Publications by authors named "Graham P Holloway"

111 Publications

Adipose Tissue Inflammation Is Directly Linked to Obesity-Induced Insulin Resistance, while Gut Dysbiosis and Mitochondrial Dysfunction Are Not Required.

Function (Oxf) 2020 25;1(2):zqaa013. Epub 2020 Aug 25.

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada.

Obesity is associated with adipose tissue hypertrophy, systemic inflammation, mitochondrial dysfunction, and intestinal dysbiosis. Rodent models of high-fat diet (HFD)-feeding or genetic deletion of multifunctional proteins involved in immunity and metabolism are often used to probe the etiology of obesity; however, these models make it difficult to divorce the effects of obesity, diet composition, or immunity on endocrine regulation of blood glucose. We, therefore, investigated the importance of adipose inflammation, mitochondrial dysfunction, and gut dysbiosis for obesity-induced insulin resistance using a spontaneously obese mouse model. We examined metabolic changes in skeletal muscle, adipose tissue, liver, the intestinal microbiome, and whole-body glucose control in spontaneously hyperphagic C57Bl/6J mice compared to lean littermates. A separate subset of lean and obese mice was subject to 8 weeks of obesogenic HFD feeding, or to pair feeding of a standard rodent diet. Hyperphagia, obesity, adipose inflammation, and insulin resistance were present in obese mice despite consuming a standard rodent diet, and these effects were blunted with caloric restriction. However, hyperphagic obese mice had normal mitochondrial respiratory function in all tissues tested and no discernable intestinal dysbiosis relative to lean littermates. In contrast, feeding mice an obesogenic HFD altered the composition of the gut microbiome, impaired skeletal muscle mitochondrial bioenergetics, and promoted poor glucose control. These data show that adipose inflammation and redox stress occurred in all models of obesity, but gut dysbiosis and mitochondrial respiratory dysfunction are not always required for obesity-induced insulin resistance. Rather, changes in the intestinal microbiome and mitochondrial bioenergetics may reflect physiological consequences of HFD feeding.
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http://dx.doi.org/10.1093/function/zqaa013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8276887PMC
August 2020

Independent of mitochondrial respiratory function, dietary nitrate attenuates HFD-induced lipid accumulation and mitochondrial ROS emission within the liver.

Am J Physiol Endocrinol Metab 2021 Aug 6;321(2):E217-E228. Epub 2021 Jul 6.

Human Health and Nutritional Sciences, grid.34429.38University of Guelph, Guelph, Ontario, Canada.

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 ( < 0.0001), and within the liver, increased lipid accumulation ( < 0.0001), mitochondrial-specific reactive oxygen species emission ( = 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 ( = 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. The mechanism(s) for how dietary nitrate prevents high-fat diet (HFD)-induced glucose intolerance remain poorly defined. We show that dietary nitrate attenuates HFD-induced increases in lipid accumulation, mitochondrial-specific reactive oxygen species (ROS) emission, and markers of oxidative stress within the liver. The beneficial effects of nitrate were independent of changes 5' AMP-activated protein kinase signaling, mitochondrial content/respiratory capacity, or lipid-supported respiratory sensitivity. Combined, these data provide potential mechanisms underlying the therapeutic potential of dietary nitrate.
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http://dx.doi.org/10.1152/ajpendo.00610.2020DOI Listing
August 2021

Insulin rapidly increases skeletal muscle mitochondrial ADP sensitivity in the absence of a high lipid environment.

Biochem J 2021 Jul;478(13):2539-2553

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Canada.

Reductions in mitochondrial function have been proposed to cause insulin resistance, however the possibility that impairments in insulin signaling negatively affects mitochondrial bioenergetics has received little attention. Therefore, we tested the hypothesis that insulin could rapidly improve mitochondrial ADP sensitivity, a key process linked to oxidative phosphorylation and redox balance, and if this phenomenon would be lost following high-fat diet (HFD)-induced insulin resistance. Insulin acutely (60 min post I.P.) increased submaximal (100-1000 µM ADP) mitochondrial respiration ∼2-fold without altering maximal (>1000 µM ADP) respiration, suggesting insulin rapidly improves mitochondrial bioenergetics. The consumption of HFD impaired submaximal ADP-supported respiration ∼50%, however, despite the induction of insulin resistance, the ability of acute insulin to stimulate ADP sensitivity and increase submaximal respiration persisted. While these data suggest that insulin mitigates HFD-induced impairments in mitochondrial bioenergetics, the presence of a high intracellular lipid environment reflective of an HFD (i.e. presence of palmitoyl-CoA) completely prevented the beneficial effects of insulin. Altogether, these data show that while insulin rapidly stimulates mitochondrial bioenergetics through an improvement in ADP sensitivity, this phenomenon is possibly lost following HFD due to the presence of intracellular lipids.
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http://dx.doi.org/10.1042/BCJ20210264DOI Listing
July 2021

Supplemental Fiber Affects Body Temperature and Fecal Metabolites but Not Respiratory Rate or Body Composition in Mid-Distance Training Sled Dogs.

Front Vet Sci 2021 29;8:639335. Epub 2021 Apr 29.

Department of Animal Biosciences, University of Guelph, Guelph, ON, Canada.

