Jamie Whitfield

Omega‐3 supplementation alters mitochondrial membrane composition and respiration kinetics in human skeletal muscle

Following fish oil supplementation, omega‐3 fatty acids are incorporated into cellular membranes, which may affect lipid–protein interactions and therefore the function of embedded proteins. As the components of the electron transport chain required for oxidative phosphorylation are contained in the mitochondrial membrane, omega‐3 supplementation may alter metabolic function. We supplemented male participants for 12 weeks with fish oil [eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA)] and analysed mitochondrial function and reactive oxygen species (ROS) emissions in permeabilized muscle fibres from the vastus lateralis muscle. Supplementation incorporated EPA and DHA into mitochondrial membranes, but did not result in changes in maximal mitochondrial respiratory function or pyruvate respiration kinetics. However, the apparent Km for ADP was decreased following supplementation, and was independent of creatine, changes in the protein content of ADP synthase or ANT transporters. The propensity for ROS emissions increased with omega‐3 supplementation, although there were no changes in markers of lipid or protein oxidative damage. These results demonstrate that omega‐3 supplementation improves mitochondrial ADP kinetics, suggesting post‐translational modification of existing proteins.

Beetroot juice supplementation does not improve performance of elite 1500-m runners.

PURPOSE Dietary nitrate supplementation with beetroot juice (BR) has received widespread attention as an ergogenic aid. However, recent evidence in well-trained cyclists has not consistently reported improved cycling economy or performance. The present study examined the effects of acute and chronic BR supplementation on V˙O2 during submaximal running and 1500-m time trial (TT) performance of elite distance runners. METHODS Eight male 1500-m runners (V˙O2peak, 80 ± 5 mL·kg·min; 1500-m personal best, 3:56 ± 9 s) participated in this study. In a randomized, double-blind, crossover design, subjects supplemented with BR or a nitrate-free BR placebo (PL) for 8 d separated by at least 1 wk. On days 1 (acute) and 8 (chronic), subjects ingested 210 mL of BR (19.5-mmol nitrate) or PL and completed a submaximal treadmill run and 1500-m TT on an indoor 200-m track. RESULTS Plasma nitrate increased from 37 ± 15 to 615 ± 151 μM (acute) and 870 ± 259 μM (chronic) after BR supplementation. There were no V˙O2 differences between conditions at 50%, 65%, and 80% V˙O2peak (acute PL, 4194 ± 90 mL·min; chronic PL, 4216 ± 95 mL·min; acute BR, 4192 ± 113 mL·min; chronic BR, 4299 ± 92 mL·min). The 1500-m TT was unaffected by acute or chronic BR supplementation (acute PL, 4:10.4 min:s ± 2.5 s; chronic PL, 4:11.4 min:s ± 2.7 s; acute BR, 4:10.7 min:s ± 1.5 s; chronic BR, 4:10.5 min:s ± 2.2 s). However, two subjects improved their TT performance after acute (5.8 and 5.0 s) and chronic BR supplementation (7.0 and 0.5 s). CONCLUSIONS Acute and chronic BR supplementation did not reduce running V˙O2 or improve 1500-m TT performance of a group of elite distance runners, but two responders to BR were identified.

Beetroot juice supplementation reduces whole body oxygen consumption but does not improve indices of mitochondrial efficiency in human skeletal muscle

Oral consumption of nitrate (NO3−) in beetroot juice has been shown to decrease the oxygen cost of submaximal exercise; however, the mechanism of action remains unresolved. We supplemented recreationally active males with beetroot juice to determine if this altered mitochondrial bioenergetics. Despite reduced submaximal exercise oxygen consumption, measures of mitochondrial coupling and respiratory efficiency were not altered in muscle. In contrast, rates of mitochondrial hydrogen peroxide (H2O2) emission were increased in the absence of markers of lipid or protein oxidative damage. These results suggest that improvements in mitochondrial oxidative metabolism are not the cause of beetroot juice‐mediated improvements in whole body oxygen consumption.

Taurine and skeletal muscle function.

