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The modern scientific study of physical activity began soon after World War II and focused on the epidemic of cardiovascular disease that was beginning to engulf the Western world. Early 'exercise prescriptions' then specified intense bouts of vigorous activity as the most effective way to maintain cardiovascular fitness and 'heart health'. Doctors and other health professionals then grew concerned that progressively fewer individuals were heeding this advice at a time when physical activity from manual work was becoming less common. Evidence was also emerging in the late 1980s that the value of accumulated, moderately intense activities, now of increasing importance during leisure time, may have been overlooked, or at least underplayed, in the prevention of heart disease, diabetes and some cancers. Perhaps in population terms adherence to moderate 'lifestyle' activities would be better than to the earlier vigorous recommendations. Social policy therefore shifted in the United States in 1996; the US Surgeon General's report set out the basic public health message of '30 min of moderate activity five, and preferably all, days of the week'. This recommendation was broadly adopted throughout much of the Western world. How this change in health strategy might impact on unhealthy weight gain and the growing obesity epidemic was given little attention. Here we examine how post-war public health policy in physical activity developed in an attempt primarily to prevent cardiovascular disease. In the following article we examine why too little attention may have been given to unhealthy weight gain and investigate how this may have happened. Then we consider how much physical activity--and of what kind--is needed to prevent unhealthy weight gain.

The current guidelines for physical activity are based on the prevention of cardiovascular disease. In this article the magnitude and type of physical activity required to prevent unhealthy weight gain are assessed. Five categories of analyses are considered, ranging from the most rigorous analyses (based on D2O18 measures of energy expenditure) to socio-ecological associations. To standardize the approach, published work on the extent of exercise was expressed as a physical activity level (PAL), i.e. the ratio of total expenditure to the measured or estimated basal metabolic rate. D2O18, direct monitoring and measurements of activity patterns and detailed prospective studies of substantial population groups all suggest that a PAL of > or = 1.8 is required to limit the proportion of overweight and obese adult men. Data on women are more difficult to interpret because women are less active and the relationship with physical activity is usually less clear. Post-obese women with a PAL of >1.75 do not regain weight and other data are consistent with the need for a PAL of > or = 1.8. The analyses in both sexes are based predominantly on adults living in a Western society with the ready availability of energy-dense foods. Vigorous activity is more clearly linked to weight stability, allows a higher intensity of exercise for general activities and shortens the time needed for achieving a PAL of 1.8. This activity level is equivalent to an additional 60-90 min of brisk walking in adults who normally undertake only modest exercise. These demands are greater than the current suggested levels for cardiovascular benefit and imply the need for different environmental policies, rather than health education policies, if societies are to become generally more active and avoid unhealthy weight gain.

Total pyruvate dehydrogenase complex activity is low in white adipose tissue during the suckling period and increases markedly at weaning on to a high-carbohydrate diet. This is concomitant with an increase in the E1 alpha, E1 beta and E2 subunit protein concentration and their respective mRNAs, suggesting a pretranslational control of this phenomenon. The most marked change is seen for the E1 alpha subunit (17-fold increase in protein concentration). The changes in pyruvate dehydrogenase complex activity and subunit abundance induced by weaning on to a high-carbohydrate diet are precluded if the animals are weaned on to a high-fat diet, suggesting that the nutritional and/or related hormonal changes rather than a developmental stage are responsible for the observed adipose-tissue pyruvate dehydrogenase complex pattern.

Rat liver pyruvate dehydrogenase (PDH) kinase activator protein (KAP), a free PDH kinase readily separable from PDH complex and its intrinsic kinase, has been purified to apparent homogeneity from liver mitochondria of fed and 48-h starved rats. On SDS-PAGE an apparently single band of M(r) 45 kDa was obtained. N-Terminal amino acid sequence analyses (8-10 cycles) confirmed the presence of a single peptide in each case. The specific activity of the purified KAP from 48-h starved rats (14,413 U/mg protein) was 4.5-fold greater than that from fed rats.

