Pyruvate dehydrogenase activity in several tissues of genetically obese hyperglycemic mice

The active form (PDHa) and total activity of pyruvate dehydrogenase (PDH) were measured in homogenates from heart muscle, epididymal fat pads and liver of genetically obese hyperglycemic mice and compared with similar data derived from lean controls or Swiss albino mice. Both PDHa and total PDH activities were similar in heart muscle from all mice with a precipitous decrease in the PDHa upon fasting. Adipose tissue and liver of obese mice had a PDHa level that was almost two-fold higher than either lean control or Swiss albino mice. Fasting for 24 hours decreased the elevated activity of PDHa in adipose tissue and liver in obese mice to a value that was comparable to lean control or Swiss albino mice, fasted similarly. The elevation in both the active form and total activity of pyruvate dehydrogenase in livers from obese mice could explain the increased provision of acetyl-CoA units necessary for the accelerated hepatic lipogenesis observed with this mouse, a model for human obesity and insulin resistance.     

Pyruvate dehydrogenase activities and rates of lipogenesis during the fed-to-starved transition in liver and brown adipose tissue of the rat.

The percentages of pyruvate dehydrogenase complex (PDH) in the active form (PDHa) in two lipogenic tissues (liver and brown adipose tissue) in the fed state were 12.0% and 13.4% respectively. After acute (0.5 h) insulin treatment, PDHa activities had increased by 77% in liver and by 234% in brown fat. Significant decreases in PDHa activities were observed in both tissues by 5 h after the removal of food. The patterns of decline in PDHa activities in the two lipogenic tissues were similar in that the major decreases in activities were observed within the first 7 h of starvation. The significant decreases in PDHa activities observed after starvation for 6 h were accompanied by decreased rates of lipogenesis. Hepatic and brown-fat PDHa activities after acute (30 min) exposure to exogenous insulin were less in 6 h-starved than in fed rats, but the absolute increases in PDHa activities over the 30 min exposure period were similar in fed and 6 h-starved rats. Increases in PDHa activities were paralleled by increases in lipid synthesis in both tissues. Re-activation of PDH in response to insulin treatment or chow re-feeding after 48 h starvation occurred more rapidly in brown adipose tissue than in liver. The results are discussed in relation to the importance of the activity of the PDH complex as a determinant of the total rate of lipogenesis during the fed-to-starved transition and after insulin challenge or re-feeding.     

The activity of the pyruvate dehydrogenase complex in heart and liver from mice during the development of obesity and insulin resistance.

The amount of pyruvate dehydrogenase in the active form (PDHa) was increased 1.7-fold compared with controls in heart muscle of mice 1 week after induction of obesity with a single injection of gold-thioglucose. At 4 weeks post injection, the amount of PDHa was decreased to 32% of control, a value which was observed in later stages of the obesity syndrome. In contrast, liver PDHa was increased and remained at an increased activity during the development of obesity. Despite normal post-prandial serum insulin contents, liver membrane insulin-receptor numbers were decreased 1 week after gold-thioglucose injection, and there was no change in receptor affinity. The decrease in heart PDHa in the obese animals was reversed by a single dose of 2-tetradecylglycidic acid, but this inhibitor of mitochondrial fatty acid oxidation did not affect liver PDHa in these animals. These early and diverse changes in PDHa argue for a multifactorial aetiology in the development of the whole-body insulin resistance seen in older gold-thioglucose-treated obese animals.     

Hepatic pyruvate metabolism during liver regeneration after partial hepatectomy in the rat

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.     

Comparison of tissue pyruvate dehydrogenase activities on re-feeding rats fed ad libitum or meal-fed rats with a chow-diet meal.

Meal-fed rats and rats fed ad libitum had similar rates of hepatic glycogenesis at 60 min after the initiation of re-feeding a chow meal after 22 h starvation, but hepatic PDHa (active form of pyruvate dehydrogenase) activities were 4-fold higher in the meal-fed group. In heart, PDHa activities were 3-fold higher before re-feeding and 2-fold higher after re-feeding in the meal-fed group compared with the group fed ad lib. The blood metabolite profile suggested diminished fat oxidation in starved meal-fed rats and accelerated flux through PDH in meal-fed re-fed rats compared with the group fed ad lib.     

