Independent, but not co-supplementation, with nitrate and resveratrol improves glucose tolerance and reduces markers of cellular stress in high-fat-fed male mice.

Independent supplementation with nitrate (NIT) and resveratrol (RSV) enriches various aspects of mitochondrial biology in key metabolic tissues. Although RSV is known to activate Sirt1 and initiate mitochondrial biogenesis, the metabolic benefits elicited by dietary nitrate appear to be dependent on 5'-adenosine monophosphate-activated protein kinase (AMPK)-mediated signaling events, a process also linked to the activation of Sirt1. Although the benefits of individual supplementation with these compounds have been characterized, it is unknown if co-supplementation may produce superior metabolic adaptations. Thus, we aimed to determine if treatment with combined +NIT and +RSV (+RN) could additively alter metabolic adaptations in the presence of a high-fat diet (HFD). Both +RSV and +NIT improved glucose tolerance compared with HFD (P < 0.05); however, this response was attenuated following combined +RN supplementation. Within skeletal muscle, all supplements increased mitochondrial ADP sensitivity compared with HFD (P < 0.05), without altering mitochondrial content. Although +RSV and +NIT decreased hepatic lipid deposition compared with HFD (P < 0.05), this effect was abolished with +RN, which aligned with significant reductions in Sirt1 protein content (P < 0.05) after combined treatment, in the absence of changes to mitochondrial content or function. Within epididymal white adipose tissue (eWAT), all supplements reduced crown-like structure accumulation compared with HFD (P < 0.0001) and mitochondrial reactive oxygen species (ROS) emission (P < 0.05), alongside reduced adipocyte cross-sectional area (CSA) (P < 0.05), with the greatest effect observed after +RN treatment (P = 0.0001). Although the present data suggest additive changes in adipose tissue metabolism after +RN treatment, concomitant impairments in hepatic lipid homeostasis appear to prevent improvements in whole body glucose homeostasis observed with independent treatment, which may be Sirt1 dependent.

Reactive oxygen species-dependent regulation of pyruvate dehydrogenase kinase-4 in white adipose tissue.

Reactive oxygen species (ROS) are important signaling molecules mediating the exercise-induced adaptations in skeletal muscle. Acute exercise also drives the expression of genes involved in reesterification and glyceroneogenesis in white adipose tissue (WAT), but whether ROS play any role in this effect has not been explored. We speculated that exercise-induced ROS would regulate acute exercise-induced responses in WAT. To address this question, we utilized various models to alter redox signaling in WAT. We examined basal and exercise-induced gene expression in a genetically modified mouse model of reduced mitochondrial ROS emission [mitochondrial catalase overexpression (MCAT)]. Additionally, H2O2, various antioxidants, and the ß3-adrenergic receptor agonist CL316243 were used to assess gene expression in white adipose tissue culture. MCAT mice have reduced ROS emission from WAT, enlarged WAT depots and adipocytes, and greater pyruvate dehydrogenase kinase-4 (Pdk4) gene expression. In WAT culture, H2O2 reduced glyceroneogenic gene expression. In wild-type mice, acute exercise induced dramatic but transient increases in Pdk4 and phosphoenolpyruvate carboxykinase (Pck1) mRNA in both subcutaneous inguinal WAT and epididymal WAT depots, which was almost completely absent in MCAT mice. Furthermore, the induction of Pdk4 and Pck1 in WAT culture by CL316243 was markedly reduced in the presence of antioxidants N-acetyl-cysteine or vitamin E. Genetic and nutritional approaches that attenuate redox signaling prevent exercise- and ß-agonist-induced gene expression within WAT. Combined, these data suggest that ROS represent important mediators of gene expression within WAT.

Nitrate attenuates high fat diet-induced glucose intolerance in association with reduced epididymal adipose tissue inflammation and mitochondrial reactive oxygen species emission.

