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Insulin resistance is one of the earliest pathological features of a suite of diseases including type 2 diabetes collectively referred to as metabolic syndrome. There is a growing body of evidence from both pre-clinical studies and human cohorts indicating that reactive oxygen species, such as the superoxide radical anion and hydrogen peroxide are key players in the development of insulin resistance. Here we review the evidence linking mitochondrial reactive oxygen species generated within mitochondria with insulin resistance in adipose tissue and skeletal muscle, two major insulin sensitive tissues. We outline the relevant mitochondria-derived reactive species, how the mitochondrial redox state is regulated, and methodologies available to measure mitochondrial reactive oxygen species. Importantly, we highlight key experimental issues to be considered when studying the role of mitochondrial reactive oxygen species in insulin resistance. Evaluating the available literature on both mitochondrial reactive oxygen species/redox state and insulin resistance in a variety of biological systems, we conclude that the weight of evidence suggests a likely role for mitochondrial reactive oxygen species in the etiology of insulin resistance in adipose tissue and skeletal muscle. However, major limitations in the methods used to study reactive oxygen species in insulin resistance as well as the lack of data linking mitochondrial reactive oxygen species and cytosolic insulin signaling pathways are significant obstacles in proving the mechanistic link between these two processes. We provide a framework to guide future studies to provide stronger mechanistic information on the link between mitochondrial reactive oxygen species and insulin resistance as understanding the source, localization, nature, and quantity of mitochondrial reactive oxygen species, their targets and downstream signaling pathways may pave the way for important new therapeutic strategies. [Display omitted] • Mitochondrial reactive oxygen species likely play a causal role in insulin resistance. • Assaying mitochondrial reactive oxygen species is challenging. • Mitochondrial lipids provide a link between reactive species and insulin resistance. • Novel therapies in insulin resistance may target mitochondrial reactive species formation. [ABSTRACT FROM AUTHOR]

Ubiquinone 8 (coenzyme Q8 or Q8) mediates electron transfer within the aerobic respiratory chain, mitigates oxidative stress, and contributes to gene expression in Escherichia coli. In addition, Q8 was proposed to confer bacterial osmotolerance by accumulating during growth at high osmotic pressure and altering membrane stability. The osmolyte trehalose and membrane lipid cardiolipin accumulate in E. coli cells cultivated at high osmotic pressure. Here,Q8deficiency impaired E. coli growth at low osmotic pressure and rendered growth osmotically sensitive. The Q8 deficiency impeded cellular O2 uptake and also inhibited the activities of two proton symporters, the osmosensing transporter ProP and the lactose transporter LacY. Q8 supplementation decreased membrane fluidity in liposomes, but did not affect ProP activity in proteoliposomes, which is respiration- independent. Liposomes and proteoliposomes prepared with E. coli lipids were used for these experiments. Similar oxygen uptake rates were observed for bacteria cultivated at low and high osmotic pressures. In contrast, respiration was dramatically inhibited when bacteria grown at the same low osmotic pressure were shifted to high osmotic pressure. Thus, respiration was restored during prolonged growth of E. coli at high osmotic pressure. Of note, bacteria cultivated at low and high osmotic pressures had similar Q8 concentrations. The protection of respiration was neither diminished by cardiolipin deficiency nor conferred by trehalose overproduction during growth at low osmotic pressure, but rather might be achieved by Q8-independent respiratory chain remodeling. We conclude that osmotolerance is conferred through Q8-independent protection of respiration, not by altering physical properties of the membrane. [ABSTRACT FROM AUTHOR]

Heme oxygenases are composed of two isozymes, Hmox1 and Hmox2, that catalyze the degradation of heme to carbon monoxide (CO), ferrous iron, and biliverdin, the latter of which is subsequently converted to bilirubin. While initially considered to be waste products, CO and biliverdin/bilirubin have been shown over the last 20 years to modulate key cellular processes, such as inflammation, cell proliferation, and apoptosis, as well as antioxidant defense. This shift in paradigm has led to the importance of heme oxygenases and their products in cell physiology now being well accepted. The identification of the two human cases thus far of heme oxygenase deficiency and the generation of mice deficient in Hmox1 or Hmox2 have reiterated a role for these enzymes in both normal cell function and disease pathogenesis, especially in the context of cardiovascular disease. This review covers the current knowledge on the function of both Hmox1 and Hmox2 at both a cellular and tissue level in the cardiovascular system. Initially, the roles of heme oxygenases in vascular health and the regulation of processes central to vascular diseases are outlined, followed by an evaluation of the role(s) of Hmox1 and Hmox2 in various diseases such as atherosclerosis, intimal hyperplasia, myocardial infarction, and angiogenesis. Finally, the therapeutic potential of heme oxygenases and their products are examined in a cardiovascular disease context, with a focus on how the knowledge we have gained on these enzymes may be capitalized in future clinical studies. [ABSTRACT FROM AUTHOR]

