the expression of miR-210 through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injury that had been induced by H₂O₂/oxygen species. Our results provide novel evidence regarding the mechanism underlying the protective effect of insulin.     

Insulin on hydrogen peroxide-induced oxidative stress involves ROS/Ca2+ and Akt/Bcl-2 signaling pathways

Abstract

Oxidative stress is induced by excess accumulation of reactive oxygen and nitrogen species (RONS). Astrocytes are metabolically active cells in the brain and understanding astrocytic responses to oxidative stress is essential to understand brain pathologies. In addition to direct oxidative stress, exogenous hydrogen peroxide (H₂O₂) can penetrate biological membranes and enhance formation of other RONS. The present study was carried out to examine the role of insulin in H₂O₂-induced oxidative stress in rat astrocytic cells. To measure changes in the viability of astrocytes at different concentrations of H₂O₂ for 3 h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay was used and 500 μM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 3 h of 500 μM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS) and calcium ion (Ca²⁺) in C6 cells, with insulin able to effectively diminish H₂O₂-induced oxidative damage to C6 cells. Western blotting studies showed that insulin treatment of astrocytes increased the levels of phosphorylated Akt and magnified the decrease in total Bcl-2 protein. The protective effect of insulin treatment on H₂O₂-induced oxidative stress in astrocytes by reducing apoptosis may relate to the PI3K/Akt pathway.     

Acute administration of 3,5-diiodo-L-thyronine to hypothyroid rats stimulates bioenergetic parameters in liver mitochondria

The role of 3,5-diiodo-L-thyronine (T₂), initially considered only a 3,3′,5-triiodo-L-thyronine (T₃) catabolite, in the bioenergetic metabolism is of growing interest. In this study we investigated the acute effects (within 1 h) of T₂ administration to hypothyroid rats on liver mitochondria fatty acid uptake and β-oxidation rate, mitochondrial efficiency (by measuring proton leak) and mitochondrial oxidative damage (by determining H₂O₂ release). Fatty acid uptake into mitochondria was measured assaying carnitine palmitoyl transferase (CPT) I and II activities, and fatty acid β-oxidation using palmitoyl-CoA as a respiratory substrate. Mitochondrial fatty acid pattern was defined by gas-liquid chromatography. In hypothyroid + T₂ vs hypothyroid rats we observed a raise in the serum level of nonesterified fatty acids (NEFA), in the mitochondrial CPT system activity and in the fatty acid β-oxidation rate. A parallel increase in the respiratory chain activity, mainly from succinate, occurs. When fatty acids are chelated by bovine serum albumin, a T₂-induced increase in both state 3 and state 4 respiration is observed, while, when fatty acids are present, mitochondrial uncoupling occurs together with increased proton leak, responsible for mitochondrial thermogenesis. T₂ administration decreases mitochondrial oxidative stress as determined by lower H₂O₂ production. We conclude that in rat liver mitochondria T₂ acutely enhances the rate of fatty acid β-oxidation, and the activity of the downstream respiratory chain. The T₂-induced increase in proton leak may contribute to mitochondrial thermogenesis and to the reduction of oxidative stress. Our results strengthen the previously reported ability of T₂ to reduce adiposity, dyslipidemia and to prevent liver steatosis.     

Dehydroepiandrosterone ameliorates H2O2-induced Leydig cells oxidation damage and apoptosis through inhibition of ROS production and activation of PI3K/Akt pathways

Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement, and administration of DHEA produces a number of beneficial effects in the elderly. Many researchers have suggested that DHEA exerts it function after conversion into more biologically active hormones in peripheral target cells. The actions of DHEA in Leydig cells, a major target cell of DHEA biotransformation in males, are not clear. The present study found that DHEA increased cell viability and decreased reactive oxygen species (ROS) and malondialdehyde contents in H₂O₂-induced Leydig cells. DHEA significantly increased the activities of superoxide dismutase, catalase and peroxidase, and decreased the DNA damage in H₂O₂-induced Leydig cells. Apoptosis was significant decreased in H₂O₂-induced Leydig cells after DHEA treatment. DHEA inhibited the loss of mitochondrial membrane potential (ΔΨm) and the upregulation of the caspase-3 protein level induced by H₂O₂ in Leydig cells. DHEA also reversed the decrease in PI3K and p-Akt protein levels induced by H₂O₂. These data showed that DHEA could ameliorate H₂O₂-induced oxidative damage by increasing anti-oxidative enzyme activities, which resulted in reduced ROS content, and decreased apoptosis, mainly by preventing the loss of ΔΨm and inhibiting caspase-3 protein levels via activation of PI3K/Akt signaling pathways. These results increase our understanding of the molecular mechanism of the anti-ageing effect of DHEA.     