KEY POINTS: Dietary nitrate is a prominent therapeutic strategy to mitigate some metabolic deleterious effects related to obesity. Mitochondrial dysfunction is causally linked to adipose tissue inflammation and insulin resistance. Whole-body glucose tolerance is prevented by nitrate independent of body weight and energy expenditure. Dietary nitrate reduces epididymal adipose tissue inflammation and mitochondrial reactive oxygen species emission while preserving insulin signalling. Metabolic beneficial effects of nitrate consumption are associated with improvements in mitochondrial redox balance in hypertrophic adipose tissue. ABSTRACT: Evidence has accumulated to indicate that dietary nitrate alters energy expenditure and the metabolic derangements associated with a high fat diet (HFD), but the mechanism(s) of action remain incompletely elucidated. Therefore, we aimed to determine if dietary nitrate (4 mm sodium nitrate via drinking water) could prevent HFD-mediated glucose intolerance in association with improved mitochondrial bioenergetics within both white (WAT) and brown (BAT) adipose tissue in mice. HFD feeding caused glucose intolerance (P < 0.05) and increased body weight. As a result of higher body weight, energy expenditure increased proportionally. HFD-fed mice displayed greater mitochondrial uncoupling and a twofold increase in uncoupling protein 1 content within BAT. Within epididymal white adipose tissue (eWAT), HFD increased cell size (i.e. hypertrophy), mitochondrial H2 O2 emission, oxidative stress, c-Jun N-terminal kinase phosphorylation and leucocyte infiltration, and induced insulin resistance. Remarkably, dietary nitrate consumption attenuated and/or mitigated all these responses, including rendering mitochondria more coupled within BAT, and normalizing mitochondrial H2 O2 emission and insulin-mediated Akt-Thr308 phosphorylation within eWAT. Intriguingly, the positive effects of dietary nitrate appear to be independent of eWAT mitochondrial respiratory capacity and content. Altogether, these data suggest that dietary nitrate attenuates the development of HFD-induced insulin resistance in association with attenuating WAT inflammation and redox balance, independent of changes in either WAT or BAT mitochondrial respiratory capacity/content.

Ablating the Rab-GTPase activating protein TBC1D1 predisposes rats to high-fat diet-induced cardiomyopathy.

KEY POINTS: Although the role of TBC1D1 within the heart remains unknown, expression of TBC1D1 increases in the left ventricle following an acute infarction, suggesting a biological importance within this tissue. We investigated the mechanistic role of TBC1D1 within the heart, aiming to establish the consequences of attenuating TBC1D1 signalling in the development of diabetic cardiomyopathy, as well as to determine potential sex differences. TBC1D1 ablation increased plasma membrane fatty acid binding protein content and myocardial palmitate oxidation. Following high-fat feeding, TBC1D1 ablation dramatically increased fibrosis and induced end-diastolic dysfunction in both male and female rats in the absence of changes in mitochondrial bioenergetics. Altogether, independent of sex, ablating TBC1D1 predisposes the left ventricle to pathological remodelling following high-fat feeding, and suggests TBC1D1 protects against diabetic cardiomyopathy. ABSTRACT: TBC1D1, a Rab-GTPase activating protein, is involved in the regulation of glucose handling and substrate metabolism within skeletal muscle, and is essential for maintaining pancreatic ß-cell mass and insulin secretion. However, the function of TBC1D1 within the heart is largely unknown. Therefore, we examined the role of TBC1D1 in the left ventricle and the functional consequence of ablating TBC1D1 on the susceptibility to high-fat diet-induced abnormalities. Since mutations within TBC1D1 (R125W) display stronger associations with clinical parameters in women, we further examined possible sex differences in the predisposition to diabetic cardiomyopathy. In control-fed animals, TBC1D1 ablation did not alter insulin-stimulated glucose uptake, or echocardiogram parameters, but increased accumulation of a plasma membrane fatty acid transporter and the capacity for palmitate oxidation. When challenged with an 8 week high-fat diet, TBC1D1 knockout rats displayed a four-fold increase in fibrosis compared to wild-type animals, and this was associated with diastolic dysfunction, suggesting a predisposition to diet-induced cardiomyopathy. Interestingly, high-fat feeding only induced cardiac hypertrophy in male TBC1D1 knockout animals, implicating a possible sex difference. Mitochondrial respiratory capacity and substrate sensitivity to pyruvate and ADP were not altered by diet or TBC1D1 ablation, nor were markers of oxidative stress, or indices of overt heart failure. Altogether, independent of sex, ablation of TBC1D1 not only increased the susceptibility to high-fat diet-induced diastolic dysfunction and left ventricular fibrosis, independent of sex, but also predisposed male animals to the development of cardiac hypertrophy. These data suggest that TBC1D1 may exert cardioprotective effects in the development of diabetic cardiomyopathy.

