Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies

It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production. respiration rate; parallel activation; oxidative phosphorylation; metabolic control analysis; flux control coefficient; muscle contraction     

Regulation of oxidative phosphorylation in intact mammalian heart in vivo

A dynamic computer model of oxidative phosphorylation in intact heart was developed by modifying the model of oxidative phosphorylation in intact skeletal muscle published previously. Next, this model was used for theoretical studies on the regulation of oxidative phosphorylation in intact heart in vivo during transition between different work intensities. It is shown that neither a direct activation of ATP usage alone nor a direct activation of both ATP usage and substrate dehydrogenation, including the calcium-activated tricarboxylate acid cycle dehydrogenases, can account for the constancy of [ADP], [PCr], [P(i)] and [NADH] during a significant increase in oxygen consumption and ATP turnover encountered in intact heart in vivo. Only a direct activation of all oxidative phosphorylation complexes in parallel with a stimulation of ATP usage and substrate dehydrogenation enabled to reproduce the experimental data concerning the constancy of metabolite concentrations. The molecular background of the differences between heart and skeletal muscle in the kinetic behaviour of the oxidative phosphorylation system is also discussed.     

A model of oxidative phosphorylation in mammalian skeletal muscle

A dynamic computer model of oxidative phosphorylation in oxidative mammalian skeletal muscle was developed. The previously published model of oxidative phosphorylation in isolated skeletal muscle mitochondria was extended by incorporation of the creatine kinase system (creatine kinase plus phosphocreatine/creatine pair), cytosolic proton production/consumption system (proton production/consumption by the creatine kinase-catalysed reaction, efflux/influx of protons), physiological size of the adenine nucleotide pool and some additional minor changes. Theoretical studies performed by means of the extended model demonstrated that the CK system, which allows for large changes in P(i) in relation to isolated mitochondria system, has no significant influence on the kinetic properties of oxidative phosphorylation, as inorganic phosphate only slightly modifies the relationship between the respiration rate and [ADP]. Computer simulations also suggested that the second-order dependence of oxidative phosphorylation on [ADP] proposed in the literature refers only to the ATP synthesis flux, but not to the oxygen consumption flux (the difference between these two fluxes being due to the proton leak). Next, time courses of changes in fluxes and metabolite concentrations during transition between different steady-states were simulated. The model suggests, in accordance with previous theoretical predictions, that activation of oxidative phosphorylation by an increase in [ADP] can (roughly) explain the behaviour of the system only at low work intensities, while at higher work intensities parallel activation of different steps of oxidative phosphorylation is involved.     

Regulation of ATP supply in mammalian skeletal muscle during resting state-->intensive work transition

In the present debating paper, the problem how the rate of ATP supply by oxidative phosphorylation in mitochondria is adjusted to meet a greatly increased demand for ATP during intensive exercise of skeletal muscle is discussed. Different experimental results are collected from different positions of the literature and confronted with five conceptual models of the regulation of the oxidative phosphorylation system. The previously performed computer simulations using a dynamic model of oxidative phosphorylation are also discussed in this context. The possible regulatory mechanisms considered in the present article are: (A) output activation: an external effector activates directly only the output of the system (ATP turnover); (B) input/output activation: an external effector activates directly the output (ATP usage) and input (substrate dehydrogenation) of the system; (C) removal of substrate shortage: only ATP consumption and substrate supply by blood are directly activated; (D) removal of oxygen shortage: only ATP consumption and oxygen supply by blood are directly activated; (E) each step activation: an external effector activates both the ATP-consuming subsystem and all the steps in the ATP-producing subsystem (particular enzymes/carriers/blocks of oxidative phosphorylation, substrate supply, oxygen supply). The performed confrontation of the considered mechanisms with the presented results leads to the conclusion that only the each step activation model is quantitatively consistent with the whole set of experimental data discussed. It is therefore postulated that a universal effector/regulatory mechanism of a still unknown nature which activates all steps of oxidative phosphorylation should exist and be discovered. A possible nature of such an effector is shortly discussed.     

