Total ATP Turnover Research Articles

Contribution of D-(-)-3-Hydroxybutyrate to the Energy Expenditure of Neonatal Pigs

In vivo oxidation rate of arterially infused D-(-)-3-hydroxybutyrate (3HB) was measured in 1-2-d-old-piglets. Twelve piglets (1.4 kg) were randomly assigned to a 12 h continuous infusion of 3HB at 19.5, 37.8, 55.8 or 74.5 µmol/min along with ∼31 kBq/h of [3-14C]3HB. Piglets were housed in respiration chambers allowing collection of total expired CO2 over 20-min intervals for the 12 h infusion and 6 h washout. Oxidation of 3HB was calculated from the quantity and specific radioactivity of expired CO2 for 20-min collection periods at 6, 9 and 12 h for each piglet and collectively plotted against plasma 3HB concentration measured in blood drawn during those 20-min periods. A Lineweaver-Burk plot of these data yielded a Km of 0.62 ± 0.07 mmol/L and Vmax of 0.74 ± 0.02 mmol ATP equivalents/(min·kg0.75) (parameter estimate ± SD), which could account for 32% of the piglet mean total ATP turnover of 2.3 mmol/(min·kg0.75). These data show that 3HB oxidation is a linear function of plasma concentration in the physiologic range measured in piglets (0.006 mmol/L to 0.1 mmol/L) and within this range would account for 0.3% to 4.5% of piglet energy requirement. Oxidation of 3HB can meet a maximum of 30 to 40% of piglet energy requirement at unphysiologically high 3HB concentrations (> mmol/L).

The root of the matter: nitrate-rich beetroot juice reduces skeletal muscle O2 uptake during exercise.

The root of the matter: nitrate-rich beetroot juice reduces skeletal muscle O2 uptake during exercise.

A higher mitochondrial content is associated with greater oxidative damage, oxidative defenses, protein synthesis and ATP turnover in resting skeletal muscle.

An unavoidable consequence of aerobic metabolism is the production of reactive oxygen species (ROS). Mitochondria have historically been considered the primary source of ROS; however, recent literature has highlighted the uncertainty in primary ROS production sites and it is unclear how variation in mitochondrial density influences ROS-induced damage and protein turnover. Fish skeletal muscle is composed of distinct, highly aerobic red muscle and anaerobic white muscle, offering an excellent model system in which to evaluate the relationship of tissue aerobic capacity and ROS-induced damage under baseline conditions. The present study used a suite of indices to better understand potential consequences of aerobic tissue capacity in red and white muscle of the pinfish, Lagodon rhomboides. Red muscle had a 7-fold greater mitochondrial volume density than white muscle, and more oxidative damage despite also having higher activity of the antioxidant enzymes superoxide dismutase and catalase. The dominant protein degradation system appears to be tissue dependent. Lysosomal degradation markers and autophagosome volume density were greater in white muscle, while ubiquitin expression and 20S proteasome activity were significantly greater in red muscle. However, ubiquitin ligase expression was significantly higher in white muscle. Red muscle had a more than 2-fold greater rate of translation and total ATP turnover than white muscle, results that may be due in part to the higher mitochondrial density and the associated increase in oxidative damage. Together, these results support the concept that an elevated aerobic capacity is associated with greater oxidative damage and higher costs of protein turnover.

Quantifying H+ exchange from muscle cytosolic energy catabolism using metabolite flux and H+ coefficients from multiple competitive cation binding: New evidence for consideration in established theories.