Starvation increased pyruvate dehydrogenase (PDH) kinase activity in extracts of freshly excised rat soleus 2.2-fold (from 0.6 min-1 in fed rats to 1.31 min-1 in 48-h-starved rats). In fed rats, activities were unchanged following 24 h of culture in medium 199, but increased 2.1-fold on 24 h of culture with 50 microM dibutyryl cAMP plus 1 mM n-octanoate and 1.6-1.7-fold with either agent alone. Approx. 70% of the increase in PDH kinase induced by starvation was lost following 24 h of culture in medium 199; the loss was prevented by 50 microM dibutyryl cAMP plus 1 mM n-octanoate. cAMP concentrations in fresh soleus muscle were 1 nmol/g (fed rats) and 1.6 nmol/g (starved rats). After 20-60 min of culture the fed-starved difference disappeared and [cAMP] fell to 0.4 nmol/g. Calcitonin-gene-related peptide (CGRP) increased cAMP 3-fold; the increase was maintained throughout 24 h of culture, but was readily reversed at 30 min or 24 h of culture by 60-min incubation with CGRP-free medium. Starvation of the rat (48 h) had no effect on the sensitivity of soleus towards the [cAMP]-increasing effect of CGRP. It is concluded that culture may reverse effects of starvation on PDH kinase activity by lowering cAMP and by removal from the in vivo effects of circulating free fatty acids; and that starvation and CGRP had no detectable long-term effects on the cAMP system in soleus muscle.

It is shown here that rat liver pyruvate dehydrogenase (PDH) kinase activator protein (KAP) catalyses ATP-dependent inactivation and [32P]phosphorylation of pig heart PDHE1 and of yeast (Saccharomyces cerevisiae) PDH complex devoid of PDH kinase activity, that fluorosulphonylbenzoyladenosine inactivates rat liver KAP and the intrinsic PDH kinase of rat liver PDH complex, and that KAP, like PDH kinase, is inactivated by thiol-reactive reagents. It is concluded that KAP is a free PDH kinase.

Rat soleus muscle strips cultured for 24 h in medium 199 were well preserved in terms of electron microscopy; ATP and creatine phosphate concentrations; rates of glucose utilization, glycogen and protein synthesis, and effects of insulin thereon. Culture led to modest changes in fluid spaces and intracellular (K+); increased basal glucose utilization up to two-fold; had no effect on the maximum response to insulin; and had no effect on sensitivity to insulin except in the presence of adenosine deaminase. Thus in vitro neither denervation nor absence of insulin had any marked effects in 24 h to decrease responses to insulin.

The activity of pyruvate dehydrogenase (PDH) complex and PDH kinase were measured in brown adipose tissue (BAT) of 4-week-gold thioglucose (GTG)-obese mice. The proportion of PDH complex in the active dephosphorylated form was 2-fold higher in BAT of post-absorptive obese mice compared with lean controls. This result was consistent with the higher circulating insulin concentration observed in GTG-obese mice. In both obese and lean mice the PDH-complex activity in BAT decreased after 24 h starvation and increased in response to supraphysiological insulin injection, indicating that the PDH complex is insulin-responsive in BAT of GTG-obese mice. There was no difference in the PDH kinase activity of BAT in post-absorptive or insulin-injected lean and obese mice, suggesting that the higher PDH-complex activity in obese mice was not due to decreased PDH kinase activity. There is no evidence for a decreased activity of PDH complex contributing to insulin resistance in BAT of 4-week-GTG-obese mice.

The increased activity of pyruvate dehydrogenase (PDH) kinase induced in hearts of rats by starvation for 48 h was maintained following preparation of cardiac myocytes, and it was also maintained, though at a decreased level, after 25 h of culture in medium 199. This loss of PDH kinase activity was not prevented by n-octanoate, dibutyryl cyclic AMP or glucagon. The PDH kinase activity of myocytes from fed rats was increased to that of starved rats after 25 h of culture with n-octanoate, dibutyryl cyclic AMP or both agents together.

The phosphorylation of sites additional to an inactivating site inhibits the formation of active pig heart pyruvate dehydrogenase complex from inactive pyruvate dehydrogenase phosphate complex by pig heart pyruvate dehydrogenase phosphate phosphatase.

