Increase In Pyruvate Dehydrogenase Kinase Activity Research Articles

Carbohydrate refeeding after a high-fat diet rapidly reverses the adaptive increase in human skeletal muscle PDH kinase activity

Pyruvate dehydrogenase (PDH) regulates oxidative carbohydrate disposal in skeletal muscle and is downregulated by reversible phosphorylation catalyzed by PDH kinase (PDK). Previous work has demonstrated increased PDK activity and PDK4 expression in human skeletal muscle following a high-fat low-carbohydrate (HF) diet, which leads to decreased PDH in the active form (PDHa activity) and carbohydrate oxidation. The purpose of this study was to examine the time course of changes in PDK and PDHa activities with refeeding of carbohydrates after an HF diet in human skeletal muscle. Healthy male volunteers (n = 8) consumed a standardized 3-day Pre-diet with the same energy content as their habitual diet, followed by a eucaloric 6-day HF diet (Pre-diet: 50:30:20%; HF diet: 5:75:20%; carbohydrate/fat/protein). Muscle biopsies were taken before and after the HF diet and at 45 min and 3 h after carbohydrate refeeding with a single high-glycemic index carbohydrate meal (88:5:7% carbohydrate/fat/protein) representing approximately one third of the individual subject's habitual energy intake. PDK activity increased from 0.08 +/- 0.01 Pre- to 0.25 +/- 0.02 min (P < 0.001) Post-HF diet, and decreased with carbohydrate refeeding to 0.17 +/- 0.05 (P = 0.014) and 0.11 +/- 0.01 min (P = 0.006) at 45 min and 3 h, respectively. PDHa decreased from 0.89 +/- 0.20 to 0.32 +/- 0.05 (P = 0.007) mmol x min(-1) x kg wet wt(-1) following the HF diet, and was increased transiently with refeeding at 45 min, but returned to lower values by 3 h (P = 0.025 compared with Pre). The potential mechanism(s) for this attenuation of PDHa activity remains unclear. These data demonstrate that in human skeletal muscle, the adaptive increase in PDK activity following an HF diet is rapidly reversed to Pre-diet activity levels within 45 min to 3 h, and this is accompanied by a short-term increase in PDHa activity.

Elevated n-3 fatty acids in a high-fat diet attenuate the increase in PDH kinase activity but not PDH activity in human skeletal muscle

Elevated n-3 fatty acids in a high-fat diet attenuate the increase in PDH kinase activity but not PDH activity in human skeletal muscle

Elevated n-3 fatty acids in a high-fat diet attenuate the increase in PDH kinase activity but not PDH activity in human skeletal muscle

We tested the hypothesis that a high-fat diet (75% fat; 5% carbohydrates; 20% protein), for which 15% of the fat content was substituted with n-3 fatty acids, would not exhibit the diet-induced increase in pyruvate dehydrogenase kinase (PDK) activity, which is normally observed in human skeletal muscle. The fat content was the same in both the regular high-fat diet (HF) and in the n-3-substituted diet (N3). PDK activity increased after both high-fat diets, but the increase was attenuated after the N3 diet (0.051 +/- 0.007 and 0.218 +/- 0.047 min(-1) for pre- and post-HF, respectively; vs. 0.073 +/- 0.016 and 0.133 +/- 0.032 min(-1) for pre- and post-N3, respectively). However, the active form of pyruvate dehydrogenase (PDHa) activity decreased to a similar extent in both conditions (0.93 +/- 0.17 and 0.43 +/- 0.09 mmol/kg wet wt pre- and post-HF; vs. 0.87 +/- 0.19 and 0.39 +/- 0.05 mmol/kg wet wt pre- and post-N3, respectively). This suggested that the difference in PDK activity did not affect PDHa activation in the basal state, and it was regulated by intramitochondrial effectors, primarily muscle pyruvate concentration. Muscle glycogen content was consistent throughout the study, before and after both diet conditions, whereas muscle glucose-6-phosphate, glycerol-3-phosphate, lactate, and pyruvate were decreased after the high-fat diets. Plasma triglycerides decreased after both high-fat diets but decreased to a greater extent after the N3, whereas plasma free fatty acids increased after both diets, but to a lesser extent after the N3. In summary, PDK activity is decreased after a high-fat diet that is rich in n-3 fatty acids, although PDHa activity was unaltered. In addition, our data demonstrated that the hypolipidemic effect of n-3 fatty acids occurs earlier (3 days) than previously reported and is evident even when the diet has 75% of its total energy derived from fat.

