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 increaseḋ V O 2 max (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.

Assessment of mitochondrial ADP-stimulated respiratory kinetics in permeabilized skeletal myofibres (PmFB) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (∼20-300 μM) and tend to overestimate respiration at rest. Noting PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. Blebbistatin (BLEB), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >∼250 and ∼80 μM in red and white rat PmFB, respectively. In the absence of BLEB, PmFB contracted and the Km for ADP decreased by ∼2 to 10-fold in a temperature-dependent manner. PmFB were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30°C but not 37°C. In PmFB from humans, contraction elicited high sensitivity to ADP (m <100 μM) whereas blocking contraction (+BLEB) and including PCr:Cr = 2 to mimic the resting energetic state yielded a Km for ADP = ∼1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.

High-intensity aerobic interval training (HIIT) is a compromise between time-consuming moderate-intensity training and sprint-interval training requiring all-out efforts. However, there are few data regarding the ability of HIIT to increase the capacities of fat and carbohydrate oxidation in skeletal muscle. Using untrained recreationally active individuals, we investigated skeletal muscle and whole-body metabolic adaptations that occurred following 6 weeks of HIIT (~1 h of 10 x 4 min intervals at ~90% of peak oxygen consumption (VO2 peak), separated by 2 min rest, 3 d.week-1). A VO2 peak test, a test to exhaustion (TE) at 90% of pre-training VO2 peak, and a 1 h cycle at 60% of pre-training VO2 peak were performed pre- and post-HIIT. Muscle biopsies were sampled during the TE at rest, after 5 min, and at exhaustion. Training power output increased by 21%, and VO2 peak increased by 9% following HIIT. Muscle adaptations at rest included the following: (i) increased cytochrome c oxidase IV content (18%) and maximal activities of the mitochondrial enzymes citrate synthase (26%), beta-hydroxyacyl-CoA dehydrogenase (29%), aspartate-amino transferase (26%), and pyruvate dehydrogenase (PDH; 21%); (ii) increased FAT/CD36, FABPpm, GLUT 4, and MCT 1 and 4 transport proteins (14%-30%); and (iii) increased glycogen content (59%). Major adaptations during exercise included the following: (i) reduced glycogenolysis, lactate accumulation, and substrate phosphorylation (0-5 min of TE); (ii) unchanged PDH activation (carbohydrate oxidation; 0-5 min of TE); (iii) ~2-fold greater time during the TE; and (iv) increased fat oxidation at 60% of pre-training VO2 peak. This study demonstrated that 18 h of repeated high-intensity exercise sessions over 6 weeks (3 d.week-1) is a powerful method to increase whole-body and skeletal muscle capacities to oxidize fat and carbohydrate in previously untrained individuals.

A growing body of research is investigating the potential contribution of mitochondrial function to the etiology of type 2 diabetes. Numerous in vitro, in situ, and in vivo methodologies are available to examine various aspects of mitochondrial function, each requiring an understanding of their principles, advantages, and limitations. This review provides investigators with a critical overview of the strengths, limitations and critical experimental parameters to consider when selecting and conducting studies on mitochondrial function. In vitro (isolated mitochondria) and in situ (permeabilized cells/tissue) approaches provide direct access to the mitochondria, allowing for study of mitochondrial bioenergetics and redox function under defined substrate conditions. Several experimental parameters must be tightly controlled, including assay media, temperature, oxygen concentration, and in the case of permeabilized skeletal muscle, the contractile state of the fibers. Recently developed technology now offers the opportunity to measure oxygen consumption in intact cultured cells. Magnetic resonance spectroscopy provides the most direct way of assessing mitochondrial function in vivo with interpretations based on specific modeling approaches. The continuing rapid evolution of these technologies offers new and exciting opportunities for deciphering the potential role of mitochondrial function in the etiology and treatment of diabetes.