Purpose of reviewTo discuss the recent work examining the importance of taurine in skeletal muscle and outline the discrepancy that exists between research findings in rodent vs. human skeletal muscle. Recent findingsThere is clear evidence that a normal taurine level is important for the normal functioning of skeletal muscle. Taurine is believed to be involved in many cellular functions, but in skeletal muscle its main roles are to facilitate Ca2+ dependent excitation–contraction processes, contribute to the regulation of cellular volume, and aid in antioxidant defense from stress responses. Most research has studied the importance of taurine in rodent skeletal muscle by downregulating and upregulating the muscle taurine content and examining the effects on the functioning of skeletal muscle at rest and during the stress of contractions (exercise). One successful research approach is to supplement the diet with taurine, which leads to increases in muscle taurine content and contractile function in rodents. However, this approach does not work in human skeletal muscle as the processes involved in the transport of taurine into the muscle are resistant to large and prolonged increases in plasma taurine following oral taurine supplementation. At present, attempts to influence muscle function with taurine supplementation can only occur through interactions outside the muscle cell in humans. SummaryFuture research should target the mechanisms responsible for the transport of taurine into human skeletal muscle and determine why the muscle defends the normal taurine content in the face of elevated plasma taurine levels, as opposed to the results in rodent muscle. This may lead to more fruitful usage of taurine as a skeletal muscle enhancing nutrient in athletic and clinical populations.

Variable effects of 12 weeks of omega-3 supplementation on resting skeletal muscle metabolism.

Omega-3 supplementation has been purported to improve the function of several organs in the body, including reports of increased resting metabolic rate (RMR) and reliance on fat oxidation. However, the potential for omega-3s to modulate human skeletal muscle metabolism has received little attention. This study examined the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation on whole-body RMR and the content of proteins involved in fat metabolism in human skeletal muscle. Recreationally active males supplemented with 3.0 g/day of EPA and DHA (n = 21) or olive oil (n = 9) for 12 weeks. Resting muscle biopsies were sampled in a subset of 10 subjects before (pre) and after (post) omega-3 supplementation. RMR significantly increased (5.3%, p = 0.040) following omega-3 supplementation (Pre, 1.33 ±0.05; Post, 1.40 ±0.04 kcal/min) with variable individual responses. When normalizing for body mass, this effect was lost (5.2%, p = 0.058). Omega-3s did not affect whole-body fat oxidation, and olive oil did not alter any parameter assessed. Omega-3 supplementation did not affect whole muscle, sarcolemmal, or mitochondrial FAT/CD36, FABPpm, FATP1 or FATP4 contents or mitochondrial electron chain and PDH proteins, but did increase the long form of UCP3 by 11%. In conclusion, supplementation with a high dose of omega-3s for 12 weeks increased RMR in a small and variable manner in a group of healthy young men. Omega-3 supplementation also had no effect on several proteins involved in skeletal muscle fat metabolism and did not cause mitochondrial biogenesis.

Ablating the protein TBC1D1 impairs contraction-induced sarcolemmal glucose transporter 4 redistribution but not insulin-mediated responses in rats

TBC1 domain family member 1 (TBC1D1), a Rab GTPase-activating protein and paralogue of Akt substrate of 160 kDa (AS160), has been implicated in both insulin- and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-mediated glucose transporter type 4 (GLUT4) translocation. However, the role of TBC1D1 in contracting muscle remains ambiguous. We therefore explored the metabolic consequence of ablating TBC1D1 in both resting and contracting skeletal muscles, utilizing a rat TBC1D1 KO model. Although insulin administration rapidly increased (p < 0.05) plasma membrane GLUT4 content in both red and white gastrocnemius muscles, the TBC1D1 ablation did not alter this response nor did it affect whole-body insulin tolerance, suggesting that TBC1D1 is not required for insulin-induced GLUT4 trafficking events. Consistent with findings in other models of altered TBC1D1 protein levels, whole-animal and ex vivo skeletal muscle fat oxidation was increased in the TBC1D1 KO rats. Although there was no change in mitochondrial content in the KO rats, maximal ADP-stimulated respiration was higher in permeabilized muscle fibers, which may contribute to the increased reliance on fatty acids in resting KO animals. Despite this increase in mitochondrial oxidative capacity, run time to exhaustion at various intensities was impaired in the KO rats. Moreover, contraction-induced increases in sarcolemmal GLUT4 content and glucose uptake were lower in the white gastrocnemius of the KO animals. Altogether, our results highlight a critical role for TBC1D1 in exercise tolerance and contraction-mediated translocation of GLUT4 to the plasma membrane in skeletal muscle.

