Rate Of Oxidative ATP Synthesis Research Articles

245-OR: Age and Diabetes Interact to Decrease Muscle Mitochondrial Function

People with T2D have lower cardiorespiratory fitness (CRF) than those without diabetes contributing to increased cardiovascular morbidity and mortality. People with T2D have impaired oxidative flux associated with lower CRF. Yet, reports on muscle mitochondrial dysfunction in T2D are mixed. As diabetes duration and age are predictors of poor outcomes, we hypothesized that T2D and increased age associate with impaired mitochondrial function. We assessed post-exercise calf mitochondrial function in overweight sedentary participants (45 with and 54 without T2D) ages 30-50 and 51-70y. Mitochondrial function was measured in vivo by 31Phosphorous magnetic resonance spectroscopy (31P-MRS) before, during and after 90 seconds of isotonic calf press at 70% maximal force and ex vivo by mitochondrial respiration in muscle fibers using the Oroboros Oxygraph-2k. There was no significant effect of age or T2D on ex vivo measures. MRS-measured phosphocreatine synthesis, apparent maximum rate of oxidative ATP synthesis and ADP time constant were worse in those with T2D or age >50 (Figure) . Age and T2D interact to significantly decrease apparent maximum rate of oxidative ATP synthesis. These data suggest that age and T2D decrease in vivo oxidative capacity. Given that impairment is only found in in vivo mitochondrial measures, factors such as micro- and macrovascular function and blood flow, combined with cardiac function, are likely implicated. Disclosure L.Abushamat: None. J.G.Regensteiner: None. R.L.Scalzo: None. D.Ramirez: None. I.E.Schauer: None. L.Knaub: None. E.Clark: None. Y.Garcia reyes: None. M.Cree-green: None. J.Reusch: Advisory Panel; Medtronic. Funding American Diabetes Association (1-21-CMF-003) (1-12-CT-64) ; Department of Veterans Affairs (BX002046, CX001532)

Single-leg exercise training augments in vivo skeletal muscle oxidative flux and vascular content and function in adults with type 2 diabetes.

People with type 2 diabetes (T2D) have impaired skeletal muscle oxidative flux due to limited oxygen delivery. In the current study, this impairment in oxidative flux in people with T2D was abrogated with a single-leg exercise training protocol. Additionally, single-leg exercise training increased skeletal muscle CD31 content, calf blood flow and state 4 mitochondrial respiration in all participants. Cardiorespiratory fitness is impaired in type 2 diabetes (T2D), conferring significant cardiovascular risk in this population; interventions are needed. Previously, we reported that a T2D-associated decrement in skeletal muscle oxidative flux is ameliorated with acute use of supplemental oxygen, suggesting that skeletal muscle oxygenation is rate-limiting to in vivo mitochondrial oxidative flux during exercise in T2D. We hypothesized that single-leg exercise training (SLET) would improve the T2D-specific impairment in in vivo mitochondrial oxidative flux during exercise. Adults with (n=19) and without T2D (n=22) with similar body mass indexes and levels of physical activity participated in two weeks of SLET. Following SLET, in vivo oxidative flux measured by 31 P-MRS increased in participants with T2D, but not people without T2D, measured by the increase in initial phosphocreatine synthesis (P=0.0455 for the group × exercise interaction) and maximum rate of oxidative ATP synthesis (P=0.0286 for the interaction). Additionally, oxidative phosphorylation increased in all participants with SLET (P=0.0209). After SLET, there was no effect of supplemental oxygen on any of the in vivo oxidative flux measurements in either group (P>0.02), consistent with resolution of the T2D-associated oxygen limitation previously observed at baseline in subjects with T2D. State 4 mitochondrial respiration also improved in muscle fibres ex vivo. Skeletal muscle vasculature content and calf blood flow increased in all participants with SLET (P<0.0040); oxygen extraction in the calf increased only in T2D (P=0.0461). SLET resolves the T2D-associated impairment of skeletal muscle in vivo mitochondrial oxidative flux potentially through improved effective blood flow/oxygen delivery.

Rates of oxidative ATP synthesis are not augmented beyond the pH threshold in human vastus lateralis muscles during a stepwise contraction protocol.