The proportion of pyruvate dehydrogenase complex in the active, dephosphorylated form was decreased (compared with lean controls) in heart muscle in gold thioglucose-treated obese hyperinsulinaemic mice, and the extent of enzyme inactivation was significantly linearly correlated with both body weight and body fat content. A single oral dose (25 mg/kg body wt.) of the beta-oxidation inhibitor 2-tetradecylglycidic acid to obese animals restored pyruvate dehydrogenase complex activity to that of lean controls. It is suggested that increased fatty acid oxidation may be a major factor in mediating the phosphorylation and inactivation of pyruvate dehydrogenase complex in mouse heart muscle in obesity, and this may represent an important mechanism in the development and/or expression of insulin resistance in respect of abnormalities of cellular glucose homoeostasis in these animals.

In tissue culture of hepatocytes, insulin (0.1-1 munits/ml for 4 h) reversed completely the effects of starvation of rats to decrease the activity of pyruvate dehydrogenase (PDH) complex and to increase the activities of PDH kinase and PDH kinase activator protein. It had no effect in hepatocytes from fed rats. Significant effects of insulin were detected with 0.01 munit/ml after 4 h, and in 1-2 h with 1 munit/ml.

The work investigated the effects of surgical stress on the activities of cardiac and hepatic pyruvate dehydrogenase complex (active form, PDHa) in fed rats. PDHa activities in heart and liver were decreased within 4h of surgery with maximum inhibition at 24h after surgery. PDHa activities remained low until the fourth (liver) and eighth (heart) post-operative days. The decreased activities found at 4h and 24h after surgery were associated with increased plasma fatty acid concentrations, and inhibition of lipolysis resulted in reactivation of the enzyme complex. The results are discussed with reference to the control of pyruvate dehydrogenase activities by the oxidation of fat fuels and multisite phosphorylation in stress states, and its possible importance in glucose conservation after surgery and trauma.

The activities of pyruvate dehydrogenase (PDH) kinase and of PDH kinase activator protein (KAP) were increased 2-2.4-fold during 25 h of culture of hepatocytes from fed rats with glucagon plus n-octanoate. PDH kinase activity in hepatocytes from starved rats (initially 2.2 x fed control) fell during 25 h of culture in medium 199 (to 1.5 x fed control), but was maintained by glucagon plus octanoate. Dibutyryl or 8-bromo cyclic AMP increased PDH kinase activity 2-2.2-fold in hepatocytes from fed rats, but phenylephrine and isoproterenol (isoprenaline) were without effect. Insulin blocked the action of glucagon to increase PDH kinase activity and decreased the effect of octanoate and octanoate plus glucagon. It is suggested that the effects of starvation to increase activities of PDH kinase and of KAP in liver are mediated by alterations in circulating concentrations of glucagon, fatty acids and insulin and in hepatic cyclic AMP.

The proportion of pyruvate dehydrogenase (PDH) complex in the active dephosphorylated form was decreased (compared with fed lean control mice) in heart muscle mitochondria after the induction of obesity with gold-thioglucose (by 54%) or starvation of lean mice for 48 h (by 81%). The effects of obesity to inactivate PDH complex were demonstrable 4 weeks after administration of gold-thioglucose, and occurred despite significant hyperinsulinaemia in obese animals. Phosphorylation and inactivation of PDH complex in mouse heart muscle in starvation was attributed to a stable increase (2.7-fold) in the activity of PDH kinase as measured in extracts of mitochondria mediated by increased specific activity of a protein activator of PDH kinase (KAP) [Denyer, Kerbey & Randle (1986) Biochem. J. 239, 347-354]. In obese mice no such increase in kinase activity was observed, and we conclude that phosphorylation and inactivation of PDH complex in heart muscle in obesity is not mediated by KAP, but rather is a consequence of increased lipid oxidation.

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.