The elucidation of the effect of ammonium chloride on pyruvate distribution and pyruvate dehydrogenase interconversion in isolated rat hepatocytes

The distribution of pyruvate between cell compartments measured in isolated hepatocytes in the presence of lactate was in agreement with delta pH across plasma and mitochondrial membranes. In isolated liver mitochondria NH4Cl decreased the transmembrane potential (delta psi) by about 14 mV, whereas no change of delta pH was observed. In the presence of lactate or alanine NH4Cl increased the mitochondrial pyruvate concentration presumably due to the inhibition of the flux through pyruvate carboxylase. In the presence of lactate or alanine changes in the amount of the active form of pyruvate dehydrogenase (PDHa) were correlated with the mitochondrial pyruvate concentration, NH4Cl increased the amount of PDHa by lowering the mitochondrial ATP/ADP and NADH/NAD+ ratios.     

Effects of administration of tri-iodothyronine on the response of cardiac and renal pyruvate dehydrogenase complex to starvation for 48 h.

Effects of administration of tri-iodothyronine (T3) on activities of cardiac and renal pyruvate dehydrogenase complex (active form, PDHa) were investigated. In fed rats, T3 treatment did not affect cardiac or renal PDHa activity, although blood non-esterified fatty acid and ketone-body concentrations were increased. Starvation (48 h) of both control and T3-treated rats resulted in similar increases in the steady-state concentrations of fatty acids and ketone bodies, but inactivation of cardiac and renal pyruvate dehydrogenase complex activities was diminished by T3 treatment. Inhibition of lipolysis increased renal and cardiac PDHa in control but not in T3-treated 48 h-starved rats, despite decreased fatty acid and ketone-body concentrations in both groups. The results suggest that hyperthyroidism influences the response of cardiac and renal PDHa activities to starvation through changes in the metabolism of lipid fuels in these tissues.     

Sensitivity of inhibition of rat liver mitochondrial outer-membrane carnitine palmitoyltransferase by malonyl-CoA to chemical- and temperature-induced changes in membrane fluidity.

In the fed state, hyperthyroidism increased glucose utilization indices (GUIs) of skeletal muscles containing a lower proportion of oxidative fibres. Glycogen concentrations were unchanged, but active pyruvate dehydrogenase (PDHa) activities were decreased. Hyperthyroidism attenuated the effects of 48 h of starvation to decrease muscle GUI. Glycogen concentrations and PDHa activities after 48 h of starvation were low and similar in euthyroid and hyperthyroid rats. The increase in glucose uptake and phosphorylation relative to oxidation and storage in skeletal muscle induced by hyperthyroidism may contribute to increased glucose re-cycling in the fed hyperthyroid state and to glucose turnover in the starved hyperthyroid state.     

Pyruvate dehydrogenase activities during the fed-to-starved transition and on re-feeding after acute or prolonged starvation.

We investigated the temporal relationship between hepatic glycogen depletion and cardiac and hepatic PDH (pyruvate dehydrogenase complex) activities during the acute phase of starvation. There was a striking correlation between the decline in hepatic glycogen and PDH inactivation during the first 10 h of starvation. Re-feeding after 6 h starvation was associated with complete re-activation of PDH in liver and re-activation to approx. 75% of the fed value in heart, whereas in rats previously starved for 24-48 h re-activation was delayed in liver and diminished in heart. The results are discussed with reference to the fate of dietary carbohydrate after re-feeding.     

Time courses of the responses of pyruvate dehydrogenase activities to short-term starvation in diaphragm and selected skeletal muscles of the rat.

In the fed state, the percentages of the pyruvate dehydrogenase complex (PDH) in the active form (PDHa) in diaphragm and a selection of skeletal muscles (adductor longus, soleus, extensor digitorum longus, tibialis anterior, gastrocnemius) ranged from 8% (soleus) to 38% (gastrocnemius). Major decreases in PDHa activities in all of these muscles were observed after 15 h of starvation, by which time activities were less than 40% of the fed values. In general, the response to starvation was observed more rapidly in muscles of high oxidative capacity. The patterns of changes in skeletal-muscle PDH activities during the fed-to-starved transition are discussed in relation to changes in lipid-fuel supply and oxidation.     