Dietary fiber affects canine physiology in many ways, such as increasing colonic absorption of water and improving gut health, both of which may positively impact exercise performance. The objectives of this study were to investigate the effects of increased dietary soluble fiber and incremental training on respiratory rate (RR), internal body temperature (BT), body composition, and fecal metabolites in mid-distance training sled dogs. Fourteen dogs (12 Siberian and 2 Alaskan Huskies) were blocked by age, sex, and body weight (BW) and then randomly allocated into one of two diet groups. Seven dogs were fed a dry extruded control diet (Ctl) with an insoluble:soluble fiber ratio of 4:1 (0.74% soluble fiber on a dry-matter basis), and seven dogs were fed a dry extruded treatment diet (Trt) with an insoluble:soluble fiber ratio of 3:1 (2.12% soluble fiber on a dry-matter basis). Fecal samples were taken once a week. All dogs underwent 9 weeks of incremental exercise conditioning where the running distance was designed to increase each week. Every 3 weeks, external telemetry equipment was used to non-invasively measure and record RR and internal BT at resting, working, and post-exercise recovery states. Body composition was measured on weeks -1 and 9 using quantitative magnetic resonance. Body composition, RR, BT, and fecal metabolites were analyzed using a mixed model with dog as a random effect and week and diet group as fixed effects. Dogs on Trt had lower working and post-exercise BT than Ctl ( < 0.05). In addition, Trt dogs had lower recovery BT at weeks 2 and 5 than Ctl dogs ( < 0.05). Treatment dogs had greater fecal short-chain fatty acid concentrations than Ctl ( < 0.05). Diet had no effect on RR or body composition ( > 0.10), but exercise resulted in an overall 7% increase in lean and 3.5% decrease in fat mass ( < 0.05). These data suggest that increasing dietary soluble fiber may positively influence BT and gut health; however, it has no effect on RR or body composition. Soluble fiber did not negatively impact any measures of overall health and performance and should be considered for use in performance dogs.
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http://dx.doi.org/10.3389/fvets.2021.639335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8116954PMC
April 2021

Endurance and Sprint Training Improve Glycemia and V˙O2peak but only Frequent Endurance Benefits Blood Pressure and Lipidemia.

Med Sci Sports Exerc 2021 Jun;53(6):1194-1205

Human Performance and Health Research Laboratory, Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, CANADA.

Purpose: Sprint interval training (SIT) has gained popularity as a time-effective alternative to moderate-intensity endurance training (END). However, whether SIT is equally effective for decreasing cardiometabolic risk factors remains debatable, as many beneficial effects of exercise are thought to be transient, and unlike END, SIT is not recommended daily. Therefore, in line with current exercise recommendations, we examined the ability of SIT and END to improve cardiometabolic health in overweight/obese males.

Methods: Twenty-three participants were randomized to perform 6 wk of constant workload SIT (3 d·wk-1, 4-6 × 30 s ~170% Wpeak, 2 min recovery, n = 12) or END (5 d·wk-1, 30-40 min, ~60% Wpeak, n = 11) on cycle ergometers. Aerobic capacity (V˙O2peak), body composition, blood pressure (BP), arterial stiffness, endothelial function, glucose and lipid tolerance, and free-living glycemic regulation were assessed pre- and posttraining.

Results: Both END and SIT increased V˙O2peak (END ~15%, SIT ~5%) and glucose tolerance (~20%). However, only END decreased diastolic BP, abdominal fat, and improved postprandial lipid tolerance, representing improvements in cardiovascular risk factors that did not occur after SIT. Although SIT, but not END, increased endothelial function, arterial stiffness was not altered in either group. Indices of free-living glycemic regulation were improved after END and trended toward an improvement after SIT (P = 0.06-0.09). However, glycemic control was better on exercise compared with rest days, highlighting the importance of exercise frequency. Furthermore, in an exploratory nature, favorable individual responses (V˙O2peak, BP, glucose tolerance, lipidemia, and body fat) were more prevalent after END than low-frequency SIT.

Conclusion: As only high-frequency END improved BP and lipid tolerance, free-living glycemic regulation was better on days that participants exercised, and favorable individual responses were consistent after END, high-frequency END may favorably improve cardiometabolic health.
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http://dx.doi.org/10.1249/MSS.0000000000002582DOI Listing
June 2021

Are Alterations in Skeletal Muscle Mitochondria a Cause or Consequence of Insulin Resistance?

Int J Mol Sci 2020 Sep 22;21(18). Epub 2020 Sep 22.

Institute for Health and Sport (iHeS), Victoria University, Melbourne 8001, Australia.

As a major site of glucose uptake following a meal, skeletal muscle has an important role in whole-body glucose metabolism. Evidence in humans and animal models of insulin resistance and type 2 diabetes suggests that alterations in mitochondrial characteristics accompany the development of skeletal muscle insulin resistance. However, it is unclear whether changes in mitochondrial content, respiratory function, or substrate oxidation are central to the development of insulin resistance or occur in response to insulin resistance. Thus, this review will aim to evaluate the apparent conflicting information placing mitochondria as a key organelle in the development of insulin resistance in skeletal muscle.
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http://dx.doi.org/10.3390/ijms21186948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554894PMC
September 2020

In vitro ketone-supported mitochondrial respiration is minimal when other substrates are readily available in cardiac and skeletal muscle.

J Physiol 2020 11 19;598(21):4869-4885. Epub 2020 Aug 19.

Department of Human Health and Nutritional Science, University of Guelph, Guelph, Ontario, Canada.

Key Points: Ketone bodies are proposed to represent an alternative fuel source driving energy production, particularly during exercise. Biologically, the extent to which mitochondria utilize ketone bodies compared to other substrates remains unknown. We demonstrate in vitro that maximal mitochondrial respiration supported by ketone bodies is low when compared to carbohydrate-derived substrates in the left ventricle and red gastrocnemius muscle from rodents, and in human skeletal muscle. When considering intramuscular concentrations of ketone bodies and the presence of other carbohydrate and lipid substrates, biological rates of mitochondrial respiration supported by ketone bodies are predicted to be minimal. At the mitochondrial level, it is therefore unlikely that ketone bodies are an important source for energy production in cardiac and skeletal muscle, particularly when other substrates are readily available.