The oxygen cost of high-intensity exercise at power outputs above an individual's lactate threshold (LT) is greater than would be predicted by the linear oxygen consumption-power relationship observed below the LT. However, whether these augmentations are caused by an increased ATP cost of force generation (ATPCOST ) or an increased oxygen cost of ATP synthesis is unclear. We used 31 P-MRS to measure changes in cytosolic [ADP] (intramyocellular marker of oxidative metabolism), oxidative ATP synthesis (ATPOX ) and ATPCOST during a 6-stage, stepwise knee extension protocol. ATPCOST was unchanged across stages. The relationship between [ADP] and muscle power output was augmented at workloads above the pH threshold (pHT ; proxy for LT), whereas increases in ATPOX were attenuated. These results suggest the greater oxygen cost of contractions at workloads beyond the pHT is not caused by mechanisms that increase ATPCOST , but rather mechanisms that alter intrinsic mitochondrial function or capacity. Increases in skeletal muscle metabolism and oxygen consumption are linearly related to muscle power output for workloads below the lactate threshold (LT), but are augmented (i.e. greater rate of increase relative to workload) thereafter. Presently, it is unclear whether these metabolic augmentations are caused by increases in the ATP cost of force generation (ATPCOST ) or changes in the efficiency of mitochondrial oxygen consumption and oxidative ATP synthesis (ATPOX ). To partition these two hypotheses in vivo, we used 31 P-MRS to calculate slopes relating step-changes in muscle work to concurrent changes in cytosolic phosphates and ATPOX before and after the pH threshold (pHT ; used here as a proxy for LT) within the vastus lateralis muscle of eight young adults during a stepwise knee extension test. Changes in muscle phosphates and ATPOX were linearly related to workload below the pHT . However, slopes above the pHT were greater for muscle phosphates (P<0.05) and lower for ATPOX (P<0.05) than were the slopes observed below the pHT . The maximal capacity for ATPOX ( ) and ADP-specific ATPOX also declined beyond the pHT (P<0.05), whereas ATPCOST was unchanged (P=0.10). These results oppose the hypothesis that high-intensity contractions increase ATPCOST and suggest that greater oxidative metabolism at workloads beyond the pHT is caused by mechanisms that affect intrinsic mitochondrial function or capacity, such as alterations in substrate selection or electron entry into the electron transport chain, temperature-mediated changes in mitochondrial permeability to protons, or stimulation of mitochondrial uncoupling by reactive oxygen species generation.

Skeletal Muscle Mitochondrial Adaptations to Maximal Strength Training in Older Adults.

Maximal strength training (MST) results in robust improvements in skeletal muscle force production, efficiency, and mass. However, the effects of MST on muscle mitochondria are still unknown. Accordingly, the purpose of this study was to examine, from the molecular level to whole-muscle, mitochondrial adaptations induced by 8 weeks of knee-extension MST in the quadriceps of 10 older adults using immunoblotting, spectrophotometry, high-resolution respirometry in permeabilized muscle fibers, in vivo 31P magnetic resonance spectroscopy (31P-MRS), and gas exchange. As anticipated, MST resulted in an increased isometric knee-extensor force from 133 ± 36 to 147 ± 49 Nm (p < .05) and quadriceps muscle volume from 1,410 ± 103 to 1,555 ± 455 cm3 (p < .05). Mitochondrial complex (I-V) protein abundance and citrate synthase activity were not significantly altered by MST. Assessed ex vivo, maximal ADP-stimulated respiration (state 3CI+CII, PRE: 23 ± 6 and POST: 14 ± 5 ρM·mg-1·s-1, p < .05), was decreased by MST, predominantly, as a result of a decline in complex I-linked respiration (p < .05). Additionally, state 3 free-fatty acid linked respiration was decreased following MST (PRE: 19 ± 5 and POST: 14 ± 3 ρM·mg-1·s-1, p < .05). Assessed in vivo, MST slowed the PCr recovery time constant (PRE: 49 ± 13 and POST: 57 ± 16 seconds, p < .05) and lowered, by ~20% (p = .055), the quadriceps peak rate of oxidative ATP synthesis, but did not significantly alter the oxidation of lipid. Although these, likely qualitative, mitochondrial adaptations are potentially negative in terms of skeletal muscle energetic capacity, they need to be considered in light of the many improvements in muscle function that MST affords older adults.