The purpose of this investigation was to present calculations of fractional H+ exchange (~H+ e) from the chemical reactions of non‐mitochondrial energy catabolism. Data of muscle pH and metabolite accumulation were based on published research for intense exercise to contractile failure within ~3 min, from which capacities and time profiles were modeled. Data were obtained from prior research for multiple competitive cation dissociation constants of metabolites and the chemical reactions of non‐mitochondrial energy catabolism, and pH dependent calculations of ~H+ e from specific chemical reactions. Data revealed that the 3 min of intense exercise incurred a total ATP turnover of 142.5 mmol L−1, with a total intramuscular ~H+ exchange (‐‘ve = release) of −187.9 mmol L−1. Total ~H+ metabolic consumption was 130.6 mmol L−1, revealing a net total ~H+ e (~H+ te) of −57.3 mmol L−1. Lactate production had a ~H+ te of 44.2 mmol L−1 (for a peak accumulation = 45 mmol L−1). The net ~H+ te for the sum of the CK, AK, and AMPD reactions was 36.33 mmol L−1. The ~H+ te from ATP turnover equaled −47.5 mmol L−1. The total ~H+ release to lactate ratio was 4.3 (187.9/44). Muscle ~H+ release during intense exercise is up to ~4‐fold larger than previously assumed based on the lactic acid construct.

Abstract 2407: Extracellular acidosis inhibits glucose starvation-induced lung cancer cell death by suppressing overall metabolic rate

Abstract Bioenergetic tumor metabolism is essential to meet ATP requirements for energy homeostasis. Cancer cells utilize aerobic glycolysis and proliferate continuously beyond the capacity of blood supply. These phenotypic alterations lead to various microenvironmental stresses, such as hypoxia, nutrient (glucose) deprivation, and extracellular acidosis, to which cancer cells must metabolically adapt in order to survive and proliferate. However, compared to hypoxia, relatively little is known about the metabolic adaptations in response to glucose deprivation and acidosis. In the present study, we explored the effects of extracellular acidosis on cancer metabolism under glucose deprivation stress, as extracellular acidosis and insufficient glucose supply usually co-exist in the tumor microenvironment. To this aim, A549 lung cancer cells were treated with acidosis, glucose deprivation, or a combination. Then, cell survival, total cellular ATP turnover, relative glycolytic and mitochondrial dependency and capacity, and de novo rRNA and protein synthesis were determined. We found that acidosis (pH 6.8) inhibited ATP depletion and cell death induced by glucose deprivation. The cytoprotective effect of acidosis against glucose deprivation-induced cell death was mediated by two distinct mechanisms. First, acidosis shifted cellular ATP production from glycolysis to OXPHOS that became independent of glucose uptake but dependent on glutaminolysis and fatty acid β-oxidation. Second, acidosis reduced ATP consumption by inhibiting de novo rRNA and protein synthesis that utilizes ATP. In contrast, the protective effect of acidosis on glucose starvation-induced cell death was independent of AMPK, PI3K, ERK, and p38 MAPK. Based on this mechanistic knowledge of acidosis-dependent metabolic alterations, we tested the effect of the mitochondrial complex I inhibitor metformin on death of lung cancer cells. Metformin and a combination of rotenone and AICAR mimicking an action of metformin induced ATP depletion and cell death in glucose-free medium, but under acidosis the cytotoxic effect of metformin was significantly attenuated because overall ATP turnover was reduced under low pH. These results suggest that during acidosis, cells are forced to a reduced glycolytic energy production, but the decrease in protein synthesis and ATP requirement serves as an adaptive response to glucose deprivation stress. This acidosis-dependent, glucose starvation-resistant phenotype may provide an adaptive strategy in an energy-restricted tumor microenvironment. Our findings highlight the importance of considering the two factors, acidosis and nutrient starvation in combination to understand their contributions to tumor metabolism. Citation Format: Yuki Iwai, Ryota Kikuchi, Yasutaka Watanabe, Nobuyuki Koyama, Koichi Hagiwara, Hiroyuki Nakamura, Kazutetsu Aoshiba. Extracellular acidosis inhibits glucose starvation-induced lung cancer cell death by suppressing overall metabolic rate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2407.

Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro.

