Sepsis alters pyruvate dehydrogenase kinase activity in skeletal muscle

Chronic sepsis promotes a stable increase in pyruvate dehydrogenase kinase (PDHK) activity in skeletal muscle. PDHK is found tightly bound to the pyruvate dehydrogenase (PDH) complex and as free kinase. We investigated the ability of sepsis to modify the activity of the PDHK intrinsic to the PDH and free PDHK. Sepsis was induced by the intraabdominal introduction of a fecal-agar pellet infected with E. coli and B. fragilis. Five days later, mitochondria were isolated from skeletal muscle and PDHK measured in mitochondrial extracts. Sepsis caused an approximate 2-fold stimulation of PDHK. The mitochondrial extracts from control and septic rats were fractionated by gel chromatography on Sephacryl S-300 to separate PDHK intrinsic to PDH complex and free PDHK. PDH complex eluted at void volume and was assayed for PDHK intrinsic to the complex. The activity of PDHK intrinsic to PDH complex was a significantly increased 3 fold during sepsis. Free PDHK activity eluted after the PDH complex and its activity was enhanced by 70% during sepsis. Incubation of PDHK intrinsic to PDH with dichloroactate, an uncompetitive inhibitor of PDHK, showed the PDHK from septic rats relatively less sensitive to inhibition than controls. These results indicate that sepsis induces stable changes in PDHK in skeletal muscle.     

Ribonucleoprotein complexes of pyruvate kinase from human skeletal muscle

Summary About 25% of total pyruvate kinase activity in muscle appears in a bound form which is insoluble in water or diluted salt solutions at pH 5.8. That activity is associated with the ribonuc-leoprotein complexes and is soluble at high ionic strength. A procedure is described for the purification and crystallization of this enzyme form herein called pyruvate kinase M_B and water soluble form M_A.Crystalline nucleoproteins are composed of active and inactive RNA-protein complexes with varying RNA content. By fractional crystallization and gel filtration a number of crystalline complexes were separated, two of them highly purified. One preparation was homogenous, contained 0.5% RNA and had a specific activity of 265 U/mg protein, the other one 10% RNA and 200 U/mg protein respectively.Forms M_A and M_B share the same protein as shown in immunodiffusion test with the anti-M_A sera. They differ in solubility and stability in diluted solutions. In Tris-HCl buffer, pH 7.6 form M_B is rapidly inactivated whereas form M_A is quite stable under the same conditions. Both forms have different K_m for phosphoenolpyruvate and ADP and V_max as well.Digestion of pyruvate kinase M_B with RNase was without marked effect on specific activity of the enzyme.The presence of numerous ribonucleoprotein complexes with a polynucleotide content in the range of 0.5 to 20% and specific activity of 160–220 U/mg protein suggests the control by RNA binding of pyruvate kinase activity from human skeletal muscle.This work was supported by a grant from the Biochemical and Biophysical Committee of the Polish Academy of Sciences.     

Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise.

Prolonged moderate-intensity exercise is characterized by a progressive reduction in carbohydrate oxidation and concomitant increase in fat oxidation. Pyruvate dehydrogenase (PDH) controls the entry of pyruvate into oxidative pathways and is a rate-limiting enzyme for carbohydrate metabolism. PDH is controlled by the activities of a kinase (PDK, inhibitory) and phosphatase (stimulatory). To test the hypothesis that increased PDK activity was associated with decreased PDH activity and carbohydrate oxidation during an acute exercise bout, seven recreationally active men completed 4 h of cycle exercise at 55% peak oxygen consumption. Muscle samples were obtained before and at 10 min and 4 h of exercise for the measurement of PDH activity and the extraction of intact mitochondria for the measurements of PDK activity and PDK-2 and PDK-4 protein expression. Carbohydrate oxidation was reduced (P < 0.05) with exercise duration. Muscle glycogen content was lower (P < or = 0.05) at 4 h compared with rest and there was no change in muscle pyruvate content from 10 to 240 min during exercise (10 min: 0.28 +/- 0.05; 240 min: 0.35 +/- 0.09 mmol/kg dry muscle). PDH activity increased (P < 0.05) above resting values at 10 min (2.86 +/- 0.26 mmol.min(-1).kg wet muscle(-1)), but was lower than 10 min after 4 h (2.23 +/- 0.24 mmol.min(-1).kg wet muscle(-1)) of exercise. PDK-2 and PDK-4 protein expression was not different from rest at 10 min and 4 h of exercise. PDK activity at rest averaged 0.081 +/- 0.016 min(-1), was similar at 10 min, and increased (P < 0.05) to 0.189 +/- 0.013 min(-1) at 4 h. Although reduced glycolytic flux may have played a role in decreasing carbohydrate oxidation, the results suggest that increased PDK activity contributed to the reduction in PDH activity and carbohydrate oxidation late in prolonged exercise. The increased PDK activity was independent of changes in intra-mitochondrial effectors, and PDK-2 and PDK-4 protein content, suggesting that it was caused by a change in the specific activity of the existing kinases.     