Abstract: Ketone bodies (KB) have recently gained popularity as an alternative fuel source to support mitochondrial oxidative phosphorylation and enhance exercise performance. However, given the low activity of ketolytic enzymes and potential inhibition from carbohydrate oxidation, it remains unknown if KBs can contribute to energy production. We therefore determined the ability of KBs (sodium dl-β-hydroxybutyrate, β-HB; lithium acetoacetate, AcAc) to stimulate in vitro mitochondrial respiration in the left ventricle (LV) and red gastrocnemius (RG) of rats, and in human vastus lateralis. Compared to pyruvate, the ability of KBs to maximally drive respiration was low in isolated mitochondria and permeabilized fibres (PmFb) from the LV (∼30-35% of pyruvate), RG (∼10-30%), and human vastus lateralis (∼2-10%). In PmFb, the concentration of KBs required to half-maximally drive respiration (LV: 889 µm β-HB, 801 µm AcAc; RG: 782 µm β-HB, 267 µm AcAc) were greater than KB content representative of the muscle microenvironment (∼100 µm). This would predict low rates (∼1-4% of pyruvate) of biological KB-supported respiration in the LV (8-14 pmol s mg ) and RG (3-6 pmol s mg ) at rest and following exercise. Moreover, KBs did not increase respiration in the presence of saturating pyruvate, submaximal pyruvate (100 µm) reduced the ability of physiological β-HB to drive respiration, and addition of other intracellular substrates (succinate + palmitoylcarnitine) decreased maximal KB-supported respiration. As a result, product inhibition is likely to limit KB oxidation. Altogether, the ability of KBs to drive mitochondrial respiration is minimal and they are likely to be outcompeted by other substrates, compromising their use as an important energy source.
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http://dx.doi.org/10.1113/JP280032DOI Listing
November 2020

Exercise but Not Supplemental Dietary Tryptophan Influences Heart Rate and Respiratory Rate in Sled Dogs.

Vet Sci 2020 Jul 23;7(3). Epub 2020 Jul 23.

Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada.

Tryptophan (Trp), an indispensable amino acid for dogs, is the precursor of serotonin, a neurotransmitter with a variety of effects throughout the body, including the ability to modulate cardiac and pulmonary activity. This study aimed to investigate the effects of a 12-week incremental exercise regimen and supplemental dietary Trp on heart rate (HR) and respiratory rate (RR) in client-owned sled dogs. Sixteen Siberian huskies were randomly allocated to either treatment or control diet groups. Both groups were fed a control diet (Trp to large neutral amino acid ratio of 0.047:1); however, treatment dogs received a Trp supplement to achieve a Trp to large neutral amino acid ratio of 0.075:1. Every three weeks, external telemetry equipment was used to non-invasively measure and record HR and RR at a resting, working, and post-exercise state in a controlled exercise challenge. A mixed model was used to test differences between diet, activity parameter, and week. Dietary Trp supplementation had no effect on HR or RR. Independent of diet, resting, working, post-exercise HR, and time to recover post-exercise HR decreased from week -1 to week 11 ( < 0.05). Resting HR had the greatest reduction from week -1 to week 11 (21%, < 0.05). Working RR did not change with exercise ( > 0.10), but rRR and postRR decreased from week -1 to week 11 ( < 0.05). These data suggest that the exercise regimen the dogs were subjected to may have positively impacted the dogs' capacity to sustain aerobic exercise, whereas Trp supplementation had no effect on HR or RR.
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http://dx.doi.org/10.3390/vetsci7030097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7559096PMC
July 2020

Long-term, high-fat feeding exacerbates short-term increases in adipose mitochondrial reactive oxygen species, without impairing mitochondrial respiration.

Am J Physiol Endocrinol Metab 2020 08 16;319(2):E376-E387. Epub 2020 Jun 16.

Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada.

White adipose tissue (WAT) dysfunction in obesity is implicated in the onset of whole body insulin resistance. Alterations in mitochondrial bioenergetics, namely impaired mitochondrial respiration and increased mitochondrial reactive oxygen species (mtROS) production, have been suggested to contribute to this metabolic dysregulation. However, techniques investigating mitochondrial function are classically normalized to tissue weight, which may be confounding when considering obesity-related adipocyte hypertrophy. Furthermore, the effect of long-term high-fat diet (HFD) on mtROS in WAT has yet to be elucidated. Therefore, we sought to determine the HFD-mediated temporal changes in mitochondrial respiration and mtROS emission in WAT. C57BL/6N mice received low-fat diet or HFD for 1 or 8 wk and changes in inguinal WAT (iWAT) and epididymal WAT (eWAT) were assessed. While tissue weight-normalized mitochondrial respiration was reduced in iWAT following 8-wk HFD-feeding, this effect was mitigated when adipocyte cell size and/or number were considered. These data suggest HFD does not impair mitochondrial respiratory capacity per adipocyte within WAT. In support of this assertion, within eWAT compensatory increases in lipid-supported and maximal succinate-supported respiration occurred at 8 wk despite cell hypertrophy and increases in WAT inflammation. Although these data suggest impairments in mitochondrial respiration do not contribute to HFD-mediated WAT phenotype, lipid-supported mtROS emission increased following 1-wk HFD in eWAT, while both lipid and carbohydrate-supported mtROS were increased at 8 wk in both depots. Combined, these data establish that while HFD does not impair adipocyte mitochondrial respiratory capacity, increased mtROS is an enduring physiological occurrence within WAT in HFD-induced obesity.
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http://dx.doi.org/10.1152/ajpendo.00028.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473917PMC
August 2020

Acute insulin deprivation results in altered mitochondrial substrate sensitivity conducive to greater fatty acid transport.