Coenzyme Q (CoQ) is an essential lipid of cells present in all cellular compartments. The functions of CoQ in mitochondrial respiration and as an antioxidant are established, although the lipid likely has additional, presently unknown, roles. While the therapeutic utility of CoQ10 supplements is recognized in the rare cases of primary CoQ10 deficiencies, a potential role for CoQ10 supplements in cardiovascular disease, particularly heart failure, has also been studied for over 40 years. This review summarizes our current knowledge in these areas derived from animal studies and human trials. Current evidence for a benefit of CoQ10 supplements in diseases other than primary CoQ10 deficiencies is insufficient. [ABSTRACT FROM AUTHOR]

Ageing cells undergo changes in redox homeostasis and acquire high levels of reactive oxygen species ( ROS). Because accumulation of ROS involves a change in redox state of cells, functions that are involved in setting redox and maintaining redox homeostasis are very relevant to an understanding of the possible roles of redox homeostasis and ROS in ageing. This review discusses these aspects of ROS in relation to replicative ageing in the model organism Saccharomyces cerevisiae, with reference to ROS generated in cells; cellular responses to oxidative stress; and how cells maintain redox homeostasis in different cellular compartments. It also considers when ROS generation begins as cells age, which ROS species are relevant to ageing and which cellular compartments and processes may contribute ROS to the ageing process. The discussion also covers the heterogeneity of cells with respect to ROS accumulation at particular cell ages, and the possibility of testing the oxidative theory of ageing in yeast cells. [ABSTRACT FROM AUTHOR]

Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (EGSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between redox-, oxidative- and environmental stress response systems. EGSH values of the cytosol, mitochondrial matrix and peroxisome were determined in exponential and stationary phase in various media. These values (−340 to −350 mV) were more reducing than previously reported. Interestingly, sub-cellular redox state remained unchanged when cells were challenged with stresses previously reported to affect redox homeostasis. Only hydrogen peroxide and heat stress significantly altered organellar redox state. Hydrogen peroxide stress altered the redox state of the glutathione disulfide/glutathione couple (GSSG, 2H+/2GSH) and pH. Recovery from moderate hydrogen peroxide stress was most rapid in the cytosol, followed by the mitochondrial matrix, with the peroxisome the least able to recover. Conversely, the bulk of the redox shift observed during heat stress resulted from alterations in pH and not the GSSG, 2H+/2GSH couple. This study presents the first direct measurement of pH-adjusted redox state in sub-cellular compartments during growth and stress conditions. Redox state is distinctly regulated in organelles and data presented challenge the notion that perturbation of redox state is central in the response to many stress conditions. [ABSTRACT FROM AUTHOR]

Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for maintenance of cytosolic redox state. Mutations in these genes led to shifts in the half-cell glutathione redox potential by 75-10 mV. Interestingly, some specific oxidative stress-response processes were identified as over-represented in the data set. Further investigation of the role of oxidative stress-responsive systems in sub-cellular redox homeostasis was conducted using roGFP2 constructs targeted to the mitochondrial matrix and peroxisome and EGSH was measured in cells in exponential and stationary phase. Analyses allowed for the identification of key redox systems on a sub-cellular level and the identification of novel genes involved in the regulation of cellular redox homeostasis. [ABSTRACT FROM AUTHOR]

Abstract: Glutathione (GSH) is a key redox buffer and protectant. Growth (approx. one or two divisions) of cells lacking γ-glutamylcysteine synthetase (gsh1) in the absence of GSH led to irreversible respiratory incompetency in all cells, and after five divisions 75% of cells completely lacked mitochondrial DNA (mtDNA). The level of GSH required to allow continuous growth was distinct from that required to prevent loss of mtDNA. GSH limitation led to a change in the transcript levels of 190 genes, including 30 genes regulated by the Aft1p and/or Aft2p transcription factors, which regulate the cellular response to changes in iron availability. Disruption of AFT1 but not AFT2 in gsh1 cells afforded a protective effect on maintenance of respiratory competency, as did overexpression of GRX3 or GRX4 (encoding monothiol glutaredoxins that act as negative regulators of Aft1p). Importantly, an iron-independent mechanism (~30%) was also observed to mediate GSH-dependent mtDNA loss. Analysis of the redox environment in the cytosol, mitochondrial matrix, and intermembrane space (IMS) found that the cytosol was most severely and rapidly affected by GSH depletion. GSH may also modulate the redox environment of the IMS. The implications of altered GSH homeostasis for maintenance of mtDNA, compartmental redox, and the pathophysiology of certain diseases are discussed. [ABSTRACT FROM AUTHOR]