The role of insulin against hydrogen peroxide-induced oxidative damages in differentiated SH-SY5Y cells

Abstract

Exogenous hydrogen peroxide (H₂O₂) can easily penetrate into biological membranes and enhance the formation of other reactive oxygen species (ROS). In the present study, we have investigated the neuroprotective effects of insulin on H₂O₂-induced toxicity of retinoic acid (RA)-differentiated SH-SY5Y cells. To measure the changes in the cell viability of SH-SY5Y cells at different concentrations of H₂O₂ for 24?h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based assay was used and a 100?µM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 24?h of 100?µM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), ROS, and calcium ion (Ca²⁺) in neuronal cells, but insulin can effectively diminish the H₂O₂-induced oxidative damages to these cells. Moreover, cells treated with insulin increased H₂O₂-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of insulin treatment with SH-SY5Y cells increased the Bcl-2 levels and decreased the Akt levels. The treatment of insulin had played a protective effect on H₂O₂-induced oxidative stress related to the Akt/Bcl-2 pathways.     

An Integrated Mitochondrial ROS Production and Scavenging Model: Implications for Heart Failure

It has been observed experimentally that cells from failing hearts exhibit elevated levels of reactive oxygen species (ROS) upon increases in energetic workload. One proposed mechanism for this behavior is mitochondrial Ca²⁺ mismanagement that leads to depletion of ROS scavengers. Here, we present a computational model to test this hypothesis. Previously published models of ROS production and scavenging were combined and reparameterized to describe ROS regulation in the cellular environment. Extramitochondrial Ca²⁺ pulses were applied to simulate frequency-dependent changes in cytosolic Ca²⁺. Model results show that decreased mitochondrial Ca²⁺uptake due to mitochondrial Ca²⁺ uniporter inhibition (simulating Ru360) or elevated cytosolic Na⁺, as in heart failure, leads to a decreased supply of NADH and NADPH upon increasing cellular workload. Oxidation of NADPH leads to oxidation of glutathione (GSH) and increased mitochondrial ROS levels, validating the Ca²⁺ mismanagement hypothesis. The model goes on to predict that the ratio of steady-state [H₂O₂]_m during 3Hz pacing to [H₂O₂]_m at rest is highly sensitive to the size of the GSH pool. The largest relative increase in [H₂O₂]_m in response to pacing is shown to occur when the total GSH and GSSG is close to 1 mM, whereas pool sizes below 0.9 mM result in high resting H₂O₂ levels, a quantitative prediction only possible with a computational model.     

An Integrated Mitochondrial ROS Production and Scavenging Model: Implications for Heart Failure

It has been observed experimentally that cells from failing hearts exhibit elevated levels of reactive oxygen species (ROS) upon increases in energetic workload. One proposed mechanism for this behavior is mitochondrial Ca²⁺ mismanagement that leads to depletion of ROS scavengers. Here, we present a computational model to test this hypothesis. Previously published models of ROS production and scavenging were combined and reparameterized to describe ROS regulation in the cellular environment. Extramitochondrial Ca²⁺ pulses were applied to simulate frequency-dependent changes in cytosolic Ca²⁺. Model results show that decreased mitochondrial Ca²⁺uptake due to mitochondrial Ca²⁺ uniporter inhibition (simulating Ru360) or elevated cytosolic Na⁺, as in heart failure, leads to a decreased supply of NADH and NADPH upon increasing cellular workload. Oxidation of NADPH leads to oxidation of glutathione (GSH) and increased mitochondrial ROS levels, validating the Ca²⁺ mismanagement hypothesis. The model goes on to predict that the ratio of steady-state [H₂O₂]_m during 3Hz pacing to [H₂O₂]_m at rest is highly sensitive to the size of the GSH pool. The largest relative increase in [H₂O₂]_m in response to pacing is shown to occur when the total GSH and GSSG is close to 1 mM, whereas pool sizes below 0.9 mM result in high resting H₂O₂ levels, a quantitative prediction only possible with a computational model.     