1. Pig heart pyruvate dehydrogenase complex is inactivated by phosphorylation (MgATP2-) of an alpha-chain of the decarboxylase component. Three serine residues may be phosphorylated, one of which (site 1) is the major inactivating site. 2. The relative rates of phosphorylation are site 1 greater than 2 greater than site 3. 3. The kinetics of the inactivating phosphorylation were investigated by measuring inactivation of the complex with MgATP2-. The apparent Km for the Mg complex of ATP was 25.5 microM; ADP was a competitive inhibitor (Ki 69.8 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 2.8 microM). Inactivation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA. 4. The kinetics of additional phosphorylations (predominantly site 2 under these conditions) were investigated by measurement of 32P incorporation into non-radioactive pyruvate dehydrogenase phosphate containing 3-6% of active complex, and assumed from parrallel experiments with 32P labelling to contain 91% of protein-bound phosphate in site 1 and 9% in site 2. 5. The apparent Km for the Mg complex of ATP was 10.1 microM; ADP was a competitive inhibitor (Ki 31.5 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 1.1 mM). 6. Incorporation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA, although it was less marked at the highest ratios.

The pyruvate dehydrogenase (E1) and acetyltransferase (E2) components of pig heart and ox kidney pyruvate dehydrogenase (PDH) complex were separated and purified. The E1 component was phosphorylated (alpha-chain) and inactivated by MgATP. Phosphorylation was mainly confined to site 1. Addition of E2 accelerated phosphorylation of all three sites in E1 alpha and inactivation of E1. On the basis of histone H1 phosphorylation, E2 is presumed to contain PDH kinase, which was removed (greater than 98%) by treatment with p-hydroxymercuriphenylsulphonate. Stimulation of ATP-dependent inactivation of E1 by E2 was independent of histone H1 kinase activity of E2. The effect of E2 is attributed to conformational change(s) induced in E1 and/or E1-associated PDH kinase. PDH kinase activity associated with E1 could not be separated from it be gel filtration or DEAE-cellulose chromatography. Subunits of PDH kinase were not detected on sodium dodecyl sulphate/polyacrylamide gels of E1 or E2, presumably because of low concentration. The activity of pig heart PDH complex was increased by E2, but not by E1, indicating that E2 is rate-limiting in the holocomplex reaction. ATP-dependent inactivation of PDH complex was accelerated by E1 or by phosphorylated E1 plus associated PDH kinase, but not by E2 plus presumed PDH kinase. It is suggested that a substantial proportion of PDH kinase may accompany E1 when PDH complex is dissociated into its component enzymes. The possibility that E1 may possess intrinsic PDH kinase activity is considered unlikely, but may not have been fully excluded.

In animal tissues the pyruvate dehydrogenase complex is regulated by product inhibition and by a phosphorylation-dephosphorylation cycle catalysed by a kinase and a phosphatase. Physiologic and molecular aspects of this regulation are reviewed, and the results of recent studies are described. Insulin deficiency in the rat (diabetes or starvation) is shown to inhibit the conversion of inactive (phospho-) complex into active (dephospho-) complex by the phosphatase by an effect on the substrate for the phosphatase (phosphorylated complex). This change is stable and persists during isolation, incubation, and extraction of mitochondria or purification of phosphorylated complex. The subunit ratios in the purified pig heart pyruvate dehydrogenase complex and the stoichiometry of phosphorylations have been determined by radioamidination and incorporation of 32P. The ratios of decarboxylase tetramer (alpha 2, beta 2) : dihydrolipoyl acetyltransferase monomer : dihydrolipoly dehydrogenase monomer were 1:1:0.5. Inactivation of the complex was accomplished by incorporation of a single phosphate into one alpha subunit of the decarboxylase tetramer. Two further phosphates are then incorporated and these additional phosphorylations inhibit reactivation of the complex by the phosphate. It is suggested that multisite phosphorylations may inhibit reactivation of the complex by the phosphatase in diabetes and in starvation.