Am J Physiol Endocrinol Metab 2020 08 16;319(2):E345-E353. Epub 2020 Jun 16.

Department of Human Health & Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada.

Type 1 and type 2 diabetes are both tightly associated with impaired glucose control. Although both pathologies stem from different mechanisms, a reduction in insulin action coincides with drastic metabolic dysfunction in skeletal muscle and metabolic inflexibility. However, the underlying explanation for this response remains poorly understood, particularly since it is difficult to distinguish the role of attenuated insulin action from the detrimental effects of reactive lipid accumulation, which impairs mitochondrial function and promotes reactive oxygen species (ROS) emission. We therefore utilized streptozotocin to examine the effects of acute insulin deprivation, in the absence of a high-lipid/nutrient excess environment, on the regulation of mitochondrial substrate sensitivity and ROS emission. The ablation of insulin resulted in reductions in absolute mitochondrial oxidative capacity and ADP-supported respiration and reduced the ability for malonyl-CoA to inhibit carnitine palmitoyltransferase I (CPT-I) and suppress fatty acid-supported respiration. These bioenergetic responses coincided with increased mitochondrial-derived HO emission and lipid transporter content, independent of major mitochondrial substrate transporter proteins and enzymes involved in fatty acid oxidation. Together, these data suggest that attenuated/ablated insulin signaling does not affect mitochondrial ADP sensitivity, whereas the increased reliance on fatty acid oxidation in situations where insulin action is reduced may occur as a result of altered regulation of mitochondrial fatty acid transport through CPT-I.
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http://dx.doi.org/10.1152/ajpendo.00495.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473910PMC
August 2020

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

J Physiol 2020 08 16;598(16):3357-3371. Epub 2020 Jun 16.

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada.

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 H O 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 H O 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.
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http://dx.doi.org/10.1113/JP279455DOI Listing
August 2020

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

Appl Physiol Nutr Metab 2020 Sep 7;45(9):1049-1053. Epub 2020 May 7.

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada.

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. Dietary nitrate decreases blood pressure but does not alter left ventricular calcium-handling protein content or SERCA activity in healthy rats.
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http://dx.doi.org/10.1139/apnm-2020-0078DOI Listing
September 2020

Resting metabolic rate and skeletal muscle SERCA and Na /K ATPase activities are not affected by fish oil supplementation in healthy older adults.

Physiol Rep 2020 05;8(9):e14408

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada.

Omega-3 polyunsaturated fatty acids (PUFAs) have unique properties purported to influence several aspects of metabolism, including energy expenditure and protein function. Supplementing with n-3 PUFAs may increase whole-body resting metabolic rate (RMR), by enhancing Na /K ATPase (NKA) activity and reducing the efficiency of sarcoplasmic reticulum (SR) Ca ATPase (SERCA) activity by inducing a Ca leak-pump cycle. The purpose of this study was to examine the effects of fish oil (FO) on RMR, substrate oxidation, and skeletal muscle SERCA and NKA pump function in healthy older individuals. Subjects (n = 16 females; n = 8 males; 65 ± 1 years) were randomly assigned into groups supplemented with either olive oil (OO) (5 g/day) or FO (5 g/day) containing 2 g/day eicosapentaenoic acid and 1 g/day docosahexaenoic acid for 12 weeks. Participants visited the laboratory for RMR and substrate oxidation measurements after an overnight fast at weeks 0 and 12. Skeletal muscle biopsies were taken during weeks 0 and 12 for analysis of NKA and SERCA function and protein content. There was a main effect of time with decrease in RMR (5%) and fat oxidation (18%) in both the supplementation groups. The kinetic parameters of SERCA and NKA maximal activity, as well as the expression of SR and NKA proteins, were not affected after OO and FO supplementation. In conclusion, these results suggest that FO supplementation is not effective in altering RMR, substrate oxidation, and skeletal muscle SERCA and NKA protein levels and activities, in healthy older men and women.
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http://dx.doi.org/10.14814/phy2.14408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7186565PMC
May 2020

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

J Physiol 2020 02 4;598(4):683-697. Epub 2020 Feb 4.

Department of Human Health & Nutritional Sciences, University of Guelph, Ontario, Canada.

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 β-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.
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http://dx.doi.org/10.1113/JP279042DOI Listing
February 2020

Low-load resistance training to task failure with and without blood flow restriction: muscular functional and structural adaptations.

Am J Physiol Regul Integr Comp Physiol 2020 02 11;318(2):R284-R295. Epub 2019 Dec 11.

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada.

The application of blood flow restriction (BFR) during resistance exercise is increasingly recognized for its ability to improve rehabilitation and for its effectiveness in increasing muscle hypertrophy and strength among healthy populations. However, direct comparison of the skeletal muscle adaptations to low-load resistance exercise (LL-RE) and low-load BFR resistance exercise (LL-BFR) performed to task failure is lacking. Using a within-subject design, we examined whole muscle group and skeletal muscle adaptations to 6 wk of LL-RE and LL-BFR training to repetition failure. Muscle strength and size outcomes were similar for both types of training, despite ~33% lower total exercise volume (load × repetition) with LL-BFR than LL-RE (28,544 ± 1,771 vs. 18,949 ± 1,541 kg, = 0.004). After training, only LL-BFR improved the average power output throughout the midportion of a voluntary muscle endurance task. Specifically, LL-BFR training sustained an 18% greater power output from baseline and resulted in a greater change from baseline than LL-RE (19 ± 3 vs. 3 ± 4 W, = 0.008). This improvement occurred despite histological analysis revealing similar increases in capillary content of type I muscle fibers following LL-RE and LL-BFR training, which was primarily driven by increased capillary contacts (4.53 ± 0.23 before training vs. 5.33 ± 0.27 and 5.17 ± 0.25 after LL-RE and LL-BFR, respectively, both < 0.05). Moreover, maximally supported mitochondrial respiratory capacity increased only in the LL-RE leg by 30% from baseline ( = 0.006). Overall, low-load resistance training increased indexes of muscle oxidative capacity and strength, which were not further augmented with the application of BFR. However, performance on a muscle endurance test was improved following BFR training.
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http://dx.doi.org/10.1152/ajpregu.00243.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052604PMC
February 2020

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

Am J Physiol Cell Physiol 2020 01 13;318(1):C137-C149. Epub 2019 Nov 13.

