PDK4 mRNA Expression Research Articles

Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells

The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1-4) and pyruvate dehydrogenase phosphatase (PDP1-2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehydrogenase complex activity in response to various different metabolic conditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phosphatidylinositol 3-kinase-dependent pathway, which may involve glycogen synthase kinase-3 but does not utilize the mammalian target of rapamycin or mitogen-activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators-activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARalpha and PPARdelta agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARgamma activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle.

Energy deficit without reducing dietary carbohydrate alters resting carbohydrate oxidation and fatty acid availability

Reduced carbohydrate (CHO) availability after exercise has a potent influence on the regulation of substrate metabolism, but little is known about the impact of fat availability and/or energy deficit on fuel metabolism when dietary CHO availability is not reduced. The purpose of this study was to determine the influence of a postexercise energy deficit, independent of CHO availability, on plasma substrate concentrations and substrate oxidation. Seven moderately trained men (peak oxygen uptake: 56 +/- 2 ml.kg(-1).min(-1)) performed exhaustive cycling exercise on two separate occasions. The two trials differed only by the meals ingested after exercise: 1) a high-fat diet designed to maintain energy balance or 2) a low-fat diet designed to elicit energy deficit. The CHO and protein contents of the diets were identical. The next morning, we measured plasma substrate and insulin concentrations and CHO oxidation, and we obtained muscle biopsies from the vastus lateralis for measurement of pyruvate dehydrogenase kinase (PDK)-2 and PDK-4 mRNA expression by using RT-PCR. Despite identical blood glucose (5.0 +/- 0.1 and 4.9 +/- 0.1 mM) and insulin (7.9 +/- 1.1 and 8.4 +/- 0.9 microU/ml) concentrations, plasma fatty acid and glycerol concentrations were elevated three- to fourfold during energy deficit compared with energy balance and CHO oxidation was 40% lower (P < 0.01) the morning after energy deficit compared with energy balance (328 +/- 69 and 565 +/- 89 micromol/min). The lower CHO oxidation was accompanied by a 7.3 +/- 2.5-fold increase in PDK-4 mRNA expression after energy deficit (P < 0.05), whereas PDK-2 mRNA was similar between the trials. In conclusion, energy deficit increases fatty acid availability, increases PDK-4 mRNA expression, and suppresses CHO oxidation even when dietary CHO content is not reduced.

Energy Deficit Alters Carbohydrate Oxidation and PDK4 mRNA Independent of Carbohydrate Intake

1220 Reduced carbohydrate (CHO) availability after exercise has a potent influence on the regulation of substrate metabolism, but little is known about the impact of caloric deficit on fuel metabolism when dietary CHO availability is not reduced. PURPOSE: To determine the influence of post-exercise energy dfiit, independent of CHO availability, on CHO oxidation and expression of pyruvate dehydrogenase kinase (PDK) mRNA. METHODS: Five moderately-trained men (VO2max: 56 ± 2 ml/kg/min) performed exhaustive cycling exercise on 2 separate occasions. The two trials differed only by the meals ingested after exercise: 1) a high-fat diet designed to maintain energy balance (EB) or 2) a low-fat diet designed to elicit energy deficit (ED). The CHO content of the diets was identical. The next morning we measured CHO oxidation, blood glucose and insulin concentrations, and muscle biopsies were obtained from the vastus lateralis for measurement of muscle glycogen concentration and PDK2 and PDK4 mRNA expression. RESULTS: Despite a similar resynthesis of muscle glycogen in ED and EB (340 ± 39 and 398 ± 40 mmol/kg dry wt) and identical blood glucose (4.7 ± 0.1 and 4.8 ± 0.1 mM) and insulin (6.4 ± 1.1 and 6.5 ± 1.4 ìU/ml) concentrations, CHO oxidation was 40% lower (p<0.01) the morning after ED compared with EB (352 ± 86 and 582 ± 117 μmol/min). The lower CHO oxidation was accompanied by a 2.2 ± 0.3 fold increase in PDK4 mRNA expression after ED relative to EB (p<0.05), while PDK2 mRNA was similar between the trials. CONCLUSION: Energy deficit suppresses CHO oxidation even when dietary CHO availability is not reduced. This reduction in CHO oxidation during energy deficit may be acting through an increase in PDK4.

Pyruvate dehydrogenase activation and kinase expression in human skeletal muscle during fasting.

Fasting forces adaptive changes in whole body and skeletal muscle metabolism that increase fat oxidation and decrease the oxidation of carbohydrate. We tested the hypothesis that 40 h of fasting would decrease pyruvate dehydrogenase (PDH) activity and increase PDH kinase (PDK) isoform mRNA expression in human skeletal muscle. The putative transcriptional activators of PDK isozymes, peroxisome proliferator-activated receptor-alpha (PPAR-alpha) protein, and forkhead homolog in rhabdomyosarcoma (FKHR) mRNA were also measured. Eleven healthy adults fasted after a standard meal (25% fat, 60% carbohydrate, 15% protein) with blood and skeletal muscle samples taken at 3, 15, and 40 h postprandial. Fasting increased plasma free fatty acid, glycerol, and beta-hydroxybutyrate concentrations and decreased glucose and insulin concentrations. PDH activity decreased from 0.88 +/- 0.11 mmol acetyl-CoA. min(-1). kg wet muscle wt(-1) at 3 h to 0.62 +/- 0.10 (P = not significant) and 0.39 +/- 0.06 (P < 0.05) mmol. min(-1). kg wet mass(-1) after 15 and 40 h of fasting. Although all four PDK isoforms were expressed in human skeletal muscle, PDK-2 and -4 mRNA were the most abundant. PDK-1 and -3 mRNA abundance was approximately 1 and 15% of the PDK-2 and -4 levels, respectively. The 40-h fast had no effect on PDK-1, -2, and -3 mRNA expression. PDK-4 mRNA was significantly increased approximately 3-fold after 15 h and approximately 14-fold after 40 h of fasting. Skeletal muscle PPAR-alpha protein and FKHR mRNA abundance were unaffected by the fast. The results suggest that decreased PDH activation after 40 h of fasting may have been a function of the large increase in PDK-4 mRNA expression and possible subsequent increase in PDK protein and activity. The changes in PDK-4 expression and PDH activity did not coincide with increases in the transcriptional activators PPAR-alpha and FKHR.

Reduced PDK4 expression associates with increased insulin sensitivity in postobese patients.

The aim of this study was to verify whether changes in PDK4 mRNA expression in skeletal muscle in formerly obese subjects who underwent malabsorptive bariatric surgery [bilio-pancreatic diversion (BPD)] might be related to insulin sensitivity improvement, and if these possible modifications might correlate with a reduction of the intramyocytic lipid level. Six obese women (body mass index 46.6 +/- 8.2 kg/m(2)) were enrolled in the study. Body composition, euglycemic-hyperinsulinemic clamp and muscle biopsies for skeletal muscle lipid analysis, and semiquantitative reverse transcriptase-polymerase chain reaction were performed before and 3 years after BPD. The average weight loss observed after surgery was approximately 42%. Increased glucose uptake was accompanied by a significant decrease of PDK4 mRNA (R(2) = 0.71, p < 0.001). The amounts of intramyocytic triglycerides correlate directly with PDK4 mRNA (R(2) = 0.87, p = 0.005) and inversely with glucose uptake values (R(2) = 0.75, p < 0.001). Our results support the concept that a reduced tissue availability of fatty acids consequent to a massive lipid malabsorption influences glucose metabolism acting through the regulation of PDH complex. In fact, as shown in animals, a higher level of FFA availability is likely to induce overexpression of PDK4 also in humans.

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.