Blood flow restricted resistance exercise and reductions in oxygen tension attenuate mitochondrial H2 O2 emission rates in human skeletal muscle.

KEY POINTS: Blood flow restricted resistance exercise (BFR-RE) is capable of inducing comparable adaptations to traditional resistance exercise (RE), despite a lower total exercise volume. It has been suggested that an increase in reactive oxygen species (ROS) production may be involved in this response; however, oxygen partial pressure ( PO2 ) is reduced during BFR-RE, and the influence of PO2 on mitochondrial redox balance remains poorly understood. In human skeletal muscle tissue, we demonstrate that both maximal and submaximal mitochondrial ROS emission rates are acutely decreased 2 h following BFR-RE, but not RE, occurring along with a reduction in tissue oxygenation during BFR-RE. We further suggest that PO2 is involved in this response because an in vitro analysis revealed that reducing PO2 dramatically decreased mitochondrial ROS emissions and electron leak to ROS. Altogether, these data indicate that mitochondrial ROS emission rates are attenuated following BFR-RE, and such a response is likely influenced by reductions in PO2 . ABSTRACT: Low-load blood flow restricted resistance exercise (BFR-RE) training has been proposed to induce comparable adaptations to traditional resistance exercise (RE) training, however, the acute signalling events remain unknown. Although a suggested mechanism of BFR-RE is an increase in reactive oxygen species (ROS) production, oxygen partial pressure ( PO2 ) is reduced during BFR-RE, and the influence of O2 tension on mitochondrial redox balance remains ambiguous. We therefore aimed to determine whether skeletal muscle mitochondrial bioenergetics were altered following an acute bout of BFR-RE or RE, and to further examine the role of PO2 in this response. Accordingly, muscle biopsies were obtained from 10 males at rest and 2 h after performing three sets of single-leg squats (RE or BFR-RE) to failure at 30% one-repetition maximum. We determined that mitochondrial respiratory capacity and ADP sensitivity were not altered in response to RE or BFR-RE. Although maximal (succinate) and submaximal (non-saturating ADP) mitochondrial ROS emission rates were unchanged following RE, BFR-RE attenuated these responses by ∼30% compared to pre-exercise, occurring along with a reduction in skeletal muscle tissue oxygenation during BFR-RE (P < 0.01 vs. RE). In a separate cohort of participants, evaluation of mitochondrial bioenergetics in vitro revealed that mild O2 restriction (50 µm) dramatically attenuated maximal (∼4-fold) and submaximal (∼50-fold) mitochondrial ROS emission rates and the fraction of electron leak to ROS compared to room air (200 µm). Combined, these data demonstrate that mitochondrial ROS emissions are attenuated following BFR-RE, a response which may be mediated by a reduction in skeletal muscle PO2 .

Mitochondrial-derived reactive oxygen species influence ADP sensitivity, but not CPT-I substrate sensitivity.