Effects of a mitochondria targeted antioxidant on skeletal muscle mitochondrial function with cigarette smoke exposure and Chronic Obstructive Pulmonary Disease

Reactive oxygen species (ROS) are important signaling molecules in cells. However, the exaggerated free radical production accompanying mitochondrial dysfunction with Chronic Obstructive Pulmonary Disease (COPD) has been suggested to play a crucial role in the development of metabolic abnormalities in these patients. Consequently, selective inhibition of mitochondrial ROS could be a potential therapeutic strategy to restore mitochondrial function and subsequently attenuate exercise‐induced metabolic disturbance during exercise in COPD patients. The goal of this study was therefore to determine the effects of a mitochondria targeted antioxidant, MitoQ, on free radical levels, mitochondrial function, and skeletal muscle metabolism in six patients with moderate to very‐severe COPD and a smoking mouse model (C57/Bl6 exposed to cigarette smoke for 8 months) using electron paramagnetic spectroscopy (EPR), 31P/1H magnetic resonance spectroscopy (1H/31P MRS), high‐resolution respirometry in permeabilized skeletal muscle fiber. 4 weeks of MitoQ supplementation (MQ) decreased blood free radical levels measured by EPR (control, CTL: 4.3 ± 3.3 a.u.; MQ: 1.8 ± 1.7 a.u.; P&lt;0.05). Nevertheless, the exercise‐induced changes in intracellular O2 partial pressure, high‐energy phosphate, and pH measured by 1H/31P MRS in the plantar flexor muscles of patients with COPD were not significantly altered with MitoQ. Also, the peak rate of oxidative ATP synthesis measured by 31P‐MRS was lower with MitoQ (CTL: 19 ± 6 mM.min−1; MQ: 14 ± 4 mM.min−1, P&lt;0.05), suggesting an impaired mitochondrial function in vivo. This finding was further supported by the lower muscle respiratory capacity measured in permeabilized skeletal muscle fibers incubated with MitoQ (−43%, P&lt;0.05) from a smoking mouse model. Specifically, complex I (−42%) and free‐fatty acid (−51%) linked respiration were significantly decreased with MitoQ (P&lt;0.05). Together, these findings suggest that decreasing ROS levels using a mitochondria targeted antioxidant may, in fact, be detrimental to oxidative phosphorylation capacity in the skeletal muscle of COPD patients.Support or Funding InformationThis work was funded in part by grants from the Flight Attendant Medical Research Institute (FAMRI), NIH National Heart, Lung, and Blood Institute (HL125756)

Control of Cardiac Mitochondrial Fuel Selection by Calcium

Calcium ion concentration modulates the function of several mitochondrial enzymes. Specifically, the kinetic operations of the decarboxylating dehydrogenases pyruvate dehydrogenase, isocitrate dehydrogenase, alpha‐ketoglutarate dehydrogenase are all affected by [Ca2+]. Previous studies have shown that, despite its ability to affect the function of specific dehydrogenases, [Ca2+] does not substantially alter mitochondrial ATP synthesis in vitro or in vivo in the heart. We hypothesize that, rather than contributing to respiratory control, [Ca2+] plays a role in contributing to fuel selection. Specifically, cardiac mitochondria are able to use different primary carbon substrates (carbohydrates, fatty acids, and ketones) to synthesize ATP aerobically in the living cells. To determine if and how [Ca2+] affects the relative use of carbohydrates versus fatty acids in vitro we measured oxygen consumption and TCA cycle intermediate concentrations in suspensions of cardiac mitochondria with different combinations of pyruvate and palmitoyl‐L‐carnitine in the media at various [Ca2+] and ADP infusion rates. Stoichiometric analysis of the data reveals that when both fatty acid and carbohydrate substrates are available, fuel selection is sensitive to both the rate of ADP infusion and to the calcium concentration. With increasing both the rate of oxidative ATP synthesis and [Ca2+], the fuel utilization ratio shifts to increased fractional consumption of pyruvate. Thus, our results indicate that the effects of ATP synthesis rate and [Ca2+] are interdependent.Support or Funding InformationThis work supported by NIH grants HL122199 and T32GM008322This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Calcium and ADP Infusion Rates in Cardiac Mitochondrial Fuel Selection