Influence of substrate activation (hydrolysis of ATP by first steps of glycolysis and beta-oxidation) on the effect of enzyme deficiencies,inhibitors, substrate shortage and energy demand on oxidative phosphorylation

In intact tissues respiratory substrates (glucose, fatty acids) must be activated with the use of ATP before they may be oxidised and used for energy (ATP) production. This activation by product constitutes an example of a typical positive feedback. In the present paper, the influence of substrate activation on the effect of inborn enzyme deficiencies, inhibitors, lowered oxygen tension, respiratory fuel shortage and increased energy demand on respiration and ATP synthesis is studied with the aid of the dynamic computer model of oxidative phosphorylation in isolated mitochondria developed previously. Computer simulations demonstrate that, in the case where oxidative phosphorylation in the whole organism is partially inhibited, the necessity of substrate activation can have significant impact on the relationship between the activity of (particular steps of) oxidative phosphorylation (or the value of energy demand) and the respiration rate. Depending on the sensitivity of ATP usage to ATP concentration, substrate activation may either slightly enhance the effect of the decrease in the oxidative phosphorylation activity (increase in energy demand) or may lead to a non-stability and sudden collapse of the respiration rate and phosphorylation potential below (above) a certain threshold value of oxidative phosphorylation activity (energy demand). This theoretical finding suggests a possible causal relationship between the affinity of ATP usage to [ATP] and the tissue specificity of mitochondrial diseases.     

Regulation of pyruvate dehydrogenase in the common killifish, Fundulus heteroclitus, during hypoxia exposure

We examined the metabolic responses of the hypoxia-tolerant killifish (Fundulus heteroclitus) to 15 h of severe hypoxia and recovery with emphasis on muscle substrate usage and the regulation of the mitochondrial protein pyruvate dehydrogenase (PDH), which controls carbohydrate oxidation. Hypoxia survival involved a transient activation of substrate-level phosphorylation in muscle (decreases in [creatine phospate] and increases in [lactate]) during which time mechanisms to reduce overall ATP consumption were initiated. This metabolic transition did not affect total cellular [ATP], but had an impact on cellular energy status as indicated by large decreases in [ATP]/[ADP(free)] and [ATP]/[AMP(free)] and a significant loss of phosphorylation potential and Gibbs free energy of ATP hydrolysis (DeltafG'). The activity of PDH was rapidly (within 3 h) decreased by approximately 50% upon hypoxia exposure and remained depressed relative to normoxic samples throughout. Inactivation of PDH was primarily mediated via posttranslational modification following the accumulation of acetyl-CoA and subsequent activation of pyruvate dehydrogenase kinase (PDK). Estimated changes in cytoplasmic and mitochondrial [NAD(+)]/[NADH] did not parallel one another, suggesting the mitochondrial NADH shuttles do not function during hypoxia exposure. Large increases in the expression of PDK (PDK isoform 2) were consistent with decreased PDH activity; however, these changes in mRNA were not associated with changes in total PDK-2 protein content assessed using mammalian antibodies. No other changes in the expression of other known hypoxia-responsive genes (e.g., lactate dehydrogenase-A or -B) were observed in either muscle or liver.     

Theoretical studies on the regulation of oxidative phosphorylation in intact tissues