Estrogen down-regulates uncoupling proteins and increases oxidative stress in breast cancer

Oxidative stress has been postulated as one of the mechanisms underlying the estrogen carcinogenic effect in breast cancer. Estrogens are known to increase mitochondrial-derived reactive oxygen species (ROS) by an unknown mechanism. Given that uncoupling proteins (UCPs) are key regulators of mitochondrial energy efficiency and ROS production, our aim was to check the presence and activity of UCPs in estrogen receptor (ER)-positive and ER-negative breast cancer cells and tumors, as well as their relation to oxidative stress. Estrogen (1 nM) induced higher oxidative stress in the ER-positive MCF-7 cell line, showing increased mitochondrial membrane potential, H₂O₂ levels, and DNA and protein damage compared to ER-negative MDA-MB-231 cells. All isoforms of uncoupling proteins were highly expressed in ER-positive breast cancer cells and tumors compared to negative ones. ROS production in mitochondria isolated from MCF-7 was increased by inhibition of UCPs with GDP, but not in mitochondria from MDA-MB-231. Estrogen treatment decreased uncoupling protein and catalase levels in MCF-7 and decreased GDP-dependent ROS production in isolated mitochondria. On the whole, these results suggest that estrogens, through an ER-dependent mechanism, may increase mitochondrial ROS production by repressing uncoupling proteins, which offers a new perspective on the understanding of why estrogens are a risk factor for breast cancer.     

Fasting induces cyanide-resistant respiration and oxidative stress in the amoeba Chaos carolinensis: implications for the cubic structural transition in mitochondrial membranes

Summary. Large free-living amoeba (Chaos carolinensis) can survive in spring water without food intake for several weeks. Starvation is associated with a dramatic change in mitochondrial cristae from random tubular to ordered (paracrystalline) cubic morphology. Whole-cell polarography was used to monitor changes in respiratory activity during fasting. Basal respiration per cell decreased progressively during starvation, while the cyanide-resistant fraction increased. Spectrofluorometric assay of H₂O₂ and reactive oxygen species (ROS) in cell lysates (using the dye 2′,7′-dichlorofluorescein diacetate) indicates greater H₂O₂ and ROS generation in starved than in fed cells. Fluorescence microscopy of intact cells incubated with the same dye demonstrates that H₂O₂ and ROS tend to accumulate in vacuoles. A remarkable generation of O₂ observed with starved cells after addition of KCN may be explained by release of H₂O₂ from these compartments into the cytosol, where it can react with catalase. Together, these observations suggest that fasting increases oxidative stress in the amoeba and that this organism has several protective mechanisms to deal with it, including activation of a plantlike alternative oxidase. The hypothesis is forwarded that the cubic structural transition of the mitochondrial inner membrane represents another protective mechanism, reducing oxidative damage by enhancing the efflux of H₂O₂ and ROS and by reducing the susceptibility of membrane lipids to the oxidants. Received September 1, 2001 Accepted January 7, 2002     

Oleic Acid and Eicosapentaenoic Acid Reverse Palmitic Acid-induced Insulin Resistance in Human HepG2 Cells via the Reactive Oxygen Species/JUN Pathway