Calcium ion concentration modulates the function of several mitochondrial enzymes. Specifically, the kinetic operations of the decarboxylating dehydrogenases pyruvate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase are all affected by [Ca2+]. Previous studies have shown that, despite its ability to affect the function of specific dehydrogenases, [Ca2+] does not substantially alter mitochondrial ATP synthesis in vitro or in vivo in the heart. We hypothesize that, rather than contributing to respiratory control, [Ca2+] plays a role in contributing to fuel selection. Specifically, cardiac mitochondria are able to use different primary carbon substrates (carbohydrates, fatty acids, and ketones) to synthesize ATP aerobically in the living cells. To determine if and how [Ca2+] affects the relative use of carbohydrates versus fatty acids in vitro we measured oxygen consumption and TCA cycle intermediate concentrations in suspensions of cardiac mitochondria with different combinations of pyruvate and palmitoyl-L-carnitine in the media and at various calcium concentrations and ADP infusion rates. Stoichiometric analysis of the data reveals that when both fatty acid and carbohydrate substrates are available, fuel selection is sensitive to both the rate of ADP infusion and to the calcium concentration. Under low-flux (leak state) conditions and with zero added Ca2+, β-oxidation provides roughly 70% of acetyl-CoA for the citrate synthase reaction, with the rest coming from the pyruvate dehydrogenase reaction. With both increasing rates of oxidative ATP synthesis and with increasing [Ca2+], the fuel utilization ratio shifts to increased fractional consumption of pyruvate. The effects of ATP synthesis load and [Ca2+] are shown to be interdependent.

Mitochondrial impairment induced by postnatal ActRIIB blockade does not alter function and energy status in exercising mouse glycolytic muscle in vivo

Because it leads to a rapid and massive muscle hypertrophy, postnatal blockade of the activin type IIB receptor (ActRIIB) is a promising therapeutic strategy for counteracting muscle wasting. However, the functional consequences remain very poorly documented in vivo. Here, we have investigated the impact of 8-wk ActRIIB blockade with soluble receptor (sActRIIB-Fc) on gastrocnemius muscle anatomy, energy metabolism, and force-generating capacity in wild-type mice, using totally noninvasive magnetic resonance imaging (MRI) and dynamic(31)P-MRS. Compared with vehicle (PBS) control, sActRIIB-Fc treatment resulted in a dramatic increase in body weight (+29%) and muscle volume (+58%) calculated from hindlimb MR imaging, but did not alter fiber type distribution determined via myosin heavy chain isoform analysis. In resting muscle, sActRIIB-Fc treatment induced acidosis and PCr depletion, thereby suggesting reduced tissue oxygenation. During an in vivo fatiguing exercise (6-min repeated maximal isometric contraction electrically induced at 1.7 Hz), maximal and total absolute forces were larger in sActRIIB-Fc treated animals (+26 and +12%, respectively), whereas specific force and fatigue resistance were lower (-30 and -37%, respectively). Treatment with sActRIIB-Fc further decreased the maximal rate of oxidative ATP synthesis (-42%) and the oxidative capacity (-34%), but did not alter the bioenergetics status in contracting muscle. Our findings demonstrate in vivo that sActRIIB-Fc treatment increases absolute force-generating capacity and reduces mitochondrial function in glycolytic gastrocnemius muscle, but this reduction does not compromise energy status during sustained activity. Overall, these data support the clinical interest of postnatal ActRIIB blockade.

Insulin Resistance Is Not Associated with an Impaired Mitochondrial Function in Contracting Gastrocnemius Muscle of Goto-Kakizaki Diabetic Rats In Vivo.

Insulin resistance, altered lipid metabolism and mitochondrial dysfunction in skeletal muscle would play a major role in type 2 diabetes mellitus (T2DM) development, but the causal relationships between these events remain conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated throughout a multidisciplinary approach combining in vivo and in vitro measurements in Goto-Kakizaki (GK) rats, a non-obese T2DM model developing peripheral insulin resistant without abnormal level of plasma non-esterified fatty acids (NEFA). Wistar rats were used as controls. Mechanical performance and energy metabolism were assessed strictly non-invasively using magnetic resonance (MR) imaging and 31-phosphorus MR spectroscopy (31P-MRS). Compared with control group, plasma insulin and glucose were respectively lower and higher in GK rats, but plasma NEFA level was normal. In resting GK muscle, phosphocreatine content was reduced whereas glucose content and intracellular pH were both higher. However, there were not differences between both groups for basal oxidative ATP synthesis rate, citrate synthase activity, and intramyocellular contents for lipids, glycogen, ATP and ADP (an important in vivo mitochondrial regulator). During a standardized fatiguing protocol (6 min of maximal repeated isometric contractions electrically induced at a frequency of 1.7 Hz), mechanical performance and glycolytic ATP production rate were reduced in diabetic animals whereas oxidative ATP production rate, maximal mitochondrial capacity and ATP cost of contraction were not changed. These findings provide in vivo evidence that insulin resistance is not caused by an impairment of mitochondrial function in this diabetic model.