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada.

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, HO, various antioxidants, and the β3-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 () gene expression. In WAT culture, HO reduced glyceroneogenic gene expression. In wild-type mice, acute exercise induced dramatic but transient increases in and phosphoenolpyruvate carboxykinase () mRNA in both subcutaneous inguinal WAT and epididymal WAT depots, which was almost completely absent in MCAT mice. Furthermore, the induction of and in WAT culture by CL316243 was markedly reduced in the presence of antioxidants -acetyl-cysteine or vitamin E. Genetic and nutritional approaches that attenuate redox signaling prevent exercise- and β-agonist-induced gene expression within WAT. Combined, these data suggest that ROS represent important mediators of gene expression within WAT.
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http://dx.doi.org/10.1152/ajpcell.00313.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985839PMC
January 2020

Short-term bed rest-induced insulin resistance cannot be explained by increased mitochondrial H O emission.

J Physiol 2020 01 26;598(1):123-137. Epub 2019 Dec 26.

Human Health & Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada.

Key Points: We determined if bed rest increased mitochondrially derived reactive oxygen species and cellular redox stress, contributing to the induction of insulin resistance. Bed rest decreased maximal and submaximal ADP-stimulated mitochondrial respiration. Bed rest did not alter mitochondrial H O emission in the presence of ADP concentrations indicative of resting muscle, the ratio of H O emission to mitochondrial O consumption or markers of oxidative stress The present data suggest strongly that mitochondrial H O does not contribute to bed rest-induced insulin resistance ABSTRACT: Mitochondrial H O has been causally linked to diet-induced insulin resistance, although it remains unclear if muscle disuse similarly increases mitochondrial H O . Therefore, we investigated the potential that an increase in skeletal muscle mitochondrial H O emission, potentially as a result of decreased ADP sensitivity, contributes to cellular redox stress and the induction of insulin resistance during short-term bed rest in 20 healthy males. Bed rest led to a decline in glucose infusion rate during a hyperinsulinaemic-euglycaemic clamp (-42 ± 2%; P < 0.001), and in permeabilized skeletal muscle fibres it decreased OXPHOS protein content (-16 ± 8%) and mitochondrial respiration across a range of ADP concentrations (-13 ± 5%). While bed rest tended to increase maximal mitochondrial H O emission rates (P = 0.053), H O emission in the presence of ADP concentrations indicative of resting muscle, the ratio of H O emission to mitochondrial O consumption, and markers of oxidative stress were not altered following bed rest. Altogether, while bed rest impairs mitochondrial ADP-stimulated respiration, an increase in mitochondrial H O emission does not contribute to the induction of insulin resistance following short-term bed rest.
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http://dx.doi.org/10.1113/JP278920DOI Listing
January 2020

Assessment of Na+/K+ ATPase Activity in Small Rodent and Human Skeletal Muscle Samples.

Med Sci Sports Exerc 2019 11;51(11):2403-2409

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, CANADA.

Introduction: In skeletal muscle, the Na/K ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism; however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for the determination of NKA activity in small rodent muscle samples (5-10 mg) and in human skeletal muscle.

Methods: Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize "background noise."

Results: We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as the addition of 25 μM of blebbistatin, a specific myosin ATPase inhibitor, considerably minimized "background noise" (threefold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. The specificity of the assay was demonstrated after the addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, the addition of blebbistatin did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively.

Conclusion: The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.
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http://dx.doi.org/10.1249/MSS.0000000000002063DOI Listing
November 2019

New tools for an old question: dependence of ATP and bicarbonate for branched-chain keto acids oxidation.

Biochem J 2019 08 15;476(15):2235-2237. Epub 2019 Aug 15.

University of Guelph, Guelph, Canada

Branched-chain keto acids (BCKA) metabolism involves several well-regulated steps within mitochondria, requires cofactors, and is modulated according to the metabolic status of the cells. This regulation has made it challenging to utilize approaches to determine the contribution of branched-chain amino acid oxidation to energy production. These methodological issues were elegantly addressed in a recent publication within the Biochemical Journal. In this issue, Goldberg et al. [Biochem. J. (2019) 476, 1521-1537] demonstrated in a well-designed system the dependence of ATP and bicarbonate for BCKA full oxidation. In addition, the utilized system allowed the authors to characterize specific biochemical routes within mitochondria for each BCKA. Among them, a quantitative analysis of the participation of BCKA on mitochondrial flux was estimated between tissues. These findings are milestones with meaningful impact in several fields of metabolism.
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http://dx.doi.org/10.1042/BCJ20190368DOI Listing
August 2019

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

J Physiol 2019 08 7;597(15):3985-3997. Epub 2019 Jul 7.

Human Health & Nutritional Science, University of Guelph, Guelph, Ontario, Canada.

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 ( ) is reduced during BFR-RE, and the influence of 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 is involved in this response because an in vitro analysis revealed that reducing 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 .

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 ( ) is reduced during BFR-RE, and the influence of O 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 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 O 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 .
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http://dx.doi.org/10.1113/JP277765DOI Listing
August 2019

Incorporation of Omega-3 Fatty Acids Into Human Skeletal Muscle Sarcolemmal and Mitochondrial Membranes Following 12 Weeks of Fish Oil Supplementation.