The theoretical studies on the regulation of oxidative phosphorylation that were performed with the aid of kinetic models of this process are overviewed. A definition of the regulation of the flux through a metabolic pathway is proposed and opposed to the control exerted by particular enzymes over this flux. Different kinetic models of oxidative phosphorylation proposed in the literature are presented, of which only the model proposed by myself and co-workers was extensively used in theoretical studies on the regulation and compensation in the oxidative phosphorylation system. These theoretical studies have led to the following conclusions: (1) in isolated mitochondria, an increase in the activity of an artificial ATP-using system stimulates mitochondria mainly via changes in [ADP], while changes in [ATP] and [P(i)] play only a minor role; (2) in non-excitable tissues (e.g. liver), hormones (acting via some cytosolic factor(s)) activate directly both ATP usage and at least some enzymes of the ATP-producing block; (3) in excitable tissues (e.g. skeletal muscle), neural signals stimulate (via some cytosolic factor(s)) in parallel all the steps of oxidative phosphorylation together with ATP usage and substrate dehydrogenation; (4) the decrease in the flux through cytochrome oxidase caused by a decrease in oxygen concentration is, at least partially, compensated by a decrease in Delta p and increase in the reduction level of cytochrome c. A theoretical prediction is formulated that there should exist and be observable a universal cytosolic factor/regulatory mechanism which directly activates (at least in excitable tissues) all complexes of oxidative phosphorylation during an increased energy demand.     

Parallel activation in the ATP supply-demand system lessens the impact of inborn enzyme deficiencies, inhibitors, poisons or substrate shortage on oxidative phosphorylation in vivo

A potential kinetic impact of parallel activation of different steps during an increased energy demand on the effect of inborn enzyme deficiencies, physiological inhibitors, external poisons and substrate shortage on oxidative phosphorylation was studied in the theoretical way. Numerical simulations were performed with the aid of the previously developed computer model of oxidative phosphorylation. It was demonstrated that the parallel activation mechanism diminishes significantly changes in fluxes and metabolite concentrations occurring at a given degree of inactivation of the system by one of the above-mentioned factors. It was also shown that parallel activation decreases greatly the threshold value of the relative activity of oxidative phosphorylation, below which the oxygen consumption flux and ATP turnover flux become significantly affected. Finally, computer simulations predicted that parallel activation leads to a considerable increase in the apparent affinity of oxidative phosphorylation to oxygen, which delays the effect of inhibitors and poisons competing with oxygen for the active centre of cytochrome oxidase. It is concluded that one of possible functions of parallel direct activation of different steps of oxidative phosphorylation is to increase the resistance of the system to a decrease in the concentration/activity of different oxidative phosphorylation complexes.     

Competition between extramitochondrial and intramitochondrial ATP-consuming processes.

The relationship between intra- and extramitochondrial ATP utilization was investigated in liver mitochondria isolated from normally fed, starved and high-protein fed rats. ATP export was provoked by adding a hexokinase-glucose-trap and intramitochondrial ATP consumption by adding ammonia, bicarbonate and ornithine in order to stimulate citrulline synthesis. Both processes compete for ATP produced via oxidative phosphorylation; the rate of citrulline formation declines as the extramitochondrial [ATP]/[ADP] ratio decreases. It is concluded that ATP for adenine nucleotide translocation and that for carbamoyl phosphate synthesis are delivered from a common intramitochondrial pool of adenine nucleotides. In mitochondria from rats with a high-protein diet, citrulline synthesis greatly stimulates the rate of oxidative phosphorylation (about two thirds of state 3 respiration). Under these conditions the intramitochondrial [ATP]/[ADP] ratio is significantly reduced. The intramitochondrial [ATP]/[ADP] ratio is not in thermodynamic equilibrium with the extramitochondrial one.     

Relationships between the neuronal sodium/potassium pump and energy metabolism. Effects of K+, Na+, and adenosine triphosphate in isolated brain synaptosomes