Opposite effects of hyperoxia on mitochondrial and contractile efficiency in human quadriceps muscles.

Exercise efficiency is an important determinant of exercise capacity. However, little is known about the physiological factors that can modulate muscle efficiency during exercise. We examined whether improved O2 availability would 1) impair mitochondrial efficiency and shift the energy production toward aerobic ATP synthesis and 2) reduce the ATP cost of dynamic contraction owing to an improved neuromuscular efficiency, such that 3) whole body O2 cost would remain unchanged. We used (31)P-magnetic resonance spectroscopy, surface electromyography, and pulmonary O2 consumption (V̇o2p) measurements in eight active subjects during 6 min of dynamic knee-extension exercise under different fractions of inspired O2 (FiO2 , 0.21 in normoxia and 1.0 in hyperoxia). V̇o2p (755 ± 111 ml/min in normoxia and 799 ± 188 ml/min in hyperoxia, P > 0.05) and O2 cost (P > 0.05) were not significantly different between normoxia and hyperoxia. In contrast, the total ATP synthesis rate and the ATP cost of dynamic contraction were significantly lower in hyperoxia than normoxia (P < 0.05). As a result, the ratio of the rate of oxidative ATP synthesis from the quadriceps to V̇o2p was lower in hyperoxia than normoxia but did not reach statistical significance (16 ± 3 mM/ml in normoxia and 12 ± 5 mM/ml in hyperoxia, P = 0.07). Together, these findings reveal dynamic and independent regulations of mitochondrial and contractile efficiency as a consequence of O2 availability in young active individuals. Furthermore, muscle efficiency appears to be already optimized in normoxia and is unlikely to contribute to the well-established improvement in exercise capacity induced by hyperoxia.

Short‐term training alters the control of mitochondrial respiration rate before maximal oxidative ATP synthesis

BackgroundThere has not been a study that has directly assessed temporal changes in mitochondrial oxidative capacity and metabolic control as a consequence of short‐term exercise training in vivo.MethodsWe used 31P‐magnetic resonance spectroscopy (31P‐MRS) to examine the effect of short‐term plantar flexion exercise training on phosphocreatine (PCr) recovery kinetics and the control of respiration rate. To this aim, we investigated 12 healthy men experienced with this exercise modality (TRA) and 8 time‐control subjects (TC).ResultsAfter 5 days of training, maximum work rate during incremental plantar flexion exercise was significantly improved (P &lt; 0.01). During the recovery period, the maximal rate of oxidative ATP synthesis (PRE: 28 ± 13 mM.min−1; POST: 26 ± 15 mM.min−1) and the PCr recovery time constant (PRE: 31 ± 19 s; POST: 29 ± 16) were not significantly altered. In contrast, the Hill coefficient (nH) describing the cooperativity between respiration rate and ADP was significantly increased in TRA (PRE:nH =2.7 ± 1.4; POST: nH =3.4 ± 1.9, P &lt; 0.05). Meanwhile, we did not observe any systematic variation in any of these variables in the TC.ConclusionThis study reveals that 5 days of training induces rapid adaptation in the allosteric control of respiration rate by ADP before any substantial improvement in muscle oxidative capacity.

Exercise‐induced PCr recovery kinetics and tissue oxygenation: The role of free radicals and aging