Key point• ATP transfer from mitochondria to the cytoplasm occurs mainly through phosphate transfer to creatine by mitochondrial creatine kinase (miCK) but also by transport and/or diffusion of ADP and ATP through specific mitochondrial transport protein complexes.• Determining the effect of exercise on phosphate shuttling may require contractile signals in situ and varying creatine concentrations to alter miCK activity.• Mitochondrial respiratory sensitivity to ADP was assessed in permeabilized muscle fibre bundles (PmFBs) before and after 2 h cycling exercise in human skeletal muscle.• In relaxed PmFBs, ADP sensitivity decreased post-exercise when miCK phosphate shuttling was low (no creatine) with no change in net ADP sensitivity in the presence of creatine, whereas in contracting fibres post-exercise ADP sensitivity was higher with creatine.• This shows miCK activity is increased post-exercise, especially during contraction in PmFBs, and suggests exercise regulates phosphate shuttling, which would improve maintenance of energy homeostasis during contraction.Abstract Energy transfer between mitochondrial and cytosolic compartments is predominantly achieved by creatine-dependent phosphate shuttling (PCr/Cr) involving mitochondrial creatine kinase (miCK). However, ADP/ATP diffusion through adenine nucleotide translocase (ANT) and voltage-dependent anion carriers (VDACs) is also involved in this process. To determine if exercise alters the regulation of this system, ADP-stimulated mitochondrial respiratory kinetics were assessed in permeabilized muscle fibre bundles (PmFBs) taken from biopsies before and after 2 h of cycling exercise (60%V O 2 peak ) in nine lean males. Concentrations of creatine (Cr) and phosphocreatine (PCr) as well as the contractile state of PmFBs were manipulated in situ. In the absence of contractile signals (relaxed PmFBs) and miCK activity (no Cr), post-exercise respiratory sensitivity to ADP was reduced in situ (up to 126% higher apparent K m to ADP) suggesting inhibition of ADP/ATP diffusion between matrix and cytosolic compartments (possibly ANT and VDACs). However this effect was masked in the presence of saturating Cr (no effect of exercise on ADP sensitivity). Given that the role of ANT is thought to be independent of Cr, these findings suggest ADP/ATP, but not PCr/Cr, cycling through the outer mitochondrial membrane (VDACs) may be attenuated in resting muscle after exercise. In contrast, in contracted PmFBs, post-exercise respiratory sensitivity to ADP increased with miCK activation (saturating Cr; 33% lower apparent K m to ADP), suggesting prior exercise increases miCK sensitivity in situ. These observations demonstrate that exercise increases miCK-dependent respiratory sensitivity to ADP, promoting mitochondrial-cytosolic energy exchange via PCr/Cr cycling, possibly through VDACs. This effect may mask an underlying inhibition of Cr-independent ADP/ATP diffusion. This enhanced regulation of miCK-dependent phosphate shuttling may improve energy homeostasis through more efficient c...

The effects of training on silent mating-type information regulator 2 homolog 1 (SIRT1) activity and protein in relationship to peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) and mitochondrial content were determined in human skeletal muscle. Six weeks of high-intensity interval training ( approximately 1 h of 10 x 4 min intervals at 90% peak oxygen consumption separated by 2 min rest, 3 days per week) increased maximal activities of mitochondrial enzymes in skeletal muscle by 28% to 36% (citrate synthase, beta-hydroxyacyl-coenzyme A dehydrogenase, and cytochrome c oxidase subunit IV) and PGC-1alpha protein (16%) when measured 4 days after training. Interestingly, total muscle SIRT1 activity (31%) and activity per SIRT1 protein (58%) increased despite decreased SIRT1 protein (20%). The present data demonstrate that exercise-induced mitochondrial biogenesis is accompanied by elevated SIRT1 activity in human skeletal muscle.