Oleic acid (OA), a monounsaturated fatty acid (MUFA), has previously been shown to reverse saturated fatty acid palmitic acid (PA)-induced hepatic insulin resistance (IR). However, its underlying molecular mechanism is unclear. In addition, previous studies have shown that eicosapentaenoic acid (EPA), a ω-3 polyunsaturated fatty acid (PUFA), reverses PA-induced muscle IR, but whether EPA plays the same role in hepatic IR and its possible mechanism involved need to be further clarified. Here, we confirmed that EPA reversed PA-induced IR in HepG2 cells and compared the proteomic changes in HepG2 cells after treatment with different free fatty acids (FFAs). A total of 234 proteins were determined to be differentially expressed after PA+OA treatment. Their functions were mainly related to responses to stress and endogenous stimuli, lipid metabolic process, and protein binding. For PA+EPA treatment, the PA-induced expression changes of 1326 proteins could be reversed by EPA, 415 of which were mitochondrial proteins, with most of the functional proteins involved in oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle. Mechanistic studies revealed that the protein encoded by JUN and reactive oxygen species (ROS) play a role in OA- and EPA-reversed PA-induced IR, respectively. EPA and OA alleviated PA-induced abnormal adenosine triphosphate (ATP) production, ROS generation, and calcium (Ca²⁺) content. Importantly, H₂O₂-activated production of ROS increased the protein expression of JUN, further resulting in IR in HepG2 cells. Taken together, we demonstrate that ROS/JUN is a common response pathway employed by HepG2 cells toward FFA-regulated IR.     

High Dietary Fat Selectively Increases Catalase Expression within Cardiac Mitochondria

Obesity is a predictor of diabetes and cardiovascular disease. One consequence of obesity is dyslipidemia characterized by high blood triglycerides. It has been proposed that oxidative stress, driven by utilization of lipids for energy, contributes to these diseases. The effects of oxidative stress are mitigated by an endogenous antioxidant enzyme network, but little is known about its response to high fat utilization. Our experiments used a multiplexed quantitative proteomics method to measure antioxidant enzyme expression in heart tissue in a mouse model of diet-induced obesity. This experiment showed a rapid and specific up-regulation of catalase protein, with subsequent assays showing increases in activity and mRNA. Catalase, traditionally considered a peroxisomal protein, was found to be present in cardiac mitochondria and significantly increased in content and activity during high fat feeding. These data, coupled with the fact that fatty acid oxidation enhances mitochondrial H₂O₂ production, suggest that a localized catalase increase is needed to consume excessive mitochondrial H₂O₂ produced by increased fat metabolism. To determine whether the catalase-specific response is a common feature of physiological conditions that increase blood triglycerides and fatty acid oxidation, we measured changes in antioxidant expression in fasted versus fed mice. Indeed, a similar specific catalase increase was observed in mice fasted for 24 h. Our findings suggest a fundamental metabolic process in which catalase expression is regulated to prevent damage while preserving an H₂O₂-mediated sensing of diet composition that appropriately adjusts insulin sensitivity in the short term as needed to prioritize lipid metabolism for complete utilization.     

Overexpression of Peroxisomal Malate Dehydrogenase MDH3 Gene Enhances Cell Death on H 2O 2 Stress in the ald5 Mutant of Saccharomyces cerevisiae

Mitochondrial aldehyde dehydrogenase ALD5 of Saccharomyces cerevisiae is involved in the biosynthesis of mitochondrial electron transport chain, and the ald5 mutant is incompetent for respiration. With use of the mutant, we examined the detoxication of H₂O₂ generation by fatty acid -oxidation in peroxisome. The ald5 mutant (AKD321), as well as the 746 ⁰ mutant, was more resistant to H₂O₂ stress than the wild type. However, overexpression of the MDH3 gene that was involved in the reoxidation of NADH during fatty acid -oxidation caused a decrease in cell viability of AKD321 to H₂O₂ stress, while the 746 ⁰ mutant had no such effect. Intracellular H₂O₂ concentration increased approximately fourfold in MDH3 overexpressing ald5 strain (MD3-AKD321), compared with AKD321. The peroxisomal catalase activity of MD3-AKD321 decreased by 83% to that of AKD321. And also, the overexpression of MDH3 had only a weak effect in MDH3 overexpressing 746 ⁰ strain, decreasing by 14% to that of 746 ⁰ mutant. The increased palmitoyl CoA oxidation by overexpression of MDH3 gene was the same in both strains. Under conditions of MDH3 overexpression, peroxisomal catalase (CTA1) appears to be a limiting factor to oxidative stress. These observations point to an important, as yet unidentified, role of mitochondrial aldehyde dehydrogenase (ALD5) to endogeneous oxidative stress in peroxisome.Received: 23 September 2002 / Accepted: 24 October 2002     

