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      Moderate‐intensity training in hypoxia improves exercise performance and glycolytic capacity of skeletal muscle in horses

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          Abstract

          We investigated whether moderate‐intensity training of horses in moderate hypoxia for 4 weeks elicits greater adaptations in exercise performance, aerobic capacity, and glycolytic/oxidative metabolism in skeletal muscle compared to normoxic training. In a randomized crossover study design, seven untrained Thoroughbred horses (5.9 ± 1.1 years, 508 ± 9 kg) completed 4 weeks (3 sessions/week) of two training protocols consisting of 3‐min cantering at 70% of maximal oxygen consumption ( V ˙ O 2 max ) in hypoxia (HYP; F IO 2 = 14.7%) and normoxia (NOR; F IO 2 = 21.0%) with a 4‐month washout period. Normoxic incremental exercise tests (IET) were conducted before and after training. Biopsy samples were obtained from the middle gluteal muscle before IET and monocarboxylate transporter (MCT) protein expression and glycolytic/mitochondrial enzyme activities were analyzed. Data were analyzed using mixed models ( p < 0.05). Running speed was 7.9 ± 0.2 m/s in both groups and arterial oxygen saturation during training in NOR and HYP were 92.9 ± 0.9% and 75.7 ± 3.9%, respectively. Run time in HYP (+9.7%) and V ˙ O 2 max in both groups (NOR, +6.4%; HYP, +4.3%) at IET increased after 4 weeks of training. However, cardiac output, arterial‐mixed venous O 2 difference, and hemoglobin concentration at exhaustion were unchanged in both conditions. While MCT1 protein and citrate synthase activity did not increase in both conditions after training, MCT4 protein (+13%), and phosphofructokinase activity (+42%) increased only in HYP. In conclusion, 4 weeks of moderate‐intensity hypoxic training improves exercise performance and glycolytic capacity of skeletal muscle in horses.

          Abstract

          Four weeks of moderate‐intensity training in hypoxia (70% V ˙ O 2 max 3 min, 3 sessions/week, F IO 2 = 14.7%) increased run time and V ˙ O 2 max at incremental exercise tests, monocarboxylate transporter (MCT) 4 protein content and phosphofructokinase (PFK) activity of skeletal muscle in horses, whereas the same training in normoxia only increased V ˙ O 2 max .

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          Cell-cell and intracellular lactate shuttles.

          Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilized continuously in diverse cells under fully aerobic conditions. 'Cell-cell' and 'intracellular lactate shuttle' concepts describe the roles of lactate in delivery of oxidative and gluconeogenic substrates as well as in cell signalling. Examples of the cell-cell shuttles include lactate exchanges between between white-glycolytic and red-oxidative fibres within a working muscle bed, and between working skeletal muscle and heart, brain, liver and kidneys. Examples of intracellular lactate shuttles include lactate uptake by mitochondria and pyruvate for lactate exchange in peroxisomes. Lactate for pyruvate exchanges affect cell redox state, and by itself lactate is a ROS generator. In vivo, lactate is a preferred substrate and high blood lactate levels down-regulate the use of glucose and free fatty acids (FFA). As well, lactate binding may affect metabolic regulation, for instance binding to G-protein receptors in adipocytes inhibiting lipolysis, and thus decreasing plasma FFA availability. In vitro lactate accumulation upregulates expression of MCT1 and genes coding for other components of the mitochondrial reticulum in skeletal muscle. The mitochondrial reticulum in muscle and mitochondrial networks in other aerobic tissues function to establish concentration and proton gradients necessary for cells with high mitochondrial densities to oxidize lactate. The presence of lactate shuttles gives rise to the realization that glycolytic and oxidative pathways should be viewed as linked, as opposed to alternative, processes, because lactate, the product of one pathway, is the substrate for the other.
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            Repeated transient mRNA bursts precede increases in transcriptional and mitochondrial proteins during training in human skeletal muscle.