BackgroundThe tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect), which weakens the spatial linkage between hypoxia and acidosis.MethodsFive different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15) were treated with hypoxia, acidosis (pH 6.3), or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein.ResultsMicroarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe), genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), and Ribosomal protein L37 (RPL37). Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa) and protein synthesis (both cell lines) was observed when hypoxia and low pHe were combined.ConclusionsWe demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de novo protein synthesis, depending on whether the two factors induced alone or overlapping, and as such it is important for in vivo studies to take this into account.

Efficiency of energy transfer during exercise: what are the limiting factors?

Efficiency of energy transfer during exercise: what are the limiting factors?

A microscale mathematical model for metabolic symbiosis: Investigating the effects of metabolic inhibition on ATP turnover in tumors.

Cancer cells are notorious for their metabolic adaptations to hypoxic and acidic conditions, and especially for highly elevated glycolytic rates in tumor tissues. An end product of glycolysis is lactate, a molecule that cells can utilize instead of glucose to fuel respiration in the presence of oxygen. This could be beneficial to those cells that do not have sufficient oxygen as it conserves glucose for glycolysis. To better quantify this phenomenon we develop a diffusion-reaction mathematical model for nutrient concentrations in cancerous tissue surrounding a single cylindrical microvessel. We use our model to analyze the interdependence between cell populations' metabolic behaviors on a microscopic scale, specifically the emerging paradigm of metabolic symbiosis that exists between aerobic and glycolytic cells. The ATP turnover rates are calculated as a function of distance from the blood vessel, which exhibit a lactate-consuming population at intermediate distances from the vessel. We also consider the ramifications of the Warburg effect where cells utilize aerobic glycolysis along with this lactate-consuming respiration. We also investigate the effect of inhibiting metabolic pathways on cancer cells since insufficient ATP can trigger cell apoptosis. Effects that could be induced by metabolic inhibitors are analyzed by calculating the total ATP turnover in a unit tissue annulus in various parameter regimes that correspond to treatment conditions where specific metabolic pathways are knocked out. We conclude that therapies that target glycolysis, e.g. lactate dehydrogenase inhibitors or glycolytic enzyme inhibition, are the keys to successful metabolic repression.

Skeletal muscle ATP turnover by 31P magnetic resonance spectroscopy during moderate and heavy bilateral knee extension.

During constant-power high-intensity exercise, the expected increase in oxygen uptake (V̇O2) is supplemented by a V̇O2slow component (V̇O2 sc ), reflecting reduced work efficiency, predominantly within the locomotor muscles. The intracellular source of inefficiency is postulated to be an increase in the ATP cost of power production (an increase in P/W). To test this hypothesis, we measured intramuscular ATP turnover with (31)P magnetic resonance spectroscopy (MRS) and whole-body V̇O2 during moderate (MOD) and heavy (HVY) bilateral knee-extension exercise in healthy participants (n=14). Unlocalized (31)P spectra were collected from the quadriceps throughout using a dual-tuned ((1)H and (31)P) surface coil with a simple pulse-and-acquire sequence. Total ATP turnover rate (ATPtot) was estimated at exercise cessation from direct measurements of the dynamics of phosphocreatine (PCr) and proton handling. Between 3 and 8min during MOD, there was no discernable V̇O2 sc (mean±SD, 0.06±0.12lmin(-1)) or change in [PCr] (30±8 vs. 32±7mm) or ATPtot (24±14 vs. 17±14mmmin(-1); each P=n.s.). During HVY, the V̇O2 sc was 0.37±0.16lmin(-1) (22±8%), [PCr] decreased (19±7 vs. 18±7mm, or 12±15%; P<0.05) and ATPtot increased (38±16 vs. 44±14mmmin(-1), or 26±30%; P<0.05) between 3 and 8min. However, the increase in ATPtot (ΔATPtot) was not correlated with the V̇O2 sc during HVY (r(2)=0.06; P=n.s.). This lack of relationship between ΔATPtot and V̇O2 sc , together with a steepening of the [PCr]-V̇O2 relationship in HVY, suggests that reduced work efficiency during heavy exercise arises from both contractile (P/W) and mitochondrial sources (the O2 cost of ATP resynthesis; P/O).