Activation of AMPKα2 Is Not Required for Mitochondrial FAT/CD36 Accumulation during Exercise

Exercise has been shown to induce the translocation of fatty acid translocase (FAT/CD36), a fatty acid transport protein, to both plasma and mitochondrial membranes. While previous studies have examined signals involved in the induction of FAT/CD36 translocation to sarcolemmal membranes, to date the signaling events responsible for FAT/CD36 accumulation on mitochondrial membranes have not been investigated. In the current study muscle contraction rapidly increased FAT/CD36 on plasma membranes (7.5 minutes), while in contrast, FAT/CD36 only increased on mitochondrial membranes after 22.5 minutes of muscle contraction, a response that was exercise-intensity dependent. Considering that previous research has shown that AMP activated protein kinase (AMPK) α2 is not required for FAT/CD36 translocation to the plasma membrane, we investigated whether AMPK α2 signaling is necessary for mitochondrial FAT/CD36 accumulation. Administration of 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) induced AMPK phosphorylation, and resulted in FAT/CD36 accumulation on SS mitochondria, suggesting AMPK signaling may mediate this response. However, SS mitochondrial FAT/CD36 increased following acute treadmill running in both wild-type (WT) and AMPKα 2 kinase dead (KD) mice. These data suggest that AMPK signaling is not required for SS mitochondrial FAT/CD36 accumulation. The current data also implicates alternative signaling pathways that are exercise-intensity dependent, as IMF mitochondrial FAT/CD36 content only occurred at a higher power output. Taken altogether the current data suggests that activation of AMPK signaling is sufficient but not required for exercise-induced accumulation in mitochondrial FAT/CD36.

Beetroot juice increases human muscle force without changing Ca2+-handling proteins

Dietary inorganic nitrate (NO3−) supplementation improves skeletal muscle (SkM) contractile efficiency, and although rodent literature has suggested improvements in calcium handling or redox modifications as likely explanations, the direct mechanism of action in humans remains unknown. Purpose This study aimed to examine the effects of 7 d of beetroot juice (BRJ) supplementation on SkM contractile characteristics and function. Methods Recreationally active males (n = 8) underwent transcutaneous electrical muscle stimulation of the vastus lateralis for the evaluation of contractile characteristics before and after 7 d of BRJ supplementation (280 mL·d−1, ~26 mmol NO3−). An additional group of individuals (n = 8) followed the same supplementation protocol but underwent SkM biopsies pre- and post-supplementation for the determination of proteins associated with calcium handling via Western blotting, and the ratio of reduced/oxidized glutathione (GSH:GSSG), an indicator of cellular redox state, via high-performance liquid chromatography (HPLC). Results After supplementation, there was no change in maximal voluntary force production (602 ± 50 vs 596 ± 56 N) or electrically induced tetanic contractions. By contrast, force production was increased at 10 Hz electrical stimulation (41.1% ± 2.3% vs 37.6% ± 2.4% of peak force, P < 0.05), as was peak twitch tension (164.0 ± 12.5 vs 136.5 ± 7.2 N, P < 0.01) and maximal rates of force development and relaxation (3582.8 ± 382.3 vs 2575.7 ± 196.2 and −2752.4 ± 423.9 vs −2104.4 ± 249.0 N·s−1, respectively, P < 0.05). Despite these measurements implicating a change in calcium handling, the content of associated proteins (SERCA1a, SERCA2a, dihydropyradine receptor, ryanodine receptor, and calsequestrin) and the GSH:GSSG ratio were unaltered by BRJ. Conclusion BRJ supplementation increases force production at low-stimulation frequencies; however, in human SkM, this is independent of changes in redox stress or the expression of protein targets associated with calcium handling.

Glucagon receptor knockout mice are protected against acute olanzapine-induced hyperglycemia

OBJECTIVES To determine if glucagon is involved in mediating the increase in blood glucose levels caused by the second-generation antipsychotic drug olanzapine. MATERIALS AND METHODS Whole body glucagon receptor deficient mice (Gcgr-/-) or WT littermate controls were injected with olanzapine (5mg/kg BW IP) and changes in blood glucose measured over the following 120min. Separate cohorts of mice were treated with olanzapine and changes in pyruvate tolerance, insulin tolerance and whole body substrate oxidation were determined. RESULTS Olanzapine treatment increased serum glucagon and lead to rapid increases in blood glucose concentrations in WT mice. Gcgr-/- mice were protected against olanzapine-induced increases in blood glucose but this was not explained by differences in terminal serum insulin concentrations, enhanced AKT phosphorylation in skeletal muscle, adipose tissue or liver or differences in RER. In both genotypes olanzapine induced an equivalent degree of insulin resistance as measured using an insulin tolerance test. Olanzapine treatment led to an exaggerated glucose response to a pyruvate challenge in WT but not Gcgr-/- mice and this was paralleled by reductions in the protein content of PEPCK and G6Pase in livers from Gcgr-/- mice. CONCLUSIONS Gcgr-/- mice are protected against olanzapine-induced increases in blood glucose. This is likely a result of reductions in liver glucose output, perhaps secondary to decreases in PEPCK and G6Pase protein content. Our findings highlight the central role of the liver in mediating olanzapine-induced disturbances in glucose homeostasis.

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

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.