Effect of triamcinolone administration on content of flavins in rabbit liver

Triamcinoline acetonide (10 mg per kg of body weight a day) was administered to rabbit fed on a laboratory chow diet. The content of flavins in liver but not in kidney, muscle and brain started to decrease 24 h after a single dose. The activities of enzymes in the liver were determined: the activities of pyruvate dehydrogenase complex, lipoamide dehydrogenase (NADH : lipoamide oxidoreductase EC 1.6.4.3), NADH dehydrogenase (NADH : (acceptor) oxidoreductace EC 1.6.99.3) and -amino acid oxidase ( -amino acid : oxygen oxidoreductase (deaminating) EC 1.4.3.3) were decreased but those of succinate dehydrogenase (succinate : (acceptor) oxidoreductase EC 1.3.99.1) and xanthine oxidase (xanthine : oxygen oxidoreductase EC 1.2.3.2) remained unchanged. The activities of enzymes in the kidney, however, remained unchanged except the decrease in the activity of pyruvate dehydrogenase complex.     

Effect of triamcinolone administration on content of flavins in rabbit liver.

Triamcinoline acetonide (10 mg per kg of body weight a day) was administered to rabbit fed on a laboratory chow diet. The content of flavins in liver but not in kidney, muscle and brain started to decrease 24 h after a single dose. The activities of enzymes in the liver were determined: the activities of pyruvate dehydrogenase complex, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3), NADH dehydrogenase (NADH : (acceptor) oxidoreductase EC 1.6.99.3) and D-amino acid oxidase (D-amino acid: oxygen oxidoreductase (deaminating) EC 1.4.3.3) were decreased but those of succinate dehydrogenase (succinate : (acceptor) oxidoreductase EC 1.3.99.1) and xanthine oxidase (xanthine : oxygen oxidoreductase EC 1.2.3.2) remained unchanged. The activities of enzymes in the kidney, however, remained unchanged except the decrease in the activity of pyruvate dehydrogenase complex.     

The relationship between changes in lipid fuel availability and tissue fructose 2,6-bisphosphate concentrations and pyruvate dehydrogenase complex activities in the fed state.

An elevated concentration of non-esterified fatty acids in the fed state elicited inhibition of cardiac, but not hepatic, pyruvate dehydrogenase complex (PDH). There was a modest decline in fructose 2,6-bisphosphate (Fru-2,6-P2) concentration in heart, and, to a lesser extent, in liver. Surgical stress decreased PDH activities and Fru-2,6-P2 concentrations in both heart and liver. Only the former response was abolished if postoperative lipolysis was inhibited. Surgery also decreased the [Fru-2,6-P2] in gastrocnemius: this response was abolished if lipolysis was inhibited.     

Properties and Regional Distribution of Pyruvate Dehydrogenase Kinase in Rat Brain

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.     

Effects of fasting and training on pyruvate dehydrogenase activation during exercise

1. The effect of exercise (2 hr treadmill running at 28 m/min) on PDHa (the activity of the active form of pyruvate dehydrogenase) in untrained rats, trained rats (2 hr/d at 25 m/min for 4 wk), and in 24 hr fasted rats was determined. 2. Exercise increased PDHa activity approximately 2 fold in fed-untrained rats. 3. Fasting decreased PDHa activity in sedentary rats to approximately half the activity in fed rats. 4. The increase in PDHa activity during exercise was less in fasted than fed rats. 5. Training did not change the total activity of PDH (phosphorylated plus nonphosphorylated forms) but the percent of PDH in the active form was increased in muscle of trained-rested rats. 6. PDHa activity was unchanged by acute exercise (2.5 hr at 40 m/min) in the trained rats.     

Inactivation of rat adipocyte pyruvate dehydrogenase by palmitate. Protection against this effect by insulin in the presence of glucose.