High‐intensity interval training alters ATP pathway flux during maximal muscle contractions in humans

High-intensity interval training (HIT) results in potent metabolic adaptations in skeletal muscle; however, little is known about the influence of these adaptations on energetics in vivo. We used magnetic resonance spectroscopy to examine the effects of HIT on ATP synthesis from net PCr breakdown (ATPCK ), oxidative phosphorylation (ATPOX ) and non-oxidative glycolysis (ATPGLY ) in vivo in vastus lateralis during a 24-s maximal voluntary contraction (MVC). Eight young men performed 6 sessions of repeated, 30-s 'all-out' sprints on a cycle ergometer; measures of muscle energetics were obtained at baseline and after the first and sixth sessions. Training increased peak oxygen consumption (35.8 ± 1.4 to 39.3 ± 1.6 mL min(-1) kg(-1) , P = 0.01) and exercise capacity (217.0 ± 11.0 to 230.5 ± 11.7 W, P = 0.04) on the ergometer, with no effects on total ATP production or force-time integral during the MVC. While ATP production by each pathway was unchanged after the first session, 6 sessions increased the relative contribution of ATPOX (from 31 ± 2 to 39 ± 2% of total ATP turnover, P < 0.001) and lowered the relative contribution from both ATPCK (49 ± 2 to 44 ± 1%, P = 0.004) and ATPGLY (20 ± 2 to 17 ± 1%, P = 0.03). These alterations to muscle ATP production in vivo indicate that brief, maximal contractions are performed with increased support of oxidative ATP synthesis and relatively less contribution from anaerobic ATP production following training. These results extend previous reports of molecular and cellular adaptations to HIT and show that 6 training sessions are sufficient to alter in vivo muscle energetics, which likely contributes to increased exercise capacity after short-term HIT.

Short‐term, high‐intensity interval training (HIT) alters ATP pathway fluxes during maximal muscle contractions in humans

Phosphorus magnetic resonance spectroscopy (MRS) was used to examine the effects of 2 weeks of cycling training on bioenergetics of vastus lateralis in vivo. Eight healthy, untrained men (27 ± 4 yr) performed 6 sessions of HIT (4–6 bouts of 30‐s all‐out cycling). At baseline, and after the 1st and the 6th training sessions, rates of ATP synthesis from net PCr breakdown (ATPCK), glycolysis (ATPGLY) and oxidative phosphorylation (ATPOX) were measured during a 24‐s maximal voluntary isometric contraction (MVIC) in a 4T magnet. Training increased peak whole‐body oxygen uptake (36 ± 4 to 39 ± 5ml·kg−1·min−1, p=0.01) and exercise capacity (225 ± 32 to 239 ± 36 W, p=0.04) on the cycle ergometer, with no effects on total ATP production or force‐time integral during the MVIC. While ATP production through the 3 pathways remained unchanged after the first training session, 6 training sessions increased the relative contribution of ATPOX (31 ± 2 vs. 39 ± 2% of total ATP turnover, p&lt;0.001), and lowered the relative contribution from ATPCK (49 ± 2 vs. 44 ± 1%, p=0.004) and ATPGLY (20 ± 2 vs. 17 ± 1%, p=0.03). Thus, 6 sessions of HIT alters muscle energetics, such that maximal contractions are performed with increased support from oxidative ATP synthesis and relatively less contribution from anaerobic ATP production, which may contribute to improved exercise capacity.Support: NIH K02 AG023582

Dietary nitrate supplementation enhances muscle contractile efficiency during knee-extensor exercise in humans