Salsalate is a prodrug of salicylate that lowers blood glucose in patients with type 2 diabetes (T2D) and reduces nonalcoholic fatty liver disease (NAFLD) in animal models; however, the mechanism mediating these effects is unclear. Salicylate directly activates AMPK via the β1 subunit, but whether salsalate requires AMPK-β1 to improve T2D and NAFLD has not been examined. Therefore, wild-type (WT) and AMPK-β1-knockout (AMPK-β1KO) mice were treated with a salsalate dose resulting in clinically relevant serum salicylate concentrations (~1 mmol/L). Salsalate treatment increased VO 2 , lowered fasting glucose, improved glucose tolerance, and led to an ~55% reduction in liver lipid content. These effects were observed in both WT and AMPK-β1KO mice. To explain these AMPK-independent effects, we found that salicylate increases oligomycin-insensitive respiration (state 4o) and directly increases mitochondrial proton Corresponding author: Gregory R. Steinberg, gsteinberg@mcmaster.ca. Duality of Interest. No potential conflicts of interest relevant to this article were reported.Author Contributions. B.K.S. and G.R.S. designed research studies, analyzed data, and wrote the manuscript. B.K.S., R.J.F., E.M.D., A.E.G., M.C.H., V.P.H., E.A.D., K.M., J.D.C., E.P.M., and C.G.R.P. conducted experiments and analyzed data. B.K.S., R.J.F., E.M.D., A.E.G., V.P.H., E.A.D., K.M., J.D.C., E.P.M., B.E.K., M.A.T., and G.R.S. edited the manuscript. G.R.S. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0564/-/DC1. Diabetes. Author manuscript; available in PMC 2017 January 12.Published in final edited form as:Diabetes. 2016 November ; 65(11): 3352-3361. doi:10.2337/db16-0564. CIHR Author Manuscript CIHR Author Manuscript CIHR Author Manuscriptconductance at clinical concentrations. This uncoupling effect is tightly correlated with the suppression of de novo lipogenesis. Salicylate is also able to stimulate brown adipose tissue respiration independent of uncoupling protein 1. These data indicate that the primary mechanism by which salsalate improves glucose homeostasis and NAFLD is via salicylate-driven mitochondrial uncoupling.Nonalcoholic fatty liver disease (NAFLD) is considered an important contributing factor to the development of insulin resistance and type 2 diabetes (T2D) (1). Despite the rising prevalence of NAFLD and importance for the development of T2D, there are currently no pharmacological approaches for the treatment of this disease (2).Salsalate is a prodrug of salicylate and is hydrolyzed in the small intestine to produce two molecules of salicylate (3,4). The circulating concentration of salicylate in humans administered salsalate in T2D clinical trials is ~1 mmol/L (5-8). Salsalate has also been shown to improve symptoms of NAFLD (9) and nonalcoholic steatohepatitis in...

We examined the relationship between the pressure-time product (Pdt) of the inspiratory muscles and the O2 cost of breathing (VO2 resp) in five normal subjects breathing through an external inspiratory resistance with a tidal volume of 800 ml at a constant end-expiratory lung volume [functional residual capacity, (FRC)]. Each subject performed 30-40 runs, each of approximately 30 breaths, with inspiratory flow rates ranging from 0.26 +/- 0.01 to 0.89 +/- 0.04 l/s (means +/- SE) and inspiratory mouth pressures ranging from 10 +/- 1 to 68 +/- 4% of the maximum inspiratory pressure at FRC. In all subjects VO2 resp was linearly related to Pdt when mean inspiratory flow (VI) was constant, but the slope of this relationship increased with increasing VI. Therefore, Pdt is an accurate index of VO2 resp only when VI is constant. There was a linear relationship between the VO2 resp and the work rate across the external resistance (W) for all runs in each subject over the range of W 10 +/- 1 to 137 +/- 21 J/min. Thus, at a constant tidal volume the VO2 resp was related to the mean inspiratory pressure, independent of flow or inspiratory duration. If the VO2 resp were determined mainly during inspiration, then for a given rate of external work or O2 consumption, VI would be inversely related to mean inspiratory pressure. Efficiency (E) was 2.1 +/- 0.2% and constant over a large range of VI, pressure, work rate, or resistance and was not altered by the presence of a potentially fatiguing load. The constant E over such a wide range of conditions implies a complex integration of the recruitment, mechanical function, and energy consumption of the muscles utilized in breathing.