The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity

Pyruvate dehydrogenase (PDH) is a mitochondrial enzyme responsible for regulating the conversion of pyruvate to acetyl-CoA for use in the tricarboxylic acid cycle. PDH is regulated through phosphorylation and inactivation by PDH kinase (PDK) and dephosphorylation and activation by PDH phosphatase (PDP). The effect of endurance training on PDK in humans has been investigated; however, to date no study has examined the effect of endurance training on PDP in humans. Therefore, the purpose of this study was to examine differences in PDP activity and PDP1 protein content in human skeletal muscle across a range of muscle aerobic capacities. This association is important as higher PDP activity and protein content will allow for increased activation of PDH, and carbohydrate oxidation. The main findings of this study were that 1) PDP activity (r(2) = 0.399, P = 0.001) and PDP1 protein expression (r(2) = 0.153, P = 0.039) were positively correlated with citrate synthase (CS) activity as a marker for muscle aerobic capacity; 2) E1α (r(2) = 0.310, P = 0.002) and PDK2 protein (r(2) = 0.229, P =0.012) are positively correlated with muscle CS activity; and 3) although it is the most abundant isoform, PDP1 protein content only explained ～ 18% of the variance in PDP activity (r(2) = 0.184, P = 0.033). In addition, PDP1 in combination with E1α explained ～ 38% of the variance in PDP activity (r(2) = 0.383, P = 0.005), suggesting that there may be alternative regulatory mechanisms of this enzyme other than protein content. These data suggest that with higher muscle aerobic capacity (CS activity) there is a greater capacity for carbohydrate oxidation (E1α), in concert with higher potential for PDH activation (PDP activity).     

Kinetic properties of human muscle pyruvate kinase

Summary The steady-state kinetics of human skeletal muscle pyruvate kinase (M_A) and its RNA-complex (M_B) has been examined and compared. Kinetic studies revealed significant differences in kinetic properties with respect to free and complex form of pyruvate kinase.The M_A form follows a simple Michaelis-Menten kinetics in contrast with the M_B form, which displays a negative cooperativity with respect to ADP. V_max for the complex is 40–60% that for free enzyme. Heterologous RNA is a noncompetitive inhibitor of free enzyme but the kinetics of the complex (M_B) is not affected.In presence of 1.0 mM ATP in an assay mixture the kinetic constants of the complex were unchanged except for V_max, which increased by nearly 60%. Aged preparations of free enzyme (M_A) were activated by 100% and more, but the native enzyme was inhibited by 22%.Inorganic phosphate is a potent activator of both forms of pyruvate kinase. In presence of 50 mM K-phosphate the apparent Michaelis constant and interaction coefficient are unchanged, but V_max for free enzyme increases by 35% and for the complex by 70%, respectively. The specific activity of aged M_A form can be restored to the original value after incubation of the enzyme in 50 mM K-phosphate, pH 7.6, or by addition of ATP (1.0 mM) to the assay mixture.     

Differential coupling of smooth and skeletal muscle pyruvate kinase to creatine kinase.

The interaction of pyruvate kinase from skeletal (SKPK) and smooth (SMPK) muscle with MM-creatine kinase (MMCK) and BB-creatine kinase (BBCK) was assessed using temporal absorbance changes, variations in absorbance at different wavelengths, concentration dependence, association in an electric field, and PK kinetic activity. SKPK exhibits a time course of absorbance increase in the presence of MMCK with a time constant of 29.5 min. This increase occurs at all wavelength from 240 to 1000 nm. At 195 nm, the combination of SKPK and MMCK produces a decrease in absorption with electric fields of both 0 and 204 V/cm. The change in SKPK-MMCK is saturable. SKPK activity is significantly increased by the presence of MMCK in solutions of 0-32% ethanol. These results indicate specific SKPK-MMCK interaction. SMPK and BBCK did not exhibit similar coupling when the BBCK concentration dependence of absorbance or SMPK activity in solutions of 0-32% ethanol was determined. Both MMCK and BBCK increased SKPK activity; neither MMCK nor BBCK increased SMPK activity. The ability to form diazymatic complexes with creatine kinase appears to reside in SKPK. This coupling may account for the increased flux through PK without significant substrate changes seen during skeletal muscle activation. This coupling will not occur in smooth muscle.     

The acute effects of differential dietary fatty acids on human skeletal muscle pyruvate dehydrogenase activity.