The relationships between Na/K pump activity and adenosine triphosphate (ATP) production were determined in isolated rat brain synaptosomes. The activity of the enzyme was modulated by altering [K+]e, [Na+]i, and [ATP]i while synaptosomal oxygen uptake and lactate production were measured simultaneously. KCl increased respiration and glycolysis with an apparent Km of about 1 mM which suggests that, at the [K+]e normally present in brain, 3.3-4 mM, the pump is near saturation with this cation. Depolarization with 6-40 mM KCl had negligible effect on ouabain-sensitive O2 uptake indicating that at the voltages involved the activity of the Na/K ATPase is largely independent of membrane potential. Increases in [Na+]i by addition of veratridine markedly enhanced glycoside-inhibitable respiration and lactate production. Calculations of the rates of ATP synthesis necessary to support the operation of the pump showed that greater than 90% of the energy was derived from oxidative phosphorylation. Consistent with this: (a) the ouabain-sensitive Rb/O2 ratio was close to 12 (i.e., Rb/ATP ratio of 2); (b) inhibition of mitochondrial ATP synthesis by Amytal resulted in a decrease in the glycoside-dependent rate of 86Rb uptake. Analyses of the mechanisms responsible for activation of the energy-producing pathways during enhanced Na and K movements indicate that glycolysis is predominantly stimulated by increase in activity of phosphofructokinase mediated via a rise in the concentrations of adenosine monophosphate [AMP] and inorganic phosphate [Pi] and a fall in the concentration of phosphocreatine [PCr]; the main moving force for the elevation in mitochondrial ATP generation is the decline in [ATP]/[ADP] [Pi] (or equivalent) and consequent readjustments in the ratio of the intramitochondrial pyridine nucleotides [( NAD]m/[NADH]m). Direct stimulation of pyruvate dehydrogenase by calcium appears to be of secondary importance. It is concluded that synaptosomal Na/K pump is fueled primarily by oxidative phosphorylation and that a fall in [ATP]/[ADP][Pi] is the chief factor responsible for increased energy production.     

Mechanisms of mitochondrial response to variations in energy demand in eukaryotic cells

This review focuses on the different mechanisms involved in the adjustment of mitochondrial ATP production to cellular energy demand. The oxidative phosphorylation steady state at constant mitochondrial enzyme content can vary in response to energy demand. However, such an adaptation is tightly linked to a modification in both oxidative phosphorylation yield and phosphate potential and is obviously very limited in eukaryotic cells. We describe the three main mechanisms involved in mitochondrial response to energy demand. In heart cells, a short-term adjustment can be reached mainly through metabolic signaling via phosphotransfer networks by the compartmentalized energy transfer and signal transmission. In such a complex regulatory mechanism, Ca²⁺ signaling participates in activation of matricial dehydrogenases as well as mitochondrial ATP synthase. These processes allow a large increase in ATP production rate without an important modification in thermodynamic forces. For a long-term adaptation, two main mechanisms are involved: modulation of the mitochondrial enzyme content as a function of energy demand and/or kinetic regulation by covalent modifications (phosphorylations) of some respiratory chain complex subunits. Regardless of the mechanism involved (kinetic regulation by covalent modification or adjustment of mitochondrial enzyme content), the cAMP signaling pathway plays a major role in molecular signaling, leading to the mitochondrial response. We discuss the energetic advantages of these mechanisms. yeast; C6 glioma cells; muscle; kinetic regulation     

Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

Incubation of [gamma-32P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma-32P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma-32P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide.     

Kinetic, thermodynamic, and developmental consequences of deleting creatine kinase isoenzymes from the heart. Reaction kinetics of the creatine kinase isoenzymes in the intact heart

Creatine kinase (CK) exists as a family of isoenzymes in excitable tissue. We studied isolated perfused hearts from mice lacking genes for either the main muscle isoform of CK (M-CK) or both M-CK and the main mitochondrial isoform (Mt-CK) to determine 1) the biological significance of CK isoenzyme shifts, 2) the necessity of maintaining a high CK reaction rate, and 3) the role of CK isoenzymes in establishing the thermodynamics of ATP hydrolysis. (31)P NMR was used to measure [ATP], [PCr], [P(i)], [ADP], pH, as well as the unidirectional reaction rate of PCr--> [gamma-P]ATP. Developmental changes in the main fetal isoform of CK (BB-CK) were unaffected by loss of other CK isoenzymes. In hearts lacking both M- and Mt-CK, the rate of ATP synthesis from PCr was only 9% of the rate of ATP synthesis from oxidative phosphorylation demonstrating a lack of any high energy phosphate shuttle. We also found that the intrinsic activities of the BB-CK and the MM-CK isoenzymes were equivalent. Finally, combined loss of M- and Mt-CK (but not loss of only M-CK) prevented the amount of free energy released from ATP hydrolysis from increasing when pyruvate was provided as a substrate for oxidative phosphorylation.     