1. Adipocytes from rat epididymal fat-pads were incubated for 30 min with 5 mM-glucose and concentrations of lactate, pyruvate and amino acids typical of those found in rat plasma. 2. PDHa (active form of pyruvate dehydrogenase) activity was significantly increased after incubation of the cells with insulin (200 micro-i.u./ml), and decreased by incubation with palmitate (0.5--2 mM). 3. In the presence of insulin, palmitate did not decrease PDHa activity. 4. Dichloroacetate (1 mM) increased PDHa activity in the absence of palmitate to the same extent as did insulin. In the presence of dichloroacetate but the absence of insulin, palmitate decreased PDHa activity. In the presence of dichloroacetate and insulin, palmitate again did not decrease PDHa activity. 5. It is concluded that, in the presence of glucose, insulin has a strong protective action against inactivation of adipocyte PDHa by fatty acids.     

Effects of branched chain alpha-ketoacids on the metabolism of isolated rat liver cells. I. Regulation of branched chain alpha-ketoacid metabolism.

alpha-Ketoisocaproate (ketoleucine) is shown to be metabolized to ketone bodies rapidly by isolated rat liver cells. Acetoacetate is the major end product and maximum rates were observed with 2 mM substrate. Studies with 2-tetradecylglycidic acid (an inhibitor of long chain fatty acid oxidation) showed that ketogenesis from alpha-ketoisocaproate and from endogenous fatty acids were additive. With alpha-ketoisocaproate present as soole substrate at 2 mM, leucine production was less than 10% of alpha-ketoisocaproate uptake and only 30% of the acetyl coenzyme A generated was oxidized in the citric acid cycle. Metabolism of alpha-ketoisocaproate was inhibited by fatty acids, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and pyruvate. Oxidation of acetyl-CoA generated from alpha-ketoisocaproate was suppressed by oleate and by pyruvate, but was enhanced by lactate. Metabolism between the different branched chain alpha-ketoacids was mutually competitive. When alpha-ketoisocaproate (2 mM) was added in the presence of high pyruvate concentrations (4.4 mM), flux through pyruvate dehydrogenase was decreased, and the proportion of total pyruvate dehydrogenase in the active form (PDHa) also fell. With lactate as substrate, PDHa was only 25% of total activity and was little affected by addition of alpha-ketoisocaproate. These data suggest that enhanced oxidation of acetyl-CoA from alpha-ketoisocaproate by lactate addition is caused by a low activity of pyruvate dehydrogenase combined with increased flux through the citric acid cycle in response to the energy requirements for gluconeogenesis. However, acetyl-CoA generation from pyruvate is apparently insufficiently inhibited by alpha-ketoisocaproate to cause a diversion of acetyl-CoA formed during alpha-ketoisocaproate metabolism from ketone body formation to oxidation in the citric acid cycle. Measurements of the cell contents of CoASH, acetyl-CoA, acid-soluble acyl-CoA, and acid-insoluble fatty acyl-CoA indicated that when the branched chain alpha-ketoacids were added as sole substrate, their oxidation was limited at a step distal to the branched chain alpha-ketoacid dehydrogenase. Acid-soluble acyl-CoA derivatives were depleted after oleate addition in the presence of alpha-ketoisocaproate, suggesting an inhibition of the branched chain alpha-ketoacid dehydrogenase by the elevation of the mitochondrial NADH/NAD+ ratio observed during fatty acid oxidation. This effect was not observed in the presence of oleate and 2-tetradecylglycidic acid.     