Front Physiol 2019 29;10:348. Epub 2019 Mar 29.

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada.

Fish oil (FO) supplementation in humans results in the incorporation of omega-3 fatty acids (FAs) eicosapentaenoic acid (EPA; C20:5) and docosahexaenoic acid (DHA; C20:6) into skeletal muscle membranes. However, despite the importance of membrane composition in structure-function relationships, a paucity of information exists regarding how different muscle membranes/organelles respond to FO supplementation. Therefore, the purpose of the present study was to determine the effects 12 weeks of FO supplementation (3g EPA/2g DHA daily) on the phospholipid composition of sarcolemmal and mitochondrial fractions, as well as whole muscle responses, in healthy young males. FO supplementation increased the total phospholipid content in whole muscle (57%; < 0.05) and the sarcolemma (38%; = 0.05), but did not alter the content in mitochondria. The content of omega-3 FAs, EPA and DHA, were increased (+3-fold) in whole muscle, and mitochondrial membranes, and as a result the omega-6/omega-3 ratios were dramatically decreased (-3-fold), while conversely the unsaturation indexes were increased. Intriguingly, before supplementation the unsaturation index (UI) of sarcolemmal membranes was ∼3 times lower ( < 0.001) than either whole muscle or mitochondrial membranes. While supplementation also increased DHA within sarcolemmal membranes, EPA was not altered, and as a result the omega-6/omega-3 ratio and UI of these membranes were not altered. All together, these data revealed that mitochondrial and sarcolemmal membranes display unique phospholipid compositions and responses to FO supplementation.
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http://dx.doi.org/10.3389/fphys.2019.00348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449797PMC
March 2019

Supplementation with dietary ω-3 mitigates immobilization-induced reductions in skeletal muscle mitochondrial respiration in young women.

FASEB J 2019 07 10;33(7):8232-8240. Epub 2019 Apr 10.

Department of Human Health and Nutritional Sciences, University of Guelph, Ontario, Canada.

Omega-3 (ω-3) supplementation attenuates immobilization-induced atrophy; however, the underlying mechanisms remain unclear. Since mitochondrial dysfunction and oxidative stress have been implicated in muscle atrophy, we examined whether ω-3 supplementation could mitigate disuse-mediated mitochondrial dysfunction. Healthy young women (age = 22 ± 3 yr) randomly received control ( = 9) or ω-3 supplementation ( = 11; 3 g eicosapentaenoic acid, 2 g docosahexaenoic acid) for 4 wk prior to and throughout 2 wk of single-limb immobilization. Biopsies were performed before and after 3 and 14 d of immobilization for the assessment of mitochondrial respiration, HO emission, and markers of ADP transport/lipid metabolism. In controls, immobilization rapidly (3 d) reduced (∼20%) ADP-stimulated mitochondrial respiration without altering ADP sensitivity or the abundance of mitochondrial proteins. Extending immobilization to 14 d did not further reduce mitochondrial coupled respiration; however, unlike following 3 d, mitochondrial proteins were reduced ∼20%. In contrast, ω-3 supplementation prevented immobilization-induced reductions in mitochondrial content and respiration throughout the immobilization period. Regardless of dietary supplement, immobilization did not alter mitochondrial HO emission in the presence or absence of ADP, markers of cellular redox state, mitochondrial lipid-supported respiration, or lipid-related metabolic proteins. These data highlight the rapidity of mitochondrial adaptations in response to muscle disuse, challenge the necessity for increased oxidative stress during inactivity, and establish that ω-3 supplementation preserves oxidative phosphorylation function and content during immobilization.-Miotto, P. M., McGlory, C., Bahniwal, R., Kamal, M., Phillips, S. M., Holloway, G. P. Supplementation with dietary ω-3 mitigates immobilization-induced reductions in skeletal muscle mitochondrial respiration in young women.
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http://dx.doi.org/10.1096/fj.201900095RDOI Listing
July 2019

Dietary feeding pattern does not modulate the loss of muscle mass or the decline in metabolic health during short-term bed rest.

Am J Physiol Endocrinol Metab 2019 03 15;316(3):E536-E545. Epub 2019 Jan 15.

Department of Human Biology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre+ , The Netherlands.

Short periods of bed rest lead to the loss of muscle mass and quality. It has been speculated that dietary feeding pattern may have an impact upon muscle protein synthesis rates and, therefore, modulate the loss of muscle mass and quality. We subjected 20 healthy men (age: 25 ± 1 yr, body mass index: 23.8 ± 0.8 kg/m) to 1 wk of strict bed rest with intermittent (4 meals/day) or continuous (24 h/day) enteral tube feeding. Participants consumed deuterium oxide for 7 days before bed rest and throughout the 7-day bed rest period. Prior to and immediately after bed rest, lean body mass (dual energy X-ray absorptiometry), quadriceps cross-sectional area (CSA; CT), maximal oxygen uptake capacity (V̇o), and whole body insulin sensitivity (hyperinsulinemic-euglycemic clamp) were assessed. Muscle biopsies were collected 7 days before, 1 day before, and immediately after bed rest to assess muscle tracer incorporation. Bed rest resulted in 0.3 ± 0.3 vs. 0.7 ± 0.4 kg lean tissue loss and a 1.1 ± 0.6 vs. 0.8 ± 0.5% decline in quadriceps CSA in the intermittent vs. continuous feeding group, respectively (both P < 0.05), with no differences between groups (both P > 0.05). Moreover, feeding pattern did not modulate the bed rest-induced decline in insulin sensitivity (-46 ± 3% vs. 39 ± 3%; P < 0.001) or V̇o (-2.5 ± 2.2 vs. -8.6 ± 2.2%; P < 0.010) (both P > 0.05). Myofibrillar protein synthesis rates during bed rest did not differ between the intermittent and continuous feeding group (1.33 ± 0.07 vs. 1.50 ± 0.13%/day, respectively; P > 0.05). In conclusion, dietary feeding pattern does not modulate the loss of muscle mass or the decline in metabolic health during 1 wk of bed rest in healthy men.
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http://dx.doi.org/10.1152/ajpendo.00378.2018DOI Listing
March 2019

High intensity exercise inhibits carnitine palmitoyltransferase-I sensitivity to l-carnitine.