The purpose of this study was to elucidate the mechanistic bases for the reported reduction in the O(2) cost of exercise following short-term dietary nitrate (NO(3)(-)) supplementation. In a randomized, double-blind, crossover study, seven men (aged 19-38 yr) consumed 500 ml/day of either nitrate-rich beet root juice (BR, 5.1 mmol of NO(3)(-)/day) or placebo (PL, with negligible nitrate content) for 6 consecutive days, and completed a series of low-intensity and high-intensity "step" exercise tests on the last 3 days for the determination of the muscle metabolic (using (31)P-MRS) and pulmonary oxygen uptake (Vo(2)) responses to exercise. On days 4-6, BR resulted in a significant increase in plasma [nitrite] (mean +/- SE, PL 231 +/- 76 vs. BR 547 +/- 55 nM; P < 0.05). During low-intensity exercise, BR attenuated the reduction in muscle phosphocreatine concentration ([PCr]; PL 8.1 +/- 1.2 vs. BR 5.2 +/- 0.8 mM; P < 0.05) and the increase in Vo(2) (PL 484 +/- 41 vs. BR 362 +/- 30 ml/min; P < 0.05). During high-intensity exercise, BR reduced the amplitudes of the [PCr] (PL 3.9 +/- 1.1 vs. BR 1.6 +/- 0.7 mM; P < 0.05) and Vo(2) (PL 209 +/- 30 vs. BR 100 +/- 26 ml/min; P < 0.05) slow components and improved time to exhaustion (PL 586 +/- 80 vs. BR 734 +/- 109 s; P < 0.01). The total ATP turnover rate was estimated to be less for both low-intensity (PL 296 +/- 58 vs. BR 192 +/- 38 microM/s; P < 0.05) and high-intensity (PL 607 +/- 65 vs. BR 436 +/- 43 microM/s; P < 0.05) exercise. Thus the reduced O(2) cost of exercise following dietary NO(3)(-) supplementation appears to be due to a reduced ATP cost of muscle force production. The reduced muscle metabolic perturbation with NO(3)(-) supplementation allowed high-intensity exercise to be tolerated for a greater period of time.

Assessment of Respiration-Dependent Intra- and Extramitochondrial ATP Turnover: HepG2 Cancer Cells do not Utilize ATP from Oxidative Phosphorylation in the Cytosol

BACKGROUND: Mitochondrial ATP is transported to the cytosol in exchange for ADP through the adenine nucleotide translocator (ANT). Carboxyatractyloside (CAT) and bongkrekic acid (BA) specifically inhibit ATP delivery to the cytosol via ANT, whereas oligomycin (OL) inhibits all ATP synthesis by oxidative phosphorylation. Our AIM was to assess respiration-dependent intra and extramitochondrial ATP turnover in HepG2 human hepatocarcinoma cells and cultured rat hepatocytes stimulated by ureagenic substrates. METHODS: Overnight cultured rat hepatocytes were stimulated with ureagenic substrates (in mM: 5 Na-lactate, 5 L-ornithine, 3 NH4Cl). HepG2 cells were incubated in Hank's solution or permeabilized with digitonin in intracellular buffer plus 0.5 mM ADP and 5 mM succinate. Respiration was measured using a Seahorse XF24. RESULTS: Ureagenic substrates increased respiration by hepatocytes progressively 2 to 3-fold over an hour. Subsequent addition of OL inhibited respiration to basal levels, whereas BA and CAT inhibited ureagenic respiration by ∼65%. Partial inhibition by ANT blockers was consistent with utilization of both intra- and extramitochondrial ATP in the urea cycle. In non-permeabilized HepG2 cells, OL inhibited respiration by ∼60% but BA and CAT had no effect, whereas in permeabilized HepG2 cells, OL, BA and CAT each inhibited respiration equally. In CONCLUSION, respiration inhibited by OL reflects total ATP turnover linked to oxidative phosphorylation, whereas respiration inhibited by BA or CAT reflects extramitochondrial turnover of ATP formed by oxidative phosphorylation. The difference is intramitochondrial ATP turnover. In intact HepG2 cells, ATP generated by oxidative phosphorylation was not utilized in the cytosol, although HepG2 cells contain functional ANT.