AMP deamination delays muscle acidification during heavy exercise and hypoxia

In silico studies carried out by using a computer model of oxidative phosphorylation and anaerobic glycolysis in skeletal muscle demonstrated that deamination of AMP to IMP during heavy short term exercise and/or hypoxia lessens the acidification of myocytes. The concerted action of adenylate kinase and AMP deaminase, leading to a decrease in the total adenine nucleotide pool, constitutes an additional process consuming ADP and producing ATP. It diminishes the amount of ADP that must be converted to ATP by other processes in order to meet the rate of ADP production by ATPases (because the adenylate kinase + AMP deaminase system produces only 1 ATP per 2 ADPs used, ATP consumption is not matched by ATP production, and the reduction of the total adenine nucleotide pool occurs mostly at the cost of [ATP]). As a result, the rate of ADP consumption by other processes may be lowered. This effect concerns mostly ADP consumption by anaerobic glycolysis that is inhibited by AMP deamination-induced decrease in [ADP] and [AMP], and not oxidative phosphorylation, because during heavy exercise and/or hypoxia [ADP] is significantly greater than the Km value of this process for ADP. The resultant reduction of proton production by anaerobic glycolysis enables us to delay the termination of exercise because of fatigue and/or to diminish cell damage.     

In vitro and in vivo activation of the insulin receptor kinase in control and denervated skeletal muscle

Skeletal muscle rapidly develops severe insulin resistance following denervation, although insulin binding is unimpaired. Insulin-stimulated receptor tyrosyl kinase activity was studied in intact and 24-h denervated rat hind limb muscles using three preparations: (a) solubilized insulin receptors incubated +/- insulin with gamma-[32P]ATP and histone H2b; (b) soleus muscles prelabeled in vitro with [32P]phosphate with subsequent insulin-stimulated phosphorylation of the receptor in situ; (c) assessment of in vivo activation of muscle receptor tyrosyl kinase by insulin. The latter was achieved by solubilizing muscle insulin receptors in the presence of phosphoprotein phosphatase and kinase inhibitors and measuring receptor-catalyzed histone H2b phosphorylation in the presence of limiting (5 microM) gamma-[32P]ATP. Receptors isolated 5 and 30 min after intravenous insulin injection catalyzed 32P incorporation into histone H2b twice as fast as those from saline-treated controls; insulin stimulated histone H2b labeling exclusively on tyrosine. In vivo activation was demonstrated using solubilized and insulin-agarose-bound receptors. Autophosphorylation of the beta-subunit and receptor tyrosyl kinase activity toward histone H2b was stimulated by insulin in denervated muscles as in controls, although the biological response to insulin, in vitro and in vivo, was markedly impaired after denervation, suggesting a postreceptor defect. The method developed to assess insulin-stimulated receptor activation in vivo seems useful in characterizing mechanisms of insulin resistance.     

