The liver is particularly susceptible to the detrimental effects of a high fat diet (HFD), rapidly developing lipid accumulation and impaired cellular homeostasis. Recently, dietary nitrate has been shown to attenuate HFD-induced whole body glucose intolerance and liver steatosis, however the underlying mechanism(s) remain poorly defined. In the current study we investigated the ability of dietary nitrate to minimize possible impairments in liver mitochondrial bioenergetics following 8 wk of HFD (60% fat) in male C57BL/6J mice. Consumption of a HFD caused whole-body glucose intolerance (p<0.0001), and within the liver, increased lipid accumulation (p<0.0001), mitochondrial-specific reactive oxygen species emission (p=0.007), and markers of oxidative stress. Remarkably, dietary nitrate attenuated almost all of these pathological responses. Despite the reduction in lipid accumulation and redox stress (reduced TBARS and nitrotyrosine), nitrate did not improve insulin signaling within the liver or whole-body pyruvate tolerance (p=0.313 HFD vs HFD+nitrate). Moreover, the beneficial effects of nitrate were independent of changes in weight gain, 5' AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) signaling, mitochondrial content, mitochondrial respiratory capacity and ADP sensitivity or antioxidant protein content. Combined, these data suggest nitrate supplementation represents a potential therapeutic strategy to attenuate hepatic lipid accumulation and decrease mitochondrial ROS emission following HFD, processes linked to improvements in whole-body glucose tolerance. However, the beneficial effects of nitrate within the liver do not appear to be a result of increased oxidative capacity or mitochondrial substrate sensitivity.

Within brown adipose tissue (BAT), the brain isoform of creatine kinase (CKB) has been proposed to regulate the regeneration of ADP and phosphocreatine in a futile creatine cycle (FCC) that stimulates energy expenditure. However, the presence of FCC, and the specific creatine kinase isoforms regulating this theoretical model within white adipose tissue (WAT), remains to be fully elucidated. In the present study, creatine did not stimulate respiration in cultured adipocytes, isolated mitochondria or mouse permeabilized WAT. Additionally, while creatine kinase ubiquitous-type, mitochondrial (CKMT1) mRNA and protein were detected in human WAT, shRNA-mediated reductions in Ckmt1 did not decrease submaximal respiration in cultured adipocytes, and ablation of CKMT1 in mice did not alter energy expenditure, mitochondrial responses to pharmacological β3-adrenergic activation (CL 316, 243) or exacerbate the detrimental metabolic effects of consuming a high-fat diet. Taken together, these findings solidify CKMT1 as dispensable in the regulation of energy expenditure, and unlike in BAT, they do not support the presence of FCC within WAT.

Independent supplementation with nitrate (NIT) and resveratrol (RSV) enrich various aspects of mitochondrial biology in key metabolic tissues. While RSV is known to activate Sirt1 and initiate mitochondrial biogenesis, the metabolic benefits elicited by dietary nitrate appear to be dependent on AMPK-mediated signalling events, a process also linked to the activation of Sirt1. While the benefits of individual supplementation with these compounds has 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 to HFD (p<0.05), however, this response was attenuated following combined +RN supplementation. Within skeletal muscle, all supplements increased mitochondrial ADP-sensitivity compared to HFD (p<0.05), without altering mitochondrial content. While +RSV and +NIT decreased hepatic lipid deposition compared to HFD (p<0.05), this effect was abolished with +RN, which aligned with significant reductions in Sirt1 protein content (p<0.05) following combined treatment, in the absence of changes to mitochondrial content or function. Within eWAT, all supplements reduced crown-like structure accumulation compared to HFD (p<0.0001) and mitochondrial ROS emission (p<0.05), alongside reduced adipocyte CSA (p<0.05), with the greatest effect observed following +RN treatment (p=0.0001). While the present data suggests additive changes in adipose tissue metabolism following +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.

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Introduction: The liver is particularly susceptible to the detrimental effects of a high fat diet (HFD), and rapidly develops insulin resistance. While the underlying mechanisms associated with hepatic insulin resistance are not fully understood, mitochondria play a crucial role in regulating lipid metabolism and redox balance. Recently, dietary nitrate was shown to attenuate HFD-induced glucose intolerance, however the mechanism(s) remains unknown. Therefore, in the present study we examined the possibility that dietary nitrate increased mitochondrial lipid-supported respiration and/or attenuated reactive oxygen species (ROS) emission. Methods: Male C57Bl/6J mice (n = 22) were randomly assigned to consume a control diet (10% fat), HFD (60% fat), or HFD with 4mM sodium nitrate in drinking water for 8 weeks. Whole body glucose tolerance and energy expenditure (indirect calorimetry) were determined, and mitochondrial respiration and reactive oxygen species (ROS) emission were assessed in liver samples. Results: Compared to control animals, the consumption of HFD increased whole body fat oxidation and body mass, and induced glucose intolerance. The consumption of dietary nitrate did not affect HFD-mediated changes in energy expenditure, rates of fat oxidation or body mass, but nevertheless attenuated the induction of glucose intolerance. Within the liver, HFD-feeding in the presence or absence of dietary nitrate, did not alter pyruvate or lipid (palmitoyl-CoA) supported respiration, or the sensitivity to malonyl-CoA-mediated inhibition of lipid-supported respiration. In contrast, while HFD increased the endogenous ROS production, nitrate prevented this response. Conclusion: These data demonstrate that nitrate supplementation attenuated the HFD-mediated induction of whole-body glucose intolerance in association with a reduction in ROS emission within the liver. Future research needs to determine causality between these observations. Disclosure G. DesOrmeaux: None. H.L. Petrick: None. H. Brunetta: None. G. Holloway: None. Funding Natural Sciences and Engineering Research Council of Canada (400362)