Coenzyme Q (CoQ) is an essential redox-active lipid, and its deficiency is implicated in numerous diseases. However, it is largely unknown what regulates CoQ content. To investigate cellular CoQ regulation, we performed a genome-wide screen (~5,000 S. cerevisiae single gene mutants) to isolate genes critical for CoQ homeostasis, and measured CoQ content in each mutant using HPLC-EC detection. Some 30 knockout mutants were identified with significantly higher CoQ content compared to WT including the cho2 mutant. CHO2 encodes a phosphatidylethanolamine (PE) methyltransferase that catalyzes the conversion of PE to phosphatidylcholine (PC). Homologs of CHO2 are found in mammals (PEMT) indicating there may be role for this gene in CoQ regulation in other species. The cho2 mutant contained concentrations of CoQ five times greater than WT, and a significantly increased rate of CoQ synthesis. To investigate if Pemt–/– mice also display elevated CoQ content akin to yeast cho2 mutants, we analyzed the CoQ content in liver and adipose tissue from Pemt+/+ and Pemt–/– mice. In these tissues, CoQ content was approximately double in Pemt–/– compared to Pemt+/+. Taken together, our data suggests a novel role for phosphatidylethanolamine methyltransferases in CoQ content regulation. [ABSTRACT FROM AUTHOR]

Coenzyme Q (CoQ) is a redox-active lipid essential for mitochondrial energy generation, cellular redox regulation, antioxidant defense, apoptosis and cell death. Despite this, it is largely unknown what regulates the cellular CoQ content, how optimal CoQ content in different cellular membranes is regulated, and how this information is ‘fed back’ to the CoQ synthesis machinery. Understanding CoQ homeostasis is critical as CoQ deficiency is implicated in numerous diseases such as heart failure, and aging is associated with a decrease in tissue CoQ. Currently, CoQ supplements are essentially the only strategy available to overcome CoQ deficiencies. This strategy can be effective in cases of severe CoQ deficiency but the low bioavailability of supplemental CoQ for most tissues represents a significant limitation. Thus, a better understanding of key steps regulating CoQ content may provide the basis for novel strategies to enrich cellular CoQ content. To investigate CoQ regulation, we combined genetic and biochemical methods using the model organism S. cerevisiae. Carrying out a genome-wide screen (~6,000 mutants) to discover genes critical for CoQ content, over 100 genes were identified. Furthermore, the global transcriptional responses to changes in cellular CoQ content were determined by microarray analyses of WT and select CoQ biosynthesis mutants (coq1, 3, 7, 8 and 9). From the microarrays, coq1, 3 and 7 had similar transcriptional profiles, while coq8 and 9 clustered together indicating distinct transcriptional responses depending on how CoQ biosynthesis is disrupted. Interestingly, CoQ biosynthetic genes were not differentially expressed between the WT and coq mutants indicating that CoQ deficiency is not transcriptionally signaled. Similarly, antioxidant defense genes were not differentially expressed suggesting that in yeast CoQ is not required for steady-state maintenance of redox homeostasis. These and other results from the genome-wide screen will be presented, with the data providing novel insights into cellular CoQ regulation and function. [ABSTRACT FROM AUTHOR]