Pyruvate dehydrogenase (PDH) is an important regulator of carbohydrate oxidation during exercise, and its activity can be downregulated by an increase in dietary fat. The purpose of this study was to determine the acute metabolic effects of differential dietary fatty acids on the activation of the PDH complex (PDHa activity) at rest and at the onset of moderate-intensity exercise. University-aged male subjects (n = 7) underwent two fat-loading trials spaced at least 2 wk apart. Subjects consumed approximately 300 g saturated (SFA) or n-6 polyunsaturated fatty acid (PUFA) fat over the course of 5 h. Following this, participants cycled at 65% of their maximum oxygen uptake for 15 min. Muscle biopsies were taken before and following fat loading and at 1 min exercise. Plasma free fatty acids increased from 0.15 +/- 0.07 to 0.54 +/- 0.19 mM over 5 h with SFA and from 0.11 +/- 0.04 to 0.35 +/- 0.13 mM with n-6 PUFA and were significantly lower throughout the n-6 PUFA trial. PDHa activity was unchanged following fat loading but increased at the onset of exercise in the SFA trial, from 1.18 +/- 0.27 to 2.16 +/- 0.37 mmol x min(-1) x kg wet wt(-1). This effect was negated in the n-6 PUFA trial (1.04 +/- 0.20 to 1.28 +/- 0.36 mmol x min(-1) x kg wet wt(-1)). PDH kinase was unchanged in both trials, suggesting that the attenuation of PDHa activity with n-6 PUFA was a result of changes in the concentrations of intramitochondrial effectors, potentially intramitochondrial NADH or Ca(2+). Our findings suggest that attenuated PDHa activity contributes to the preferential oxidation of n-6 PUFA during moderate-intensity exercise.     

Pyruvate kinase from human skeletal muscle

A simple method is described for the isolation of crystalline pyruvate kinase from human skeletal muscle. The enzyme was purified by ammonium sulfate fractionation, heat treatment and crystallization. Two crystal forms of pyruvate kinase differing in solubility but not in specific activity were found. The homogenous enzyme preparations in triethanolamine buffer, pH 7.6 reveal at 25 degrees a specific activity of 245 U per mg protein, and of 340 U/mg in potassium phosphate buffer (50 mM). The enzyme is activated by inorganic phosphate and fructosediphosphate to the same extent, and inhibited non competetively by ammonium ion. The molecular weight as measured by gel filtration is 220,000 daltons and the enzyme molecule is composed of 4 subunits.     

Phosphoenolpyruvate hydrolase activity of rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase catalyzes the hydrolysis of P-enolpyruvate at the same active site which catalyzes the physiologically important kinase reaction. The hydrolase activity is lower than the kinase activity by a factor of at least 10(3). There are specific monovalent cation and divalent cation requirements. No other cofactors are required. The relative activation of the pyruvate kinase for the hydrolase reaction is: Ni(II) greater than Co(II) greater than Mg(II) greater than Mn(II). This parallels the rates of nonenzymatic hydrolysis of P-enolpyruvate (Benkovic, S.J., and Schray, K.J. (1968) Biochemistry 7, 4097-4102). The pH rate profiles of the hydrolase and kinase reactions activated by Ni(II) and Co(II) are similar, suggesting common features in their mechanisms. In contrast to the kinase reaction, the reaction velocity of the hydrolase increases at high Co(II) concentrations indicating a second mode for hydrolysis.     

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.     

Phosphorylation of Escherichia coli RNA polymerase by rabbit skeletal muscle protein kinase

Rabbit skeletal muscle protein kinase catalyzes the phosphorylation of DNA-dependent RNA polymerase of Escherichia coli in the presence of adenosine 3′,5′-monophosphate and ATP. The phosphorylation occurs on one (or more) serine residue(s) in the σ-factor under reaction conditions similar to those employed for RNA synthesis. The phosphorylation of RNA polymerase and its stimulation by protein kinase are inhibited by a specific heat-stable inhibitor from rabbit skeletal muscle. With conditions more favorable for the protein kinase reaction, phosphorylation of RNA polymerase also occurs on the β subunit of the core enzyme, but this reaction occurs at a much slower rate than the phosphorylation of the σ-factor.     

Purification and characterization of human muscle pyruvate kinase.

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.     