Inhibition of CYP2E1 attenuates myocardial dysfunction in a murine model of insulin resistance through NLRP3-mediated regulation of mitophagy

Insulin resistance leads to myocardial contractile dysfunction and deranged autophagy although the underlying mechanism or targeted therapeutic strategy is still lacking. This study was designed to examine the impact of inhibition of the cytochrome P450 2E1 (CYP2E1) enzyme on myocardial function and mitochondrial autophagy (mitophagy) in an Akt2 knockout model of insulin resistance. Adult wild-type (WT) and Akt2^?/? mice were treated with the CYP2E1 inhibitor diallyl sulfide (100?mg/kg/d, i.p.) for 4?weeks. Cardiac geometry and function were assessed using echocardiographic and IonOptix systems. Western blot analysis was used to evaluate autophagy, mitophagy, inducible NOS (iNOS), and the NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex. Akt2 deletion triggered insulin resistance, compromised cardiac contractile and intracellular Ca²⁺ property, mitochondrial ultrastructural damage, elevated O2^– production, as well as suppressed autophagy and mitophagy, accompanied with elevated levels of NLRP3 and iNOS, the effects of which were significantly attenuated or ablated by diallyl sulfide. In vitro studies revealed that the NLRP3 activator nigericin nullified diallyl sulfide-offered benefit against Akt2 knockout on cardiomyocyte mechanical function and mitophagy (using Western blot and colocalization of GFP-LC3 and MitoTracker Red). Moreover, inhibition of iNOS but not mitochondrial ROS production attenuated Akt2 deletion-induced activation of NLRP3, substantiating a role for iNOS-mediated NLRP3 in insulin resistance-induced changes in mitophagy and cardiac dysfunction. In conclusion, these data depict that insulin resistance through CYP2E1 may contribute to the pathogenesis of myopathic changes including myocardial contractile dysfunction, oxidative stress and mitochondrial injury, possibly through activation of iNOS and NLRP3 signaling.     

Reduction of hydrogen peroxide stress derived from fatty acid beta-oxidation improves fatty acid utilization in Escherichia coli

Fatty acids are a promising raw material for substance production because of their highly reduced and anhydrous nature, which can provide higher fermentation yields than sugars. However, they are insoluble in water and are poorly utilized by microbes in industrial fermentation production. We used fatty acids as raw materials for l-lysine fermentation by emulsification and improved the limited fatty acid-utilization ability of Escherichia coli. We obtained a fatty acid-utilizing mutant strain by laboratory evolution and demonstrated that it expressed lower levels of an oxidative-stress marker than wild type. The intracellular hydrogen peroxide (H₂O₂) concentration of a fatty acid-utilizing wild-type E. coli strain was higher than that of a glucose-utilizing wild-type E. coli strain. The novel mutation rpsA ^D210Y identified in our fatty acid-utilizing mutant strain enabled us to promote cell growth, fatty-acid utilization, and l-lysine production from fatty acid. Introduction of this rpsA ^D210Y mutation into a wild-type strain resulted in lower H₂O₂ concentrations. The overexpression of superoxide dismutase (sodA) increased intracellular H₂O₂ concentrations and inhibited E. coli fatty-acid utilization, whereas overexpression of an oxidative-stress regulator (oxyS) decreased intracellular H₂O₂ concentrations and promoted E. coli fatty acid utilization and l-lysine production. Addition of the reactive oxygen species (ROS) scavenger thiourea promoted l-lysine production from fatty acids and decreased intracellular H₂O₂ concentrations. Among the ROS generated by fatty-acid β-oxidation, H₂O₂ critically affected E. coli growth and l-lysine production. This indicates that the regression of ROS stress promotes fatty acid utilization, which is beneficial for fatty acids used as raw materials in industrial production.