HUMAN SKELETAL MUSCLE PDH KINASE ACTIVITY INCREASES DURING PROLONGED EXERCISE

During prolonged sub-maximal exercise, there is a shift in skeletal muscle substrate utilization from carbohydrate to fat oxidation. This decrease in carbohydrate oxidation is due at least in part to decreased activity of the active form of pyruvate dehydrogenase (PDHa). However, the exact mechanism of the observed decrease in PDHa activity is unknown. PDHa is down-regulated by an intrinsic PDH kinase (PDK) which can either be up-regulated acutely by allosteric effectors, or in an chronic manner which is independent of changes in mitochondrial effectors. PURPOSE To test the hypothesis that the observed decrease in PDHa activity during a single 4 h exercise bout is due to a chronic increase in PDK activity. METHODS Healthy male volunteers (n = 7; age, 24 ± 4 yr; mean maximal oxygen uptake or VO2max, 53.1 ± 1.6 ml/kg/min) were asked to cycle at 55% of their VO2max for 4 hours. At 0 min, 10 min, and 4 h two biopsies were taken. One biopsy was frozen immediately in liquid nitrogen for analysis of PDHa activity. A second biopsy was extracted fresh for intact mitochondria and subsequent PDK activity (calculated as the first-order rate constant of PDHa inactivation, min−1). Respiratory exchange ratio (RER) was used to calculate energy contributions from carbohydrate and fat oxidation. RESULTS RER decreased during exercise from 0.94 ± 0.01 to 0.79 ± 0.01, carbohydrate oxidation decreased from 35 ± 3 to 12.0± 1.5 kJ/min, and fat oxidation increased from 8.3 ± 0.8 to 30.2 ± 1.2 g/min. PDHa activity increased from 0 to 10 min, but was decreased at 4 h (0.79 ± 0.08, 2.86 ± 0.26, and 2.23 ± 0.24 respectively). PDK activity was increased ∼2 fold by 4 h (0 min, 0.077 ± 0.016; 10 min, 0.119 ± 0.016 and 4 h, 0.173 ± 0.019 min−1). CONCLUSION Decreased PDHa activity while cycling at 55% VO2max for 4 h is associated with a chronic increase in PDK activity. Supported by NSERC and CIHR, Canada

TIME COURSE OF A 40 HOUR FAST ON PYRUVATE DEHYDROGENASE ACTIVATION AND KINASE EXPRESSION IN HUMAN SKELETAL MUSCLE

Pyruvate dehydrogenase (PDH) activity controls the entry of carbohydrate into the tricarboxylic cycle and is regulated by PDH kinase (PDK) which phosphorylates and inactivates the enzyme and PDH phosphatase which removes phosphate and returns the enzyme to its active from (PDHa). These enzymes are acutely controlled by allosteric regulators and over the longer term by changes in the stable activity of PDK. This study was designed to examine the effects of fasting on the gene expression of the four PDK isozymes (1–4) and resting PDHa activity. Seven healthy adults fasted for 40 hr after a standard meal (25% fat, 60% carbohydrate, 15% protein) with blood and muscles samples taken at 3, 15, and 40 hr post meal. Fasting increased plasma free fatty acid, glycerol and β-hydroxybutyrate and decreased glucose and insulin concentrations. PDHa activity decreased from 0.91 ± 0.14 mmol acetyl-CoA/min/kg wet muscle at 3 hr to 0.55 ± 0.11 and 0.36 ± 0.06 mmol/min/kg wm following 15 and 40 hr of fasting. PDK2 and 4 mRNA were the most abundant isoforms in human skeletal muscle. PDK2 was unaffected by the 40 hr fast. PDK4 was also unaffected after 15 hr, but increased 13-fold after 40 hr of fasting. The abundance of PDK1 and 3 mRNA was ∼200 and 300 fold lower than PDK2, respectively, and not affected by fasting. The results suggest that the decreased PDHa activity following 15 hr of fasting was due to allosteric regulation and independent of gene expression. The decreasing insulin concentration may have reduced the activation of PDH phosphatase and caused the lower PDHa activity. Following 40 hr of fasting, the large increase in PDK4 mRNA expression suggested that a stable increase in PDK activity had occurred, which also contributed to the low PDHa activity in resting human skeletal muscle.

Human skeletal muscle pyruvate dehydrogenase kinase activity increases after a low-carbohydrate diet.

