Pyruvate Dehydrogenase Kinase-4 Protein Research Articles

LCZ696 (sacubitril/valsartan) inhibits pulmonary hypertension induced right ventricular remodeling by targeting pyruvate dehydrogenase kinase 4

BackgroundRight ventricular (RV) function is a major prognostic factor in patients with cardiopulmonary disease. Effective medical therapies are available for left heart failure, but they are usually less effective or even ineffective in right heart failure. Here, we tested the hypothesis that LCZ696 (sacubitril/valsartan) can attenuate pressure overload-induced RV remodeling by inhibiting pyruvate dehydrogenase kinase 4 (PDK4). MethodsAdult male C57 mice were subjected to transverse aortic constriction (TAC), pulmonary artery constriction (PAC), or sham surgery. Bioinformatics analysis was used to screen for common differentially expressed genes (DEGs) between TAC and PAC. Chemical compounds targeting DEGs were predicted by molecular docking analysis. Effects of LCZ696 on PAC-induced RV remodeling and the associated PDK4-related mechanisms were investigated. ResultsWe found 60 common DEGs between PAC and TAC, and Pdk4 was one of the downregulated DEGs. From 47 chemical compounds with potential cardiovascular activity and PDK4 protein binding ability, we selected LCZ696 to treat PAC-induced RV remodeling because of its high docking score for binding PDK4. Compared with vehicle-treated PAC mice, LCZ696-treated mice had significantly smaller RV wall thickness and RV diameters, less myocardial fibrosis, lower expression of PDK4 protein, and less