Neuromuscular blockade of slow twitch muscle fibres elevates muscle oxygen uptake and energy turnover during submaximal exercise in humans

We tested the hypothesis that a greater activation of fast-twitch (FT) fibres during dynamic exercise leads to a higher muscle oxygen uptake (VO2 ) and energy turnover as well as a slower muscle on-kinetics. Subjects performed one-legged knee-extensor exercise for 10 min at an intensity of 30 W without (CON) and with (CUR) arterial injections of the non-depolarizing neuromuscular blocking agent cisatracurium. In CUR, creatine phosphate (CP) was unaltered in slow twitch (ST) fibres and decreased (P < 0.05) by 28% in FT fibres, whereas in CON, CP decreased (P < 0.05) by 33% and 23% in ST and FT fibres, respectively. From 127 s of exercise, muscle VO2 was higher (P < 0.05) in CUR compared to CON (425 +/- 25 (+/- S.E.M.) versus 332 +/- 30 ml min(-1)) and remained higher (P < 0.05) throughout exercise. Using monoexponential fitting, the time constant of the exercise-induced muscle VO2 response was slower (P < 0.05) in CUR than in CON (55 +/- 6 versus 33 +/- 5 s). During CUR and CON, muscle homogenate CP was lowered (P < 0.05) by 32 and 35%, respectively, and also muscle lactate production was similar in CUR and CON (37.8 +/- 4.1 versus 35.2 +/- 6.2 mmol). Estimated total muscle ATP turnover was 19% higher (P < 0.05) in CUR than in CON (1196 +/- 90 versus 1011 +/- 59 mmol) and true mechanical efficiency was lower (P < 0.05) in CUR than in CON (26.2 +/- 2.0 versus 30.9 +/- 1.5%). In conclusion, the present findings provide evidence that FT fibres are less efficient than ST fibres in vivo at a contraction frequency of 1 Hz, and that the muscle VO2 kinetics is slowed by FT fibre activation.

Skeletal muscle ATP turnover and muscle fiber conduction velocity are elevated at higher muscle temperatures during maximal power output development in humans

The effect of temperature on skeletal muscle ATP turnover and muscle fiber conduction velocity (MFCV) was studied during maximal power output development in humans. Eight male subjects performed a 6-s maximal sprint on a mechanically braked cycle ergometer under conditions of normal (N) and elevated muscle temperature (ET). Muscle temperature was passively elevated through the combination of hot water immersion and electric blankets. Anaerobic ATP turnover was calculated from analysis of muscle biopsies obtained before and immediately after exercise. MFCV was measured during exercise using surface electromyography. Preexercise muscle temperature was 34.2 degrees C (SD 0.6) in N and 37.5 degrees C (SD 0.6) in ET. During ET, the rate of ATP turnover for phosphocreatine utilization [temperature coefficient (Q10) = 3.8], glycolysis (Q10 = 1.7), and total anaerobic ATP turnover [Q10 = 2.7; 10.8 (SD 1.9) vs. 14.6 mmol x kg(-1) (dry mass) x s(-1) (SD 2.3)] were greater than during N (P < 0.05). MFCV was also greater in ET than in N [3.79 (SD 0.47) to 5.55 m/s (SD 0.72)]. Maximal power output (Q10 = 2.2) and pedal rate (Q10 = 1.6) were greater in ET compared with N (P < 0.05). The Q10 of maximal and mean power were correlated (P < 0.05; R = 0.82 and 0.85, respectively) with the percentage of myosin heavy chain type IIA. The greater power output obtained with passive heating was achieved through an elevated rate of anaerobic ATP turnover and MFCV, possibly due to a greater effect of temperature on power production of fibers, with a predominance of myosin heavy chain IIA at the contraction frequencies reached.

Phosphotransfer dynamics in skeletal muscle from creatine kinase gene-deleted mice.