The mechanisms regulating oxidative phosphorylation during exercise remain poorly defined; however, key mitochondrial proteins, including carnitine palmitoyltransferase-I (CPT-I) and adenine nucleotide translocase, have redox-sensitive sites. Interestingly, muscle contraction has recently been shown to increase mitochondrial membrane potential and reactive oxygen species (ROS) production; therefore, we aimed to determine if mitochondrial-derived ROS influences bioenergetic responses to exercise. Specifically, we examined the influence of acute exercise on mitochondrial bioenergetics in WT (wild type) and transgenic mice (MCAT, mitochondrial-targeted catalase transgenic) possessing attenuated mitochondrial ROS. We found that ablating mitochondrial ROS did not alter palmitoyl-CoA (P-CoA) respiratory kinetics or influence the exercise-mediated reductions in malonyl CoA sensitivity, suggesting that mitochondrial ROS does not regulate CPT-I. In contrast, while mitochondrial protein content, maximal coupled respiration, and ADP (adenosine diphosphate) sensitivity in resting muscle were unchanged in the absence of mitochondrial ROS, exercise increased the apparent ADP Km (decreased ADP sensitivity) ∼30% only in WT mice. Moreover, while the presence of P-CoA decreased ADP sensitivity, it did not influence the basic response to exercise, as the apparent ADP Km was increased only in the presence of mitochondrial ROS. This basic pattern was also mirrored in the ability of ADP to suppress mitochondrial H2O2 emission rates, as exercise decreased the suppression of H2O2 only in WT mice. Altogether, these data demonstrate that while exercise-induced mitochondrial-derived ROS does not influence CPT-I substrate sensitivity, it inhibits ADP sensitivity independent of P-CoA. These data implicate mitochondrial redox signaling as a regulator of oxidative phosphorylation.

α-Linolenic acid and exercise training independently, and additively, decrease blood pressure and prevent diastolic dysfunction in obese Zucker rats.

KEY POINTS: α-linolenic acid (ALA) and exercise training both attenuate hyperlipidaemia-related cardiovascular derangements, however, there is a paucity of information pertaining to their mechanisms of action when combined. We investigated both the independent and combined effects of exercise training and ALA consumption in obese Zucker rats, aiming to determine the potential for additive improvements in cardiovascular function. ALA and exercise training independently improved cardiac output, end-diastolic volume, left ventricular fibrosis and mean blood pressure following a 4 week intervention. Combining ALA and endurance exercise yielded greater improvements in these parameters, independent of changes in markers of oxidative stress or endogenous anti-oxidants. We postulate that divergent mechanisms of action may explain these changes: ALA increases peripheral vasodilation, and exercise training stimulates angiogenesis. ABSTRACT: Although α-linolenic acid (ALA) and endurance exercise training independently attenuate hyperlipidaemia-related cardiovascular derangements, there is a paucity of information pertaining to their mechanisms of action and efficacy when combined as a preventative therapeutic approach. Therefore, we used obese Zucker rats to investigate the independent and combined effects of these interventions on cardiovascular disease. Specifically, animals were randomly assigned to one of the following groups: control diet-sedentary, ALA supplemented-sedentary, control diet-exercise trained or ALA supplemented-exercise trained. Following a 4 week intervention, although the independent and combined effects of ALA and exercise reduced (P < 0.05) the serum free/esterified cholesterol ratio, only the ALA supplemented-exercise trained animals displayed a reduction in the content of both serum free and esterified cholesterol. Moreover, although ALA and endurance training individually increased cardiac output, stroke volume and end-diastolic volume, as well as reduced left ventricle fibrosis, mean blood pressure and total peripheral resistance, these responses were all greater following the combined intervention (ALA supplemented-exercise trained). These effects occurred independent of changes in oxidative phosphorylation proteins, markers of oxidative stress or endogenous anti-oxidant capacity. We propose that the beneficial effects of a combined intervention occur as a result of divergent mechanisms of action elicited by ALA and endurance exercise because only exercise training increased the capillary content in the left ventricle and skeletal muscle, and tended to decrease protein carbonylation in the left ventricle (P = 0.06). Taken together, our data indicate that combining ALA and endurance exercise provides additional improvements in cardiovascular disease risk reduction compared to singular interventions in the obese Zucker rat.

Dietary α-linolenic acid supplementation alters skeletal muscle plasma membrane lipid composition, sarcolemmal FAT/CD36 abundance, and palmitate transport rates.