1. The proportion of active (dephosphorylated) pyruvate dehydrogenase in rat heart mitochondria was correlated with total concentration ratios of ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA. These metabolites were measured with ATP-dependent and NADH-dependent luciferases. 2. Increase in the concentration ratio of NADH/NAD+ at constant [ATP]/[ADP] and [acetyl-CoA]/[CoA] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between mitochondria incubated with 0.4mM- or 1mM-succinate and mitochondria incubated with 0.4mM-succinate+/-rotenone. 3. Increase in the concentration ratio acetyl-CoA/CoA at constant [ATP]/[ADP] and [NADH][NAD+] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between incubations in 50 micrometer-palmitotoyl-L-carnitine and in 250 micrometer-2-oxoglutarate +50 micrometer-L-malate. 4. These findings are consistent with activation of the pyruvate dehydrogenase kinase reaction by high ratios of [NADH]/[NAD+] and of [acetyl-CoA]/[CoA]. 5. Comparison between mitochondria from hearts of diabetic and non-diabetic rats shows that phosphorylation and inactivation of pyruvate dehydrogenase is enhanced in alloxan-diabetes by some factor other than concentration ratios of ATP/ADP, NADH/NAD+ or acetyl-CoA/CoA.

In mammalian tissues, two types of regulation of the pyruvate dehydrogenase complex have been described: end product inhibition by acetyl CoA and NADH: and the interconversion of an inactive phosphorylated form and an active nonphosphorylated form by an ATP requiring kinase and a specific phosphatase. This article is largely concerned with the latter type of regulation of the complex in adipose tissue by insulin (and other hormones) and in heart muscle by lipid fuels. Effectors of the two interconverting enzymes include pyruvate and ADP which inhibit the kinase, acetoin which activates the kinase and Ca2+ and Mg2+ which both activate the phosphatase and inhibit the kinase. Evidence is presented that all components of the pyruvate dehydrogenase complex including the phosphatase and kinase are located within the inner mitochondrial membrane. Direct measurements of the matrix concentration of substrates and effectors is not possible by techniques presently available. This is the key problem in the identification of the mechansims involved in the alterations in pyruvate dehydrogenase activity observed in adipose tissue and muscle. A number of indirect approaches have been used and these are reviewed. Most hopeful is the recent finding in this laboratory that in both adipose tissue and heart muscle, differences in activity of pyruvate dehydrogenase in the intact tissue persist during preparation and subsequent incubation of mitochondria.

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.

The proportion of active, dephosphorylated, pyruvate dehydrogenase complex was decreased in the mouse heart by obesity (by 56%), and this decrease in enzyme activity persisted during preparation and extraction of heart mitochondria. Phosphorylation and inactivation of pyruvate dehydrogenase may be a major factor in mediating the inhibitory effects of obesity on glucose oxidation in muscle, and this may represent an important mechanism in the development and/or expression of cellular insulin-resistance.

Extracts of heart mitochondria from fed and from 48 h starved rats subjected to gel filtration on Sephacryl S-300 gave 4 major protein peaks. Pyruvate dehydrogenase complex eluted in the void volume and was assayed for intrinsic pyruvate dehydrogenase kinase activity which was increased approximately 3-fold by 48 h starvation of the rat. A second fraction, containing peaks 2 and 3 which overlapped, enhanced the activity of the intrinsic kinase and corresponds to kinase/activator protein described previously. Its activity was increased 1.5-fold by starvation.

In animals the pyruvate dehydrogenase reaction is mainly responsible for the irreversible loss of glucose carbon by oxidation. Regulation of this reaction is shown to be a major determinant of glucose conservation in starvation and diabetes. Estimates of conservation in man in starvation and diabetes are reviewed. The pyruvate dehydrogenase complex is inhibited by products of its reactions; it is also regulated by a phosphorylation-dephosphorylation cycle catalysed by a kinase intrinsic to the complex and by a more loosely associated phosphatase. Inactivation is largely accomplished by phosphorylation of the tetrameric decarboxylase component (alpha2beta2) to alpha2Pbeta2. Complete phosphorylation produces the (alpha2P3)beta2 form. Both forms are completely reactivated by phosphatase action but the initial rate of reactivation of a complex containing alpha2Pbeta2 is approximately three times that of (alpha2P3)beta2. The proportion of active (dephosphorylated) complex is decreased in rat tissues by starvation and diabetes and in perfused rat heart by oxidation of fatty acids and ketone bodies. In adipose tissue in vitro, insulin increases the proportion of active complex and lipolytic hormones may decrease this proportion. It is suggested that rates of oxidation of lipid fuels may be a major determinant of the activity of pyruvate dehydrogenase in tissues in relation to the actions of insulin and lipolytic hormones and the effects of diabetes and starvation. Phosphorylation and inactivation of the complex are enhanced by high mitochondrial ratios of [acetyl-CoA]/[CoA], [ATP]/[ADP], [NADH]/[NAD+] and low concentrations of pyruvate, Mg2+ and Ca2+, and vice versa.