Biochem J 2019 02 8;476(3):547-558. Epub 2019 Feb 8.

Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1L 0A5

The decline in fat oxidation at higher power outputs of exercise is a complex interaction between several mechanisms; however, the influence of mitochondrial bioenergetics in this process remains elusive. Therefore, using permeabilized muscle fibers from mouse skeletal muscle, we aimed to determine if acute exercise altered mitochondrial sensitivity to (1) adenosine diphosphate (ADP) and inorganic phosphate (Pi), or (2) carnitine palmitoyltransferase-I (CPT-I) independent (palmitoylcarnitine, PC) and dependent [palmitoyl-CoA (P-CoA), malonyl-CoA (M-CoA), and l-carnitine] substrates, in an intensity-dependent manner. As the apparent ADP increased to a similar extent following low (LI) and high (HI) intensity exercise compared with sedentary (SED) animals, and Pi sensitivity was unaltered by exercise, regulation of phosphate provision likely does not contribute to the well-established intensity-dependent shift in substrate utilization. Mitochondrial sensitivity to PC and P-CoA was not influenced by exercise, while M-CoA sensitivity was attenuated similarly following LI and HI. In contrast, CPT-I sensitivity to l-carnitine was only altered following HI, as HI exercise attenuated l-carnitine sensitivity by ∼40%. Moreover, modeling the concentrations of l-carnitine and P-CoA during exercise suggests that CPT-I flux is ∼25% lower following HI, attributed equally to reductions in l-carnitine content and l-carnitine sensitivity. Altogether, these data further implicate CPT-I flux as a key event influencing metabolic interactions during exercise, as a decline in l-carnitine sensitivity in addition to availability at higher power outputs could impair mitochondrial fatty acid oxidation.
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http://dx.doi.org/10.1042/BCJ20180849DOI Listing
February 2019

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

Biochem J 2018 09 28;475(18):2997-3008. Epub 2018 Sep 28.

Human Health & Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada

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 (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 was increased only in the presence of mitochondrial ROS. This basic pattern was also mirrored in the ability of ADP to suppress mitochondrial HO emission rates, as exercise decreased the suppression of HO 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.
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http://dx.doi.org/10.1042/BCJ20180419DOI Listing
September 2018

High-Fat Diet Causes Mitochondrial Dysfunction as a Result of Impaired ADP Sensitivity.

Diabetes 2018 11 6;67(11):2199-2205. Epub 2018 Jul 6.

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada

Although molecular approaches altering mitochondrial content have implied a direct relationship between mitochondrial bioenergetics and insulin sensitivity, paradoxically, consumption of a high-fat (HF) diet increases mitochondrial content while inducing insulin resistance. We hypothesized that despite the induction of mitochondrial biogenesis, consumption of an HF diet would impair mitochondrial ADP sensitivity in skeletal muscle of mice and therefore manifest in mitochondrial dysfunction in the presence of ADP concentrations indicative of skeletal muscle biology. We found that HF consumption increased mitochondrial protein expression; however, absolute mitochondrial respiration and ADP sensitivity were impaired across a range of biologically relevant ADP concentrations. In addition, HF consumption attenuated the ability of ADP to suppress mitochondrial HO emission, further suggesting impairments in ADP sensitivity. The abundance of ADP transport proteins were not altered, but the sensitivity to carboxyatractyloside-mediated inhibition was attenuated after HF consumption, implicating alterations in adenine nucleotide translocase (ANT) ADP sensitivity in these observations. Moreover, palmitoyl-CoA is known to inhibit ANT, and modeling intramuscular palmitoyl-CoA concentrations that occur after HF consumption exacerbated the deficiency in ADP sensitivity. Altogether, these data suggest that an HF diet induces mitochondrial dysfunction secondary to an intrinsic impairment in mitochondrial ADP sensitivity that is magnified by palmitoyl-CoA.
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http://dx.doi.org/10.2337/db18-0417DOI Listing
November 2018

Impaired mitochondrial activity explains platelet dysfunction in thrombocytopenic cancer patients undergoing chemotherapy.

Haematologica 2018 09 7;103(9):1557-1567. Epub 2018 Jun 7.

Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, the Netherlands

Severe thrombocytopenia (≤50×10 platelets/L) due to hematological malignancy and intensive chemotherapy is associated with an increased risk of clinically significant bleeding. Since the bleeding risk is not linked to the platelet count only, other hemostatic factors must be involved. We studied platelet function in 77 patients with acute leukemia, multiple myeloma or malignant lymphoma, who experienced chemotherapy-induced thrombocytopenia. Platelets from all patients - independent of disease or treatment type - were to a variable extent compromised in Ca flux, integrin a β activation and P-selectin expression when stimulated with a panelof agonists. The patients' platelets were also impaired in spreading on fibrinogen. Whereas the Ca store content was unaffected, the patients' platelets showed ongoing phosphatidylserine exposure, which was not due to apoptotic caspase activity. Interestingly, mitochondrial function was markedly reduced in platelets from a representative subset of patients, as evidenced by a low mitochondrial membrane potential (<0.001) and low oxygen consumption (<0.05), while the mitochondrial content was normal. Moreover, the mitochondrial impairments coincided with elevated levels of reactive oxygen species (Spearman's rho=-0.459, =0.012). Markedly, the impairment of platelet function only appeared after two days of chemotherapy, suggesting origination in the megakaryocytes. In patients with bone marrow recovery, platelet function improved. In conclusion, our findings disclose defective receptor signaling related to impaired mitochondrial bioenergetics, independent of apoptosis, in platelets from cancer patients treated with chemotherapy, explaining the low hemostatic potential of these patients.
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http://dx.doi.org/10.3324/haematol.2017.185165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6119160PMC
September 2018

Alpha-linolenic acid and linoleic acid differentially regulate the skeletal muscle secretome of obese Zucker rats.