To characterize human skeletal muscle enzymatic adaptation to a low-carbohydrate, high-fat, and high-protein diet (LCD), subjects consumed a eucaloric diet consisting of 5% of the total energy intake from carbohydrate, 63% from fat, and 33% from protein for 6 days compared with their normal diet (52% carbohydrate, 33% fat, and 14% protein). Biopsies were taken from the vastus lateralis before and after 3 and 6 days on a LCD. Intact mitochondria were extracted from fresh muscle and analyzed for pyruvate dehydrogenase (PDH) kinase, total PDH, and carnitine palmitoyltransferase I activities and mitochondrial ATP production rate (using carbohydrate and fat substrates). beta-Hydroxyacyl CoA dehydrogenase, active PDH (PDHa), and citrate synthase activities were also measured on whole muscle homogenates. PDH kinase (PDHK) was calculated as the absolute value of the apparent first-order rate constant of the inactivation of PDH in the presence of 0.3 mM Mg2+-ATP. PDHK increased dramatically from 0.10 +/- 0.02 min-1 to 0.35 +/- 0.09 min-1 at 3 days and 0.49 +/- 0. 06 min-1 after 6 days. Resting PDHa activity decreased from 0.63 +/- 0.17 to 0.17 +/- 0.04 mmol. min-1. kg-1 after 6 days on the diet, whereas total PDH activity did not change. Activities for all other enzymes were unaltered by the LCD. In summary, severe deficiency of dietary carbohydrate combined with a twofold increase in dietary fat and protein caused a rapid three- to fivefold increase in PDHK activity in human skeletal muscle. The increased PDHK activity downregulated the amount of PDH in its active form at rest and decreased carbohydrate metabolism. However, an increase in the activities of enzymes involved in fatty acid oxidation did not occur.

TNF Binding Protein Prevents Hyperlactatemia and Inactivation of PDH Complex in Skeletal Muscle during Sepsis

Background.Hyperlactatemia is a metabolic complication of hypermetabolic, hyperdynamic sepsis. An important mechanism responsible for elevating plasma lactate concentrations in sepsis is altered regulation of the pyruvate dehydrogenase complex (PDH) in skeletal muscle. We investigated the ability of a specific tumor necrosis factor binding protein, TNFbp, to modulate lactate concentrations and skeletal muscle PDH activity in a rodent model of chronic abdominal sepsis.Materials and methods.We examined the regulation of lactate metabolism in four groups of animals: Control, Control + TNFbp, Septic, and Septic + TNFbp. Chronic (5 days) sepsis was induced by the creation of a stable intraabdominal abscess using a sterile fecal-agar pellet inoculated withE. coliplusB. Fragilisas the foreign body nidus. TNFbp (1 mg/kg/day) was injected subcutaneously daily.Results.Sepsis increased plasma and skeletal muscle lactate concentrations 2-fold compared with control. In septic rats treated with TNFbp, plasma and skeletal muscle lactate concentrations were significantly decreased compared with untreated septic rats. In skeletal muscle, sepsis resulted in a 70% decrease in the proportion of the PDH in the active form compared with controls. The sepsis-induced inhibition in the PDH complex activity was prevented by TNFbp. PDH kinase was enhanced 1.8-fold in sepsis, and the increase in PDH kinase activity was prevented by treatment with TNFbp. TNFbp treatment did not have any effects on plasma lactate or the proportion of active skeletal muscle PDH activity in control animals.Conclusions.TNFbp prevents the sepsis-induced hyperlactatemia and derangements in skeletal muscle lactate concentrations and PDH activity. These observations suggest that TNF is an important mediator responsible for lactate dyshomeostasis during sepsis.

Different mechanisms underlie the long-term regulation of pyruvate dehydrogenase kinase (PDHK) by tri-iodothyronine in heart and liver

Antibodies to purified recombinant PDHKII were used for ELISAs of PDHKII in mitochondrial extracts. In liver, hyperthyroidism elicited a 2.3-fold increase in PDHK activity ( P<0.01) which was accompanied by a significant 1.5-fold ( P<0.001) increase in the amount of mitochondrial immunoreactive PDHKII. In contrast, despite a stable 2.0-fold increase in cardiac PDHK activity ( P<0.001), the amount of mitochondrial immunoreactive PDHKII in heart was unaffected by hyperthyroidism. The mechanisms for long-term regulation of PDHK activity by thyroid hormones therefore differ fundamentally between heart and liver.