Hexose metabolism in pancreatic islets: preferential utilization of mitochondrial ATP for glucose phosphorylation

The respective contribution of exogenous and intramitochondrially formed ATP to D-glucose phosphorylation by mitochondria-bound hexokinase was examined in both rat liver and pancreatic islet mitochondria by comparing the generation of D-glucose 6-[32P]phosphate from exogenous [gamma-32P]ATP to the total rate of D-[U-14C]glucose phosphorylation. In liver mitochondria, the fractional contribution of exogenous ATP to D-glucose phosphorylation ranged from 4 to 74%, depending on the availability of endogenous ATP formed by either oxidative phosphorylation or in the reaction catalyzed by adenylate kinase. Likewise, in islet mitochondria exposed to exogenous ATP but deprived of exogenous nutrient, about 60% of D-glucose phosphorylation was supported by mitochondrial ATP. Such a fractional contribution was further increased in the presence of ADP and succinate, and suppressed by mitochondrial poisons. It is concluded that, in islet like in liver mitochondria, mitochondrial ATP is used preferentially to exogenous ATP as a substrate for D-glucose phosphorylation by mitochondria-bound hexokinase. This may favour the maintenance of a high cytosolic ATP concentration in glucose-stimulated islet cells.     

Direct evidence for the control of mitochondrial respiration by mitochondrial creatine kinase in oxidative muscle cells in situ

The efficiency of stimulation of mitochondrial respiration in permeabilized muscle cells by ADP produced at different intracellular sites, e.g. cytosolic or mitochondrial intermembrane space, was evaluated in wild-type and creatine kinase (CK)-deficient mice. To activate respiration by endogenous production of ADP in permeabilized cells, ATP was added either alone or together with creatine. In cardiac fibers, while ATP alone activated respiration to half of the maximal rate, creatine plus ATP increased the respiratory rate up to its maximum. To find out whether the stimulation by creatine is a consequence of extramitochondrial [ADP] increase, or whether it directly correlates with ADP generation by mitochondrial CK in the mitochondrial intermembrane space, an exogenous ADP-trap system was added to rephosphorylate all cytosolic ADP. Under these conditions, creatine plus ATP still increased the respiration rate by 2.5 times, compared with ATP alone, for the same extramitochondrial [ADP] of 14 microM. Moreover, this stimulatory effect of creatine, observed in wild-type cardiac fibers disappeared in mitochondrial CK deficient, but not in cytosolic CK-deficient muscle. It is concluded that respiration rates can be dissociated from cytosolic [ADP], and ADP generated by mitochondrial CK is an important regulator of oxidative phosphorylation.     

Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply

During low-to-high work transition in adult mammalian heart in vivo the concentrations of free ADP, ATP, PCr (phosphocreatine), P(i) and NADH are essentially constant, in striking contrast with skeletal muscle. The direct activation by calcium ions of ATP usage and feedback activation of ATP production by ADP (and P(i)) alone cannot explain this perfect homoeostasis. A comparison of the response to adrenaline (increase in rate-pressure product and [PCr]) of the intact beating perfused rat heart with the elasticities of the PCr producer and consumer to PCr concentration demonstrated that both the ATP/PCr-producing block and ATP/PCr-consuming block are directly activated to a similar extent during physiological heart activation. Our finding constitutes a direct evidence for the parallel-activation mechanism of the regulation of oxidative phosphorylation in heart postulated previously in a theoretical way.     

Insulin mediator causes dephosphorylation of the alpha subunit of pyruvate dehydrogenase by stimulating phosphatase activity

Insulin treatment of rats results in an increased amount or activity of insulin mediators in liver and skeletal muscle. These mediators stimulated pyruvate dehydrogenase and inhibited adenylate cyclase. The insulin-generated mediators caused dephosphorylation of the alpha subunit of pyruvate dehydrogenase in mitochondria prelabeled with [gamma-32P]ATP. An assay was developed which quantitatively measured mediator activity by determining the rate of alpha-subunit dephosphorylation. The dephosphorylation was directly proportional to the amount of mediator added and was directly related to activation of pyruvate dehydrogenase. The decrease of alpha-subunit phosphorylation resulted from stimulation of pyruvate dehydrogenase phosphatase, since it occurred in the absence of ATP and was inhibited by NaF. These data further delineate the mechanism of insulin mediator activation of pyruvate dehydrogenase.