We investigated the role of free radicals on muscle oxidative capacity and O2 availability in activity matched young (Y) and old (O) people following exercise. 39 subjects (19 Y and 20 O) performed sub‐maximal plantar flexion exercise following the consumption of either a placebo (PL) or an antioxidant (AO) cocktail. PCr resynthesis was assessed by 31P‐MRS (magnetic resonance spectroscopy), while muscle re‐oxygenation and limb blood flow were evaluated by near‐infrared spectroscopy and ultrasound Doppler, respectively. The PCr recovery time constant in O (PL: 37 ± 6; AO: 38 ± 6 s) and Y (PL: 38 ± 6; AO: 36 ± 4 s) as well as the maximal rate of oxidative ATP synthesis in O (PL: 15 ± 2; AO: 14 ± 2 mM.min−1) and Y (PL: 13 ± 2; AO: 15 ± 3 mM.min−1) were not altered by age or AO. In contrast, muscle reoxygenation rate, although not affected by age, was slower in the Y following AO, as evidenced by the time constant (P &lt; 0.05) and mean response time (PL: 49 ± 6; AO: 70 ± 10 s) of deoxy‐Hb recovery, while no difference was observed in the old. These data reveal that, when subjects are activity matched, muscle oxidative capacity is preserved with age. In addition, unlike the O, the compromising effect of the AO on O2 availability in the microcirculation of the Y, without affecting PCr recovery kinetics, implies that free radicals play a role in blood flow regulation and that O2 availability exceeds mitochondrial capacity in this population.

Mitochondria‐targeted antioxidant promotes recovery of skeletal muscle mitochondrial function after burn trauma assessed byin vivo31P nuclear magnetic resonance and electron paramagnetic resonance spectroscopy

Burn injury causes a major systemic catabolic response that is associated with mitochondrial dysfunction in skeletal muscle. We investigated the effects of the mitochondria-targeted peptide antioxidant Szeto-Schiller 31 (SS-31) on skeletal muscle in a mouse burn model using in vivo phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy to noninvasively measure high-energy phosphate levels; mitochondrial aconitase activity measurements that directly correlate with TCA cycle flux, as measured by gas chromatography mass spectrometry (GC-MS); and electron paramagnetic resonance (EPR) to assess oxidative stress. At 6 h postburn, the oxidative ATP synthesis rate was increased 5-fold in burned mice given a single dose of SS-31 relative to untreated burned mice (P=0.002). Furthermore, SS-31 administration in burned animals decreased mitochondrial aconitase activity back to control levels. EPR revealed a recovery in redox status of the SS-31-treated burn group compared to the untreated burn group (P<0.05). Our multidisciplinary convergent results suggest that SS-31 promotes recovery of mitochondrial function after burn injury by increasing ATP synthesis rate, improving mitochondrial redox status, and restoring mitochondrial coupling. These findings suggest use of noninvasive in vivo NMR and complementary EPR offers an approach to monitor the effectiveness of mitochondrial protective agents in alleviating burn injury symptoms.

The features of activation of free oxidation by α,ω-tetradecanedioic acid in liver mitochondria

It was found that α,ω-tetradecanedioic acid (TDA) at the concentration of 0–500 μM doubles the rate of nonphosphorylating respiration (free oxidation) of liver mitochondria in a dose-dependent manner. This effect of TDA is observed in the presence of the excess of EGTA, which eliminates the induction of the Ca2+-dependent nonspecific permeability of the mitochondrial inner membrane (pore opening). An unusually high concentration of cyclosporin A (10 mM) completely eliminates this effect when added to the mitochondria before or after TDA. The stimulatory effect of TDA is not accompanied by inhibition of oxidative ATP synthesis and decrease in the ADP/O ratio, in contrast to the effects of other activators of free oxidation, such as protonophore uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone and palmitic acid. It was shown that neither oligomycin, an inhibitor of H+-ATP synthase, nor ADP, ATP and Pi affected the activity of TDA. This is seen as an evidence that the effect of TDA is not associated with the influence on H+-ATP synthase and it differs from the action of membranotropic uncouplers. In the presence of the lipophilic cation tetraphenylphosphonium (TPP+) cyclosporin A does not affect the TDA-stimulated respiration of mitochondria, but carboxyatractylate and glutamate added after TDA do inhibit the respiration. In addition, under these conditions TDA decreases the rate of oxidative ATP synthesis and reduces the ADP/O ratio. It is assumed that the mechanism of the TDA-induced activation of free oxidation in liver mitochondria in the absence of TPP+ is similar to that of the so-called decouplers and is associated with the switching of the respiratory chain complexes to the idle mode (inner uncoupling).

What Do Magnetic Resonance–Based Measurements of Pi→ATP Flux Tell Us About Skeletal Muscle Metabolism?