Increased hepatic pyruvate dehydrogenase kinase activity in fed hyperthyroid rats: studies in vivo and with cultured hepatocytes

Experimental hyperthyroidism induced by the administration of tri-iodothyronine (T3; 100 μ g 100 g body wt; 3 days) increased plasma non-esterified fatty acids in the fed state in the rat. At the same time, hepatic PDH kinase responded with a persistent (1.6-fold) increase in activity. The exposure of hepatocytes from fed euthyroid rats to T3 (100 nM) in culture for 21 h increased PDH kinase activity to an extent comparable to that observed in vivo in response to hyperthyroidism. The in vitro increase in PDH kinase activity was suppressed by insulin (100 μU/ml) and by inhibition of mitochondrial fatty acid oxidation. The results demonstrate a direct hepatic action of T3 to increase PDH kinase activity, which is mediated by intramitochondrial fatty acyl-CoA or a product of β-oxidation, and facilitated by hepatic insulin resistance.

The Long‐Term Regulation of Skeletal Muscle Pyruvate Dehydrogenase Kinase by Dietary Lipid is Dependent on Fatty Acid Composition

The provision of a diet high in saturated and monounsaturated fat for 28 days evoked a significant (1.9‐fold) increase in pyruvate‐dehydrogenase kinase activity measured in isolated mitochondria from representative slow‐twitch (oxidative) skeletal muscles (pooled soleus and adductor longus muscles) from adult rats. The increase observed in response to 28 days of high‐fat feeding in slow‐twitch skeletal muscle mitochondria was similar in magnitude to that observed in heart mitochondria. Pyruvate‐dehydrogenase kinase activity was not increased in response to the provision of the high‐fat diet in mitochondria prepared from a representative fast‐twitch muscle (tibialis anterior), while the increases evoked by 28 days of high‐fat feeding in cardiac and slow‐twitch skeletal muscle were prevented by the replacement of 7% of the dietary fatty acids with long‐chain ω–3 fatty acids from marine oil. Cardiac myocytes prepared from the high‐fat‐fed rats showed impaired responses of this enzyme to n ‐octanoate (1 mM) and N6,2‐O ‐dibutyryl‐adenosine 3′,5′‐monophosphate (50 μM) individually in cultured cardiac myocytes and of glucose uptake to insulin at low concentrations in freshly prepared cardiac myocytes, compared with control rats maintained on standard low‐fat/high‐carbohydrate diet. These impairments in responses to agonists were substantially improved by the inclusion of long‐chain ω–3 fatty acids in the high‐fat diet. The results indicate that pyruvate‐dehydrogenase kinase activity in oxidative skeletal muscle is a target for longer‐term regulation by high‐fat feeding and that the fatty acid composition of the diet, rather than the fat content, is a key influence.

The Long-Term Regulation of Skeletal Muscle Pyruvate Dehydrogenase Kinase by Dietary Lipid is Dependent on Fatty Acid Composition

The provision of a diet high in saturated and monounsaturated fat for 28 days evoked a significant (1.9-fold) increase in pyruvate-dehydrogenase kinase activity measured in isolated mitochondria from representative slow-twitch (oxidative) skeletal muscles (pooled soleus and adductor longus muscles) from adult rats. The increase observed in response to 28 days of high-fat feeding in slow-twitch skeletal muscle mitochondria was similar in magnitude to that observed in heart mitochondria. Pyruvate-dehydrogenase kinase activity was not increased in response to the provision of the high-fat diet in mitochondria prepared from a representative fast-twitch muscle (tibialis anterior), while the increases evoked by 28 days of high-fat feeding in cardiac and slow-twitch skeletal muscle were prevented by the replacement of 7% of the dietary fatty acids with long-chain omega-3 fatty acids from marine oil. Cardiac myocytes prepared from the high-fat-fed rats showed impaired responses of this enzyme to n-octanoate (1 mM) and N6,2-O-dibutyryladenosine 3',5'-monophosphate (50 microM) individually in cultured cardiac myocytes and of glucose uptake to insulin at low concentrations in freshly prepared cardiac myocytes, compared with control rats maintained on standard low-fat/high-carbohydrate diet. These impairments in responses to agonists were substantially improved by the inclusion of long-chain omega-3 fatty acids in the high-fat diet. The results indicate that pyruvate-dehydrogenase kinase activity in oxidative skeletal muscle is a target for longer-term regulation by high-fat feeding and that the fatty acid composition of the diet, rather than the fat content, is a key influence.

Kinase activator protein mediates longer-term effects of starvation on activity of pyruvate dehydrogenase kinase in rat liver mitochondria.

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.