Exercise induces isoform-specific increase in 5'AMP-activated protein kinase activity in human skeletal muscle

The 5'AMP-activated protein kinase (AMPK) is stimulated by contractile activity in rat skeletal muscle. AMPK has emerged as an important signaling intermediary in the regulation of cell metabolism being linked to exercise-induced changes in muscle glucose and fatty acid metabolism. In the present study, we determined the effects of exercise on isoform-specific AMPK activity (alpha1 and alpha2) in human skeletal muscle. Needle biopsies of vastus lateralis muscle were obtained from seven healthy subjects at rest, after 20 and 60 min of cycle ergometer exercise at 70% of VO(2)max, and 30 min following the 60 min exercise bout. In comparison to the resting state, AMPK alpha2 activity significantly increased at 20 and 60 min of exercise, and remained at a higher level with 30 min of recovery. AMPK alpha1 activity tended to slightly decrease with 20 min of exercise at 70%VO(2)max; however, the change was not statistically significant. AMPK alpha1 activities were at basal levels at 60 min of exercise and 30 min of recovery. On a separate day, the same subjects exercised for 20 min at 50% of VO(2)max. Exercise at this intensity did not change alpha2 activity, and similar to exercise at 70% of VO(2)max, there was no significant change in alpha1 activity. In conclusion, exercise at a higher intensity for only 20 min leads to increases in AMPK alpha2 activity but not alpha1 activity. These results suggest that the alpha2-containing AMPK complex, rather than alpha1, may be involved in the metabolic responses to exercise in human skeletal muscle.     

Opposite pathobiochemical fate of pyruvate kinase and adenylate kinase in aged rat skeletal muscle as revealed by proteomic DIGE analysis

Sarcopenia is the drastic loss of skeletal muscle mass and strength during ageing. In order to better understand the molecular pathogenesis of age-related muscle wasting, we have performed a DIGE analysis of young adult versus old rat skeletal muscle. Proteomic profiling revealed that out of 2493 separated 2-D spots, 69 proteins exhibited a drastically changed expression. Age-dependent alterations in protein abundance indicated dramatic changes in metabolism, contractile activity, myofibrillar remodelling and stress response. In contrast to decreased levels of pyruvate kinase (PK), enolase and phosphofructokinase, the mitochondrial ATP synthase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and adenylate kinase (AK) were increased in senescent fibres. Higher expression levels of myoglobin and fatty acid binding-protein indicated a shift to more aerobic-oxidative metabolism in a slower-twitching aged fibre population. The drastic increase in alphaB-crystallin and myotilin demonstrated substantial filament remodelling during ageing. An immunoblotting survey of selected muscle proteins confirmed the pathobiochemical transition process in aged muscle metabolism. The proteomic analysis of aged muscle has identified a large cohort of new biomarkers of sarcopenia including opposite changes in PK and AK, which might be useful for the design of improved diagnostic procedures and/or therapeutic strategies to counteract ageing-induced muscle degeneration.     

Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad,Scaphiopus couchii

Both pyruvate kinase (PK) and phosphofructokinase (PFK) occur in two different forms, separable by isoelectric focusing (IEF), in skeletal muscle of the spadefoot toad Scaphiopus couchii. During estivation (aerobic dormancy) the proportions of the two forms changed compared with controls; in both cases the amount of enzyme in Peak I (pI = 5.3-5.4) decreased whereas activity in Peak II (isoelectric point = 6.2-6.4) increased. In vitro incubation of crude muscle extracts with 32P-ATP under conditions that promoted the activity of cAMP-dependent protein kinase led to strong radiolabeling associated with Peak I, but not Peak II, and reverse phase HPLC confirmed that 32P was associated with the subunits of both PK and PFK found in Peak I. Specific radiolabeling of Peak I PK and PFK by protein kinase A was further confirmed using immunoprecipitation. In total, this information allowed identification of the Peaks I and II enzymes as the phosphorylated and dephosphorylated forms, respectively, and the effect of estivation was to increase the proportion of dephosphorylated PK and PFK in muscle. Analysis of the kinetic properties of partially purified PK and PFK revealed significant kinetic differences between the two forms of each enzyme. For PK, the Peak II (low phosphate) enzyme showed a 1.6-fold higher Km for phosphoenolpyruvate and a 2.4-fold higher Ka for fructose-1,6-bisphosphate than did the Peak I (high phosphate) form. These kinetic properties suggest that Peak II PK is the less active form, and coupled with the shift to predominantly the Peak II form during estivation (87% Peak II vs. 13% Peak I), are consistent with a suppression of PK activity in estivating muscle, as part of the overall metabolic rate depression of the estivating state. A similar shift to predominantly the Peak II, low phosphate, form of PFK (75% Peak II, 25% Peak I) in muscle of estivating animals is also consistent with metabolic suppression since phosphorylation of vertebrate skeletal muscle PFK is typically stimulated during exercise to enhance enzyme binding to myofibrils in active muscle. Peak II PFK also showed reduced sensitivity to inhibition by Mg:ATP (I50 50% higher) compared with the Peak I form suggesting that the enzyme in estivating muscle is less tightly regulated by cellular adenylate status than in awake toads. The data indicate that reversible phosphorylation control over the activity states of enzymes of intermediary metabolism is an important mechanism for regulating transitions between dormant and active states in estivating species.