To assess the significance of energy supply routes in cellular energetic homeostasis, net phosphoryl fluxes catalyzed by creatine kinase (CK), adenylate kinase (AK) and glycolytic enzymes were quantified using 18O-phosphoryl labeling. Diaphragm muscle from double M-CK/ScCKmit knockout mice exhibited virtually no CK-catalyzed phosphotransfer. Deletion of the cytosolic M-CK reduced CK-catalyzed phosphotransfer by 20%, while the absence of the mitochondrial ScCKmit isoform did not affect creatine phosphate metabolic flux. Contribution of the AK-catalyzed phosphotransfer to total cellular ATP turnover was 15.0, 17.2, 20.2 and 28.0% in wild type, ScCKmit, M-CK and M-CK/ScCKmit deficient muscles, respectively. Glycolytic phosphotransfer, assessed by G-6-P 18O-phosphoryl labeling, was elevated by 32 and 65% in M-CK and M-CK/ScCKmit deficient muscles, respectively. Inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/phosphoglycerate kinase (PGK) in CK deficient muscles abolished inorganic phosphate compartmentation and redirected high-energy phosphoryl flux through the AK network. Under such conditions, AK phosphotransfer rate was equal to 86% of the total cellular ATP turnover concomitant with almost normal muscle performance. This indicates that near-equilibrium glycolytic phosphotransfer reactions catalyzed by the GAPDH/PGK support a significant portion of the high-energy phosphoryl transfer in CK deficient muscles. However, CK deficient muscles displayed aberrant ATPase-ATPsynthase communication along with lower energetic efficiency (P/O ratio), and were more sensitive to metabolic stress induced by chemical hypoxia. Thus, redistribution of phosphotransfer through glycolytic and AK networks contributes to energetic homeostasis in muscles under genetic and metabolic stress complementing loss of CK function.

Impaired Intracellular Energetic Communication in Muscles from Creatine Kinase and Adenylate Kinase (M-CK/AK1) Double Knock-out Mice

Previously we demonstrated that efficient coupling between cellular sites of ATP production and ATP utilization, required for optimal muscle performance, is mainly mediated by the combined activities of creatine kinase (CK)- and adenylate kinase (AK)-catalyzed phosphotransfer reactions. Herein, we show that simultaneous disruption of the genes for the cytosolic M-CK- and AK1 isoenzymes compromises intracellular energetic communication and severely reduces the cellular capability to maintain total ATP turnover under muscle functional load. M-CK/AK1 (MAK=/=) mutant skeletal muscle displayed aberrant ATP/ADP, ADP/AMP and ATP/GTP ratios, reduced intracellular phosphotransfer communication, and increased ATP supply capacity as assessed by 18O labeling of [Pi] and [ATP]. An analysis of actomyosin complexes in vitro demonstrated that one of the consequences of M-CK and AK1 deficiency is hampered phosphoryl delivery to the actomyosin ATPase, resulting in a loss of contractile performance. These results suggest that MAK=/= muscles are energetically less efficient than wild-type muscles, but an apparent compensatory redistribution of high-energy phosphoryl flux through glycolytic and guanylate phosphotransfer pathways limited the overall energetic deficit. Thus, this study suggests a coordinated network of complementary enzymatic pathways that serve in the maintenance of energetic homeostasis and physiological efficiency.

Knockout of Kir6.2 negates ischemic preconditioning-induced protection of myocardial energetics.