Thioacetamide-Induced Fulminant Hepatic Failure Induces Cerebral Mitochondrial Dysfunction by Altering the Electron Transport Chain Complexes

Fulminant hepatic failure (FHF) is an acute form of hepatic encephalopathy resulting from severe inflammatory or necrotic liver damage without any previously established liver damage. This develops as a complication due to viral infections, and drug abuse. FHF also occurs in acute disorders like Reye’s syndrome. Although the exact mechanisms in the etiology of FHF are not understood, elevated levels of brain ammonia have been consistently reported. Such increased ammonia levels are suggested to alter neurotransmission signals and impair cerebral energy metabolism due to mitochondrial dysfunctions. In the present study we have examined the role of cerebral electron transport chain complexes, including complex I, II, III IV, and pyruvate dehydrogenase in the non-synaptic mitochondria isolated from the cortex of the thioacetamide-induced FHF rats. Further, we have examined if the structure of mitochondria is altered. The results of the current study demonstrated a decrease in the activity of the complex I by 31 and 48% at 18 and 24 h respectively after the thioacetamide injection. Similarly, the activity of electron transport chain complex III was inhibited by 35 and 52% respectively, at 18 and 24 h, respectively. The complex II and complex IV, on the other hand, revealed unaltered activity. Further the activity of pyruvate dehydrogenase at 18 and 24 h after the induction of FHF was inhibited by 29 and 43%, respectively. Our results also suggest mitochondrial swelling in FHF induced rats. The inhibition of the respiratory complexes III and I and pyruvate dehydrogenase might lead to the increased production of free radical resulting in oxidative stress and cerebral energy disturbances thereby leading to mitochondrial swelling and further contributing to the pathogenesis of FHF.     

Activities of enzymes concerned with pyruvate and oxaloacetate metabolism in the heart and liver of developing sheep.

1. In order to assess whether the potential ability of heart ventricular muscle and liver to metabolise substrates such as alanine, aspartate and lactate varies as the sheep matures and its nutrition changes, the activities of the following enzymes were determined in tissues of lambs obtained at varying intervals between 50 days after conception to 16 weeks after birth and in livers from adult pregnant ewes: lactate dehydrogenase (EC 1.1.1.27), alanine aminotransferase (EC 2.6.1.2), pyruvate kinase (EC 2.7.1.40), pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (GTP)(EC 4.1.1.32), malate dehydrogenase (EC 1.1.1.37), aspartate aminotransferase (EC 2.6.1.1) and citrate (si)-synthase (EC 4.1.3.7). 2. In the heart a most marked increase in alanine aminotransferase activity was found throughout development. During this period the activities of citrate (si)-synthase, lactate dehydrogenase and pyruvate carboxylase also increased. There were no substantial changes in the activities of aspartate aminotransferase, malate dehydrogenase or pyruvate kinase. Pyruvate kinase activities were five times greater in the heart compared with those found in the liver. No significant activity of phosphoenolpyruvate carboxykinase (GTP) was detected in heart muscle. 3. In the liver the activities of both alanine aminotransferase and aspartate aminotransferase increased immediately following birth although the activity of alanine aminotransferase was lower in livers of pregnant ewes than in any of the lambs. As with alanine aminotransferase the highest activities of lactate dehydrogenase were found during the period of postnatal growth. No marked changes were observed in malate dehydrogenase or citrate (si)-synthase activities during development. A small decline in pyruvate kinase activity occurred whilst the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase (GTP) tended to rise during development.     

Effect of insulin on pyruvate dehydrogenase in a mixture of plasma membranes and mitochondria from normal and alloxan treated rat brains

In a mixture of plasma membranes/mitochondria from normal rat brain, pyruvate dehydrogenase (PDH) is present in the active (PDHa) and the inactive (PDHi) form; the latter is converted into the former by preincubation with Ca2+ and Mg2+ and represents about 40% of total PDH (PDHt = PDHa + PDHi). Incubation with increasing insulin concentrations activates PDHa and PDHt, the maximum being reached at 25 microU/ml insulin; inhibition appears with further insulin increase. In a mixture of plasma membranes and mitochondria from alloxan rat brain PDHa activity markedly decreases; no activation is achieved by preincubation with Ca2+ and Mg2+. However an activating effect of Ca2+ and Mg2+ appears when the mixture is added and incubated with increasing insulin concentrations. PDHa and PDHt activity reaches a maximum of stimulation at 25 microU/ml insulin; the activation is reduced at higher concentrations of insulin though no inhibition appears. ATP partially inhibits PDHa in normal and alloxan rat brain plasma membrane/mitochondria mixtures; this effect is completely cancelled by 25 microU/ml insulin.