The cellular processes influenced by consuming polyunsaturated fatty acids remains poorly defined. Within skeletal muscle, a rate-limiting step in fatty acid oxidation is the movement of lipids across the sarcolemmal membrane, and therefore, we aimed to determine the effects of consuming flaxseed oil high in α-linolenic acid (ALA), on plasma membrane lipid composition and the capacity to transport palmitate. Rats fed a diet supplemented with ALA (10%) displayed marked increases in omega-3 polyunsaturated fatty acids (PUFAs) within whole muscle and sarcolemmal membranes (approximately five-fold), at the apparent expense of arachidonic acid (-50%). These changes coincided with increased sarcolemmal palmitate transport rates (+20%), plasma membrane fatty acid translocase (FAT/CD36; +20%) abundance, skeletal muscle triacylglycerol content (approximately twofold), and rates of whole body fat oxidation (~50%). The redistribution of FAT/CD36 to the plasma membrane could not be explained by increased phosphorylation of signaling pathways implicated in regulating FAT/CD36 trafficking events (i.e., phosphorylation of ERK1/2, CaMKII, AMPK, and Akt), suggesting the increased n-3 PUFA composition of the plasma membrane influenced FAT/CD36 accumulation. Altogether, the present data provide evidence that a diet supplemented with ALA increases the transport of lipids into resting skeletal muscle in conjunction with increased sarcolemmal n-3 PUFA and FAT/CD36 contents.

Dietary Nitrate and Corresponding Gut Microbiota Prevent Cardiac Dysfunction in Obese Mice.

Impaired heart function can develop in individuals with diabetes in the absence of coronary artery disease or hypertension, suggesting mechanisms beyond hypertension/increased afterload contribute to diabetic cardiomyopathy. Identifying therapeutic approaches that improve glycemia and prevent cardiovascular disease are clearly required for clinical management of diabetes-related comorbidities. Since intestinal bacteria are important for metabolism of nitrate, we examined whether dietary nitrate and fecal microbial transplantation (FMT) from nitrate-fed mice could prevent high-fat diet (HFD)-induced cardiac abnormalities. Male C57Bl/6N mice were fed a low-fat diet (LFD), HFD, or HFD+Nitrate (4 mmol/L sodium nitrate) for 8 weeks. HFD-fed mice presented with pathological left ventricle (LV) hypertrophy, reduced stroke volume, and increased end-diastolic pressure, in association with increased myocardial fibrosis, glucose intolerance, adipose inflammation, serum lipids, LV mitochondrial reactive oxygen species (ROS), and gut dysbiosis. In contrast, dietary nitrate attenuated these detriments. In HFD-fed mice, FMT from HFD+Nitrate donors did not influence serum nitrate, blood pressure, adipose inflammation, or myocardial fibrosis. However, microbiota from HFD+Nitrate mice decreased serum lipids, LV ROS, and similar to FMT from LFD donors, prevented glucose intolerance and cardiac morphology changes. Therefore, the cardioprotective effects of nitrate are not dependent on reducing blood pressure, but rather mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis. ARTICLE HIGHLIGHTS: Identifying therapeutic approaches that prevent cardiometabolic diseases are clearly important, and nitrate represents one such potential compound given its multifactorial metabolic effects. We aimed to determine whether nitrate could prevent high-fat diet (HFD)-induced cardiac abnormalities and whether this was dependent on the gut microbiome. Dietary nitrate attenuated HFD-induced pathological changes in cardiac remodelling, left ventricle reactive oxygen species, adipose inflammation, lipid homeostasis, glucose intolerance, and gut dysbiosis. Fecal microbial transplantation from nitrate-fed mice also prevented serum dyslipidemia, left ventricle reactive oxygen species, glucose intolerance, and cardiac dysfunction. Therefore, the cardioprotective effects of nitrate are related to mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis.

Dietary nitrate does not alter cardiac function, calcium handling proteins, or SERCA activity in the left ventricle of healthy rats.

Dietary nitrate has been shown to increase cytosolic calcium concentrations within the heart, which would necessitate greater calcium sequestration for relaxation. In the present study we demonstrate that while nitrate supplementation reduced blood pressure, calcium-handling protein content, sarco(endo)plasmic reticulum Ca-ATPase 2a (SERCA) enzymatic properties, and left ventricular function were not altered. In addition, nitrite did not alter in vitro SERCA activity. Combined, these data suggest that in healthy rats, dietary nitrate does not increase left ventricle SERCA-related calcium-handling properties. Novelty Dietary nitrate decreases blood pressure but does not alter left ventricular calcium-handling protein content or SERCA activity in healthy rats.