Although ischemic preconditioning induces bioenergetic tolerance and thereby remodels energy metabolism that is crucial for postischemic recovery of the heart, the molecular components associated with preservation of cellular energy production, transfer, and utilization are not fully understood. Here myocardial bioenergetic dynamics were assessed by (18)O-assisted (31)P-NMR spectroscopy in control or preconditioned hearts from wild-type (WT) or Kir6.2-knockout (Kir6.2-KO) mice that lack metabolism-sensing sarcolemmal ATP-sensitive K(+) (K(ATP)) channels. In WT vs. Kir6.2-KO hearts, preconditioning induced a significantly higher total ATP turnover (232 +/- 20 vs. 155 +/- 15 nmol x mg protein(-1) x min(-1)), ATP synthesis rate (58 +/- 3 vs. 46 +/- 3% (18)O labeling of gamma-ATP), and ATP consumption rate (51 +/- 4 vs. 31 +/- 4% (18)O labeling of P(i)) after ischemia-reperfusion. Moreover, preconditioning preserved cardiac creatine kinase-catalyzed phosphotransfer in WT (234 +/- 26 nmol x mg protein(-1) x min(-1)) but not Kir6.2-KO (133 +/- 18 nmol x mg protein(-1) x min(-1)) hearts. In contrast with WT hearts, preconditioning failed to preserve contractile recovery in Kir6.2-KO hearts, as tight coupling between postischemic performance and high-energy phosphoryl transfer was compromised in the K(ATP)-channel-deficient myocardium. Thus intact K(ATP) channels are integral in ischemic preconditioning-induced protection of cellular energetic dynamics and associated cardiac performance.

Adenylate kinase AK1 knockout heart: energetics and functional performance under ischemia-reperfusion.

Deletion of the major adenylate kinase AK1 isoform, which catalyzes adenine nucleotide exchange, disrupts cellular energetic economy and compromises metabolic signal transduction. However, the consequences of deleting the AK1 gene on cardiac energetic dynamics and performance in the setting of ischemia-reperfusion have not been determined. Here, at the onset of ischemia, AK1 knockout mice hearts displayed accelerated loss of contractile force compared with wild-type controls, indicating reduced tolerance to ischemic stress. On reperfusion, AK1 knockout hearts demonstrated reduced nucleotide salvage, resulting in lower ATP, GTP, ADP, and GDP levels and an altered metabolic steady state associated with diminished ATP-to-P(i) and creatine phosphate-to-P(i) ratios. Postischemic AK1 knockout hearts maintained approximately 40% of beta-phosphoryl turnover, suggesting increased phosphotransfer flux through remaining adenylate kinase isoforms. This was associated with sustained creatine kinase flux and elevated cellular glucose-6-phosphate levels as the cellular energetic system adapted to deletion of AK1. Such metabolic rearrangements, along with sustained ATP-to-ADP ratio and total ATP turnover rate, maintained postischemic contractile recovery of AK1 knockout hearts at wild-type levels. Thus deletion of the AK1 gene reveals that adenylate kinase phosphotransfer supports myocardial function on initiation of ischemic stress and safeguards intracellular nucleotide pools in postischemic recovery.

Contribution by different fuels and metabolic pathways to the total ATP turnover of proliferating MCF-7 breast cancer cells.

For the past 70 years the dominant perception of cancer metabolism has been that it is fuelled mainly by glucose (via aerobic glycolysis) and glutamine. Consequently, investigations into the diagnosis, treatment and the basic metabolism of cancer cells have been directed by this perception. However, the data on cancer metabolism are equivocal, and in this study we have sought to clarify the issue. Using an innovative system we have measured the total ATP turnover of the MCF-7 breast cancer cell line, the contributions to this turnover by oxidative and glycolytic ATP production and the contributions to the oxidative component by glucose, lactate, glutamine, palmitate and oleate. The total ATP turnover over approx. 5 days was 26.8 micromol of ATP.10(7) cells(-1).h(-1). ATP production was 80% oxidative and 20% glycolytic. Contributions to the oxidative component were approx. 10% glucose, 14% glutamine, 7% palmitate, 4% oleate and 65% from unidentified sources. The contribution by glucose (glycolysis and oxidation) to total ATP turnover was 28.8%, glutamine contributed 10.7% and glucose and glutamine combined contributed 40%. Glucose and glutamine are significant fuels, but they account for less than half of the total ATP turnover. The contribution of aerobic glycolysis is not different from that in a variety of other non-transformed cell types.