            Exercise training induces mitochondrial biogenesis, but the time course of molecular sequelae that accompany repetitive training stimuli remains to be determined in human skeletal muscle. Therefore, throughout a seven-session, high-intensity interval training period that increased (12%), we examined the time course of responses of (a) mitochondrial biogenesis and fusion and fission proteins, and (b) selected transcriptional and mitochondrial mRNAs and proteins in human muscle. Muscle biopsies were obtained 4 and 24 h after the 1st, 3rd, 5th and 7th training session. PGC-1α mRNA was increased >10-fold 4 h after the 1st session and returned to control within 24 h. This 'saw-tooth' pattern continued until the 7th bout, with smaller increases after each bout. In contrast, PGC-1α protein was increased 24 h after the 1st bout (23%) and plateaued at +30-40% between the 3rd and 7th bout. Increases in PGC-1β mRNA and protein were more delayed and smaller, and did not persist. Distinct patterns of increases were observed in peroxisome proliferator-activated receptor (PPAR) α and γ protein (1 session), PPAR β/δ mRNA and protein (5 sessions) and nuclear respiratory factor-2 protein (3 sessions) while no changes occurred in mitochondrial transcription factor A protein. Citrate synthase (CS) and β-HAD mRNA were rapidly increased (1 session), followed 2 sessions later (session 3) by increases in CS and β-HAD activities, and mitochondrial DNA. Changes in COX-IV mRNA (session 3) and protein (session 5) were more delayed. Training also increased mitochondrial fission proteins (fission protein-1, >2-fold; dynamin-related protein-1, 47%) and the fusion protein mitofusin-1 (35%) but not mitofusin-2. This study has provided the following novel information: (a) the training-induced increases in transcriptional and mitochondrial proteins appear to result from the cumulative effects of transient bursts in their mRNAs, (b) training-induced mitochondrial biogenesis appears to involve re-modelling in addition to increased mitochondrial content, and (c) the 'transcriptional capacity' of human muscle is extremely sensitive, being activated by one training bout.
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              Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells.

              The assessment of mitochondrial respiratory chain (RC) enzymatic activities is essential for investigating mitochondrial function in several situations, including mitochondrial disorders, diabetes, cancer, aging and neurodegeneration, as well as for many toxicological assays. Muscle is the most commonly analyzed tissue because of its high metabolic rates and accessibility, although other tissues and cultured cell lines can be used. We describe a step-by-step protocol for a simple and reliable assessment of the RC enzymatic function (complexes I-IV) for minute quantities of muscle, cultured cells and isolated mitochondria from a variety of species and tissues, by using a single-wavelength spectrophotometer. An efficient tissue disruption and the choice for each assay of specific buffers, substrates, adjuvants and detergents in a narrow concentration range allow maximal sensitivity, specificity and linearity of the kinetics. This protocol can be completed in 3 h.
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                Author and article information

                Contributors
                mukai@equinst.go.jp
                Journal
                Physiol Rep
                Physiol Rep
                10.1002/(ISSN)2051-817X
                PHY2
                physreports
                Physiological Reports
                John Wiley and Sons Inc. (Hoboken )
                2051-817X
                10 December 2021
                December 2021
                : 9
                : 23 ( doiID: 10.1002/phy2.v9.23 )
                : e15145
                Affiliations
                [ 1 ] Sports Science Division Equine Research Institute Japan Racing Association Shimotsuke Tochigi Japan
                [ 2 ] Department of Human Sciences Kanagawa University Yokohama Kanagawa Japan
                [ 3 ] Department of Sports Sciences University of Tokyo Tokyo Japan
                Author notes
                [*] [* ] Correspondence

                Kazutaka Mukai, Equine Research Institute, Japan Racing Association, 1400‐4 Shiba, Shimotsuke, Tochigi, 329‐0412, Japan.

                Email: mukai@ 123456equinst.go.jp

                Author information
                https://orcid.org/0000-0002-1992-6634
                https://orcid.org/0000-0001-6932-2735
                https://orcid.org/0000-0001-8643-6849
                Article
                PHY215145
                10.14814/phy2.15145
                8661515
                34889527
                725832a3-f392-4ef4-a063-3bd087a93ef8
                © 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 25 November 2021
                : 05 August 2021
                : 29 November 2021
                Page count
                Figures: 6, Tables: 1, Pages: 11, Words: 6258
                Funding
                Funded by: Japan Racing Association , doi 10.13039/100015103;
                Categories
                Original Article
                Original Articles
                Custom metadata
                2.0
                December 2021
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.0.9 mode:remove_FC converted:10.12.2021

                glycolytic capacity,horse,hypoxic training,performance
                glycolytic capacity, horse, hypoxic training, performance

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