Hepatic pyruvate kinase (PK) and pyruvate dehydrogenase (PDHa) specific activities were decreased after partial hepatectomy or sham operation. The decreases were more marked and sustained after partial hepatectomy. These activity changes ensure that hepatic carbon flux after partial hepatectomy is predominantly in the direction of gluconeogenesis. The decrease in PK specific activity observed after partial hepatectomy was associated with a decreased PK activation ratio (activity measured at 0.15 mM PEP: activity measured at 5.0 mM PEP), and hepatic concentrations of PEP were increased. The low hepatic PDHa activity observed at the first day after partial hepatectomy occurred concomitantly with an increased fatty acid concentration. PDHa activity increased after inhibition of lipolysis. The results indicate that carbohydrate utilization is unimportant for hepatic energy supply during liver regeneration. There was no evidence that the control of PK or PDH in the regenerative liver after partial hepatectomy differed from that observed in the liver of the unoperated rat.

Purified pig heart pyruvate dehydrogenase complex is denuded of its intrinsic pyruvate dehydrogenase kinase activity by sedimentation from dilute solution (60 munits/ml). Kinase activity is restored by a supernatant fraction prepared by high-speed centrifugation of rat heart mitochondrial extracts; the factor responsible is referred to as kinase/activator. Kinase/activator was also assayed by its ability to accelerate NgATP-induced inactivation in dilute solutions of unprocessed complex (50 munits/ml). With this assay it has been shown that the activity of kinase/activator in heart mitochondria is increased 3-6 fold by starvation of rats for 48 h. This increase was prevented completely by cycloheximide treatment and prevented partially by puromycin treatment of rats during starvation. The concentration of kinase/activator in heart mitochondria fell during 20 h of re-feeding of 48 h-starved rats; this fall was correlated with an increase in the proportion of complex in the active form. Kinase/activator was also extracted from ox kidney mitochondria, and on gel filtration (Sephadex G-100, superfine grade) was eluted close to the void volume. Kinase/activator (ox kidney or rat heart) was thermolabile, non-diffusable on dialysis, and inactivated by trypsin. The results of this study appear to show increased cytoplasmic synthesis in starvation of pyruvate dehydrogenase kinase and/or of an activator of the kinase.

1. Pig heart pyruvate dehydrogenase phosphate complex in which all three sites of phosphorylation were completely phosphorylated was re-activated at a slower rate by phosphatase than complex predominantly phosphorylated in site 1. The ratio of initial rates of re-activation was approx. 1:5 with a comparatively crude preparation of phosphatase and with phosphatase purified by gel filtration and ion-exchange chromatography. 2. The ratio of apparent first-order rate constants during dephosphorylation of fully phosphorylated complex averaged 1/3.8/1.3 for site 1/site 2/site 3. Only site-1 dephosphorylation was linearly correlated with re-activation of the complex throughout dephosphorylation. Dephosphorylation of site 3 was linearly correlated with re-activation after an initial burst of dephosphorylation. 3. Because dephosphorylation of site 1 was always associated with dephosphorylation of site 2, it is concluded that dephosphorylation cannot be purely random. 4. The ratio of apparent first-order rate constants for dephosphorylation of site 1 (partially/fully phosphorylated complexes) averaged 1.72. This ratio is smaller than the ratio of approx. 5 for the initial rates of re-activation. Possible mechanisms involved in the diminished rate of re-activation of fully phosphorylated complex are discussed.