Magnetic resonance spectroscopy (MRS) methods offer a potentially valuable window into cellular metabolism. Measurement of flux between inorganic phosphate (Pi) and ATP using 31P MRS magnetization transfer has been used in resting muscle to assess what is claimed to be mitochondrial ATP synthesis and has been particularly popular in the study of insulin effects and insulin resistance. However, the measured Pi→ATP flux in resting skeletal muscle is far higher than the true rate of oxidative ATP synthesis, being dominated by a glycolytically mediated Pi↔ATP exchange reaction that is unrelated to mitochondrial function. Furthermore, even if measured accurately, the ATP production rate in resting muscle has no simple relationship to mitochondrial capacity as measured either ex vivo or in vivo. We summarize the published measurements of Pi→ATP flux, concentrating on work relevant to diabetes and insulin, relate it to current understanding of the physiology of mitochondrial ATP synthesis and glycolytic Pi↔ATP exchange, and discuss some possible implications of recently reported correlations between Pi→ATP flux and other physiological measures.

Implications of rapid early oxygen consumption in exercising skeletal muscle

Measured directly in vivo (Wust et al. 2011), O2 consumption rate () in dog leg muscle during the rest–exercise transition increases faster than predicted on a first-order ADP-feedback model (‘model 1’ in Wust et al. 2011). This yields a higher-order relationship between oxidative ATP synthesis rate (V) and [ADP] (‘model 3’), resembling that inferred from post-exercise PCr kinetics (Jeneson et al. 1996) and adduced as a possible explanation of what (given the low V in relation to the maximum rate Vmax) is an anomalously high [ADP] in resting human muscle (Kemp et al. 2007). However, the –[ADP] data are rather better explained by assuming a component of parallel activation (‘model 4’), approximately quantifiable as an increasing apparent Vmax from rest into exercise (Wust et al. 2011). This suggests three experimental tests, and raises a so far unanswered physiological question.

Mitochondrial capacity is affected by glycemic status in young untrained women with type 1 diabetes but is not impaired relative to healthy untrained women

In this study, we examined whether glycemic status influences aerobic function in women with type 1 diabetes and whether aerobic function is reduced relative to healthy women. To this end, we compared several factors determining aerobic function of 29 young sedentary asymptomatic women (CON) with 9 women of similar age and activity level with type 1 diabetes [DIA, HbA1c range = 6.9-8.2%]. Calf muscle mitochondrial capacity was estimated by (31)P-magnetic resonance spectroscopy. Capillarization and muscle fiber oxidative enzyme activity were assessed from vastus lateralis and soleus muscle biopsies. Oxygen uptake and cardiac output were evaluated by ergospirometry and N(2)O/SF(6) rebreathing. Calf muscle mitochondrial capacity was not different between CON and DIA, as indicated by the identical calculated maximal rates of oxidative ATP synthesis [0.0307 (0.0070) vs. 0.0309 (0.0058) s(-1), P = 0.930]. Notably, HbA1c was negatively correlated with mitochondrial capacity in DIA (R(2) = 0.475, P = 0.040). Although HbA1c was negatively correlated with cardiac output (R(2) = 0.742, P = 0.013) in DIA, there was no difference between CON and DIA in maximal oxygen consumption [2.17 (0.34) vs. 2.21 (0.32) l/min, P = 0.764], cardiac output [12.1 (1.9) vs. 12.3 (1.8) l/min, P = 0.783], and endurance capacity [532 (212) vs. 471 (119) s, P = 0.475]. There was also no difference between the two groups either in the oxidative enzyme activity or capillary-to-fiber ratio. We conclude that mitochondrial capacity depends on HbA1c in untrained women with type 1 diabetes but is not reduced relative to untrained healthy women.

Comparative determination of energy production rates and mitochondrial function using different 31P MRS quantitative methods in sedentary and trained subjects