Coenzyme Q (Qn) is a vital lipid component of the electron transport chain that functions in cellular energy metabolism and as a membrane antioxidant. In the yeast Saccharomyces cerevisiae, coq1-coq9 deletion mutants are respiratory-incompetent, sensitive to lipid peroxidation stress, and unable to synthesize Q6. The yeast coq10 deletion mutant is also respiratorydeficient and sensitive to lipid peroxidation, yet it continues to produce Q6 at an impaired rate. Thus, Coq10 is required for the function of Q6 in respiration and as an antioxidant and is believed to chaperone Q6 from its site of synthesis to the respiratory complexes. In several fungi, Coq10 is encoded as a fusion polypeptide with Coq11, a recently identified protein of unknown function required for efficient Q6 biosynthesis. Because "fused" proteins are often involved in similar biochemical pathways, here we examined the putative functional relationship between Coq10 and Coq11 in yeast. We used plate growth and Seahorse assays and LC-MS/MS analysis to show that COQ11 deletion rescues respiratory deficiency, sensitivity to lipid peroxidation, and decreased Q6 biosynthesis of the coq10Δ mutant. Additionally, immunoblotting indicated that yeast coq11Δ mutants accumulate increased amounts of certain Coq polypeptides and display a stabilized CoQ synthome. These effects suggest that Coq11 modulates Q6 biosynthesis and that its absence increases mitochondrial Q6 content in the coq10Δcoq11Δ double mutant. This augmented mitochondrial Q6 content counteracts the respiratory deficiency and lipid peroxidation sensitivity phenotypes of the coq10Δ mutant. This study further clarifies the intricate connection between Q6 biosynthesis, trafficking, and function in mitochondrial metabolism. [ABSTRACT FROM AUTHOR]

Bilirubin, a product of heme catabolism, inhibits free radical chain reactions and protects against oxidant-induced damage in vitro and ex vivo. However, direct evidence for bilirubin’s antioxidant effect in vivo remains limited. As bilirubin is formed from biliverdin by biliverdin reductase, we generated biliverdin reductase-a gene knockout (Bvra–/–) mice to assess bilirubin as an endogenous antioxidant. Bvra–/– mice are born in expected Mendelian ratios and appear normal. Compared to littermate Bvra+/+ and Bvra+/– mice, bile of Bvra–/– mice is green and their plasma concentration of bilirubin is ~100-fold lower, while plasma biliverdin is increased ~25-fold. Bvra–/– and Bvra+/+ mice have comparable plasma lipid profiles and low-molecular weight antioxidant, i.e., ascorbic acid, -tocopherol and ubiquinol-9. However, Bvra–/– mice have higher concentrations of plasma cholesteryl ester hydroperoxides, and their erythrocyte peroxiredoxin 2 is more oxidized compared to controls. This implies that the absence of bilirubin increases endogenous oxidative stress in the blood. Since plasma concentrations of the antioxidant bilirubin inversely correlate with cardiovascular and metabolic diseases, we are currently using Bvra–/– mice to assess the direct link of bilirubin to these diseases. [ABSTRACT FROM AUTHOR]

Diabetes-induced cardiac complications include left ventricular (LV) dysfunction and heart failure. We previously demonstrated that LV phosphoinositide 3-kinase p110α (PI3K) protects the heart against diabetic cardiomyopathy, associated with reduced NADPH oxidase expression and activity. Conversely, in dominant negative PI3K(p110α) transgenic mice (dnPI3K), reduced cardiac PI3K signaling exaggerated diabetes-induced cardiomyopathy, associated with upregulated NADPH oxidase. The goal was to examine whether chronic supplementation with the antioxidant coenzyme Q 10 (CoQ 10 ) could attenuate LV superoxide and diabetic cardiomyopathy in a setting of impaired PI3K signaling. Diabetes was induced in 6-week-old nontransgenic and dnPI3K male mice via streptozotocin. After 4 weeks of diabetes, CoQ 10 supplementation commenced (10 mg/kg ip, 3 times/week, 8 weeks). At study end (12 weeks of diabetes), markers of LV function, cardiomyocyte hypertrophy, collagen deposition, NADPH oxidase, oxidative stress (3-nitrotyrosine), and concentrations of CoQ 9 and CoQ 10 were determined. LV NADPH oxidase (Nox2 gene expression and activity, and lucigenin-enhanced chemiluminescence), as well as oxidative stress, were increased by diabetes, exaggerated in diabetic dnPI3K mice, and attenuated by CoQ 10 . Diabetes-induced LV diastolic dysfunction (prolonged deceleration time, elevated end-diastolic pressure, impaired E/A ratio), cardiomyocyte hypertrophy and fibrosis, expression of atrial natriuretic peptide, connective tissue growth factor, and β-myosin heavy chain were all attenuated by CoQ 10 . Chronic CoQ 10 supplementation attenuates aspects of diabetic cardiomyopathy, even in a setting of reduced cardiac PI3K protective signaling. Given that CoQ 10 supplementation has been suggested to have positive outcomes in heart failure patients, chronic CoQ 10 supplementation may be an attractive adjunct therapy for diabetic heart failure. [ABSTRACT FROM AUTHOR]