In heart muscle regulation of pyruvate dehydrogenase (PDH) complex activity by reversible phosphorylation is the major determinant of glucose oxidation under physiological conditions and in diabetes. Altered mitochondrial concentrations of effectors of PDH kinase and phosphatase (metabolites, Ca2+, H+) appear to explain effects of oxidation of lipid fuels, myocardial contraction and ischaemia on PDH complex activity. The effects of diabetes and starvation are mediated in addition by protein(s) which increase the activity of PDH kinase. End product inhibition by NADH may be important in ischaemia.

The effects of Ni(2+) on the release of amylase from rat parotids, insulin from mouse pancreatic islets and growth hormone from bovine pituitary slices were investigated. In all these secretory systems, Ni(2+) was shown to inhibit release evoked by a variety of stimuli both physiological and pharmacological. Measurements of rates of substrate oxidation and tissue concentrations of ATP and 3':5'-cyclic AMP suggest that this inhibitory action of Ni(2+) does not arise through an effect on energy metabolism or cyclic AMP metabolism. It is concluded that although some effects of Ni(2+) may involve antagonism between Ni(2+) and Ca(2+) in stimulus-secretion coupling, others appear to be independent of Ca(2+). It is suggested that Ni(2+) may block exocytosis by interfering with either secretory-granule migration or membrane fusion and microvillus formation. The possible mode of action of Ni(2+) and its potential use as a tool in the study of exocytosis are discussed.

Starvation of rats for 48 h increased the activity of PDH (pyruvate dehydrogenase) kinase 2.2-fold in extracts of liver mitochondria, 2.9-fold in PDH complex partially purified therefrom by fractional precipitation, and 5-fold in PDH complex partially purified by gel filtration on Sephacryl S-300. A protein fraction was separated from PDH complex in extracts of rat liver mitochondria by gel filtration or fractional precipitation, which increased the activity of PDH kinase in rat liver and pig heart PDH complexes. The activity of this protein fraction was increased approx. 2.5-fold by 48 h starvation of rats. With highly purified pig heart PDH complex it was shown that the protein fraction increased the Vmax. of the PDH kinase reaction 35-fold (fraction from fed rats) or 82-fold (fraction from starved rats); starvation had no effect on the concentration of protein fraction required to give 0.5 Vmax. Evidence is given that the increase in PDH kinase activity effected in extracts of liver mitochondria by starvation is due to increased activity of kinase activator protein, which is tightly bound by rat liver PDH complex and not removed by a single gel filtration. With pig heart PDH complex, increased PDH kinase activity was retained after gel filtration of an admixture with kinase activator protein from starved rats, but was restored to the control value by a second gel filtration; the alterations in PDH kinase activity were associated with obvious changes in protein bands in SDS gels.

1. When pig heart pyruvate dehydrogenase complex was phosphorylated to completion with [gamma-32P]ATP by its intrinsic kinase, three phosphorylation sites were observed. The amino acid sequences around these sites were: sequence 1, Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg; and sequence 2, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser(P)-Tyr-Arg. 2. When phosphorylated to inactivation by repetitive additions of limiting quantities of [gamma-32P]ATP, phosphate was incorporated mainly (more than 90%) into Ser-5 of sequence 2. Phosphorylation of this site thus results in activation of pyruvate dehydrogenase. 3. If Ser-5 is phosphorylated with ATP and the enzyme then incubated with [gamma-32P]ATP, phosphorylation of the remaining sites occurred. Ser-12 of sequence 2 is phosphorylated about twice as rapidly as Ser-6 of sequence 1. 4. Incubation of pyruvate dehydrogenase with excess [gamma-32P]ATP with termination of phosphorylation at about 50% complete inactivation showed that Ser-5 of sequence 2 was phosphorylated most rapidly, but also that Ser-12 of sequence 2 was significantly (15% of total) phosphorylated. Ser-6 sequence 1 contained about 1% total P. 5. These results suggest that addition of limiting amounts of ATP produces primarily phosphorylation of Ser-5 of sequence 2 (inactivating site). This also occurs during incubation with excess ATP before complete inactivation occurs, but a greater occupancy of other sites also occurs during this treatment.