Muscle energetics has been largely and quantitatively investigated using (31)P MRS. Various methods have been used to estimate the corresponding rate of oxidative ATP synthesis (ATP(ox)); however, potential differences among methods have not been investigated. In this study, we aimed to compare the rates of ATP production and energy cost in two groups of subjects with different training status using four different methods: indirect method (method 1), ADP control model (method 2) and phosphate potential control model (method 3). Method 4 was a modified version of method 3 with the introduction of a correction factor allowing for similar values to be obtained for the end-exercise oxidative ATP synthesis rate inferred from exercise measurements and the initial recovery phosphocreatine resynthesis rate. Seven sedentary and seven endurance-trained subjects performed a dynamic standardised rest-exercise-recovery protocol. We quantified the rates of ATP(ox) and anaerobic ATP synthesis (ATP(ana)) using (31)P MRS data recorded at 1.5 T. The rates of ATP(ox) over the entire exercise session were independent of the method used, except for method 4 which provided significantly higher values in both groups (p < 0.01). In addition, methods 1-3 were cross-correlated, thereby confirming their statistical agreement. The rate of ATP(ana) was significantly higher with method 1 (p < 0.01) and lower with method 4 (p < 0.01). As a result of the higher rate of ATP(ox), EC (method 4) calculated over the entire exercise session was higher and initial EC (method 1) was lower in both groups compared with the other methods. We showed in this study that the rate of ATP(ox) was independent of the calculation method, as long as no corrections (method 4) were performed. In contrast, results related to the rates of ATP(ana) were strongly affected by the calculation method and, more exactly, by the estimation of protons generated by ATP(ox). Although the absolute EC values differed between the methods, within- or between-subject comparisons are still valid given the tight relationships between them.

Magnitude and control of mitochondrial sensitivity to ADP

The transduction function for ADP stimulation of mitochondrial ATP synthesis in skeletal muscle was reconstructed in vivo and in silico to investigate the magnitude and origin of mitochondrial sensitivity to cytoplasmic ADP concentration changes. Dynamic in vivo measurements of human leg muscle phosphocreatine (PCr) content during metabolic recovery from contractions were performed by (31)P-NMR spectroscopy. The cytoplasmic ADP concentration ([ADP]) and rate of oxidative ATP synthesis (Jp) at each time point were calculated from creatine kinase equilibrium and the derivative of a monoexponential fit to the PCr recovery data, respectively. Reconstructed [ADP]-Jp relations for individual muscles containing more than 100 data points were kinetically characterized by nonlinear curve fitting yielding an apparent kinetic order and ADP affinity of 1.9 +/- 0.2 and 0.022 +/- 0.003 mM, respectively (means +/- SD; n = 6). Next, in silico [ADP]-Jp relations for skeletal muscle were generated using a computational model of muscle oxidative ATP metabolism whereby model parameters corresponding to mitochondrial enzymes were randomly changed by 50-150% to determine control of mitochondrial ADP sensitivity. The multiparametric sensitivity analysis showed that mitochondrial ADP ultrasensitivity is an emergent property of the integrated mitochondrial enzyme network controlled primarily by kinetic properties of the adenine nucleotide translocator.

Absolute quantification of phosphorus metabolite concentrations in human muscle in vivo by 31P MRS: a quantitative review

31P MRS offers a unique view of muscle metabolism in vivo, but correct quantification is important. Inter-study correlation of estimates of [Pi] and [phosphocreatine (PCr)] in a number of published studies suggest that the main technical problem in calibrated 31P MRS studies is the measurement of PCr and Pi signal intensities, rather than absolute quantification of [ATP]. For comparison, we discuss the few published biopsy studies of calf muscle and a selection of the many studies of quadriceps muscle. The ATP concentration is close to the value that we obtained in calf muscle in our own study, presented here, on four healthy subjects, by localised 31P MRS using a surface coil incorporating an internal reference and calibrated using an external phantom. However, the freeze-clamp biopsy PCr concentration is approximately 20% lower than the value obtained by 31P MRS, consistent with PCr breakdown by creatine kinase during freezing. Finally, we illustrate some consequences of uncertainty in resting [PCr] for analysis of mitochondrial function from PCr kinetics using a published 31P MRS study of exercise and recovery: the lower the assumed resting [PCr], the lower the absolute rate of oxidative ATP synthesis estimated from the PCr resynthesis rate; in addition, the lower the assumed resting [PCr], or the higher the assumed [total creatine], the higher the apparent resting [ADP], and therefore the more sigmoid the relationship between the rate of oxidative ATP synthesis and [ADP]. Correct quantification of resting metabolite concentrations is crucially important for this sort of analysis. Our own results ([PCr] = 33 +/- 2 mM, [Pi] = 4.5 +/- 0.2 mM, and [ATP] = 8.2 +/- 0.4 mM; mean +/- SEM) are close to the overall mean values of the 10 published studies on calf muscle by 'calibrated' 31P MRS (as in the present work), and of [PCr] and [Pi] in a representative selection of 'uncalibrated' 31P MRS studies (i.e. from measured PCr/ATP and Pi/ATP ratios, assuming a literature value for [ATP]).