Physiol Genomics 2018 08 4;50(8):580-589. Epub 2018 May 4.

Department of Human Health and Nutritional Sciences, University of Guelph , Guelph, Ontario , Canada.

Evidence shows that proteins secreted from skeletal muscle influence a broad range of metabolic signaling pathways. We previously reported that essential polyunsaturated fatty acids (PUFA) improved whole-body glucose homeostasis in obese Zucker rats; however, the mechanisms underlying these benefits remain enigmatic. While PUFA and obesity influence skeletal muscle function, their effects on the secretome are unknown. The aim of this work was to determine if improvements in whole-body glucose homeostasis in obese Zucker rats fed diets supplemented with either linoleic acid (LA) or alpha-linolenic acid (ALA) for 12 wk are related to changes in the skeletal muscle secretome. Secreted proteins were identified with a predictive bioinformatic analysis of microarray gene expression from red tibialis anterior skeletal muscle. Approximately 130 genes were differentially expressed (false discovery rate = 0.05) in obese rats compared with lean controls. The expression of 15 genes encoding secreted proteins was differentially regulated in obese controls, obese LA-supplemented, and obese ALA-supplemented rats compared with lean controls. Five secreted proteins ( Col3a1, Col15a1, Pdgfd, Lyz2, and Angptl4) were differentially regulated by LA and ALA. Most notably, ALA supplementation reduced Angptl4 gene expression compared with obese control and obese-LA supplemented rats and reduced circulating ANGPTL4 serum concentrations. ALA also influenced Angptl4 gene expression and ANGPTL4 secretion from differentiated rat L6 myotubes. Altogether, the present data indicate that obesity has a greater global impact on skeletal muscle gene expression than either essential PUFA; however, LA and ALA may exert their metabolic benefits in part by regulating the skeletal muscle secretome.
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http://dx.doi.org/10.1152/physiolgenomics.00038.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139631PMC
August 2018

Cytosolic reverse CrAT activity in cardiac tissue: potential importance for fuel selection.

Biochem J 2018 04 9;475(7):1267-1269. Epub 2018 Apr 9.

Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada

The movement of lipids across mitochondrial membranes represents a rate-limiting step in fatty acid oxidation within the heart. A key regulatory point in this process is flux through carnitine palmitoyltransferase-I (CPT-I), an enzyme located on the outer mitochondrial membrane. Malonyl-CoA (M-CoA) is a naturally occurring inhibitor of CPT-I; therefore, the abundance of M-CoA has long been considered a major regulator of fatty acid oxidation. A recent paper published in the by Altamimi et al. ( (2018) , 959-976) provides evidence for a novel mechanism to produce M-CoA. Specifically, these authors identified carnitine acetyltransferase within the cytosol and further show that flux in the reverse direction forms acetyl-CoA, which is the necessary substrate for the subsequent synthesis of M-CoA. The elegant study design and intriguing data presented by Altamimi et al. provide further insights into the reciprocal regulation of substrate selection within the heart, with implications for fuel utilization and the development of cardiac diseases.
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http://dx.doi.org/10.1042/BCJ20180121DOI Listing
April 2018

Exercise-induced reductions in mitochondrial ADP sensitivity contribute to the induction of gene expression and mitochondrial biogenesis through enhanced mitochondrial HO emission.

Mitochondrion 2019 05 26;46:116-122. Epub 2018 Mar 26.

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph N1G 2W1, ON, Canada. Electronic address:

Acute exercise rapidly induces mitochondrial gene expression, however, the intracellular events regulating this process remain incompletely understood. The purpose of this study was to determine whether reductions in mitochondrial ADP sensitivity during exercise have a biological role in regulating mitochondrial-derived reactive oxygen species (ROS) production and the induction of mitochondrial biogenesis. Mitochondrial creatine kinase wildtype (WT) and knockout (KO) mice have divergent responses in ADP sensitivity during exercise, and we therefore used these mice to determine the relationship between mitochondrial ADP sensitivity, ROS production, and mitochondrial adaptations to exercise. In WT mice, acute exercise reduced mitochondrial ADP respiratory sensitivity and the ability of ADP to suppress ROS production, while increasing mitochondrial gene transcription (PGC-1α, PGC-1β and PDK4). In stark contrast, in KO mice, exercise increased ADP sensitivity, reduced mitochondrial ROS emission, and did not induce gene transcription. Despite the divergence in mRNA responses, exercise similarly induced calcium/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK) phosphorylation in WT and KO mice, however only WT mice were associated with redox stress (4HNE). These data implicate acute changes in ADP sensitivity in mitochondrial adaptations to exercise. To further examine this we chronically exercise trained mice. While training increased mitochondrial content and reduced ADP sensitivity in WT mice, KO mice did not exhibit adaptations to exercise. Combined, these data suggest that exercise-induced attenuations in mitochondrial ADP sensitivity mediate redox signals that contribute to the induction of acute and chronic mitochondrial adaptations.
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http://dx.doi.org/10.1016/j.mito.2018.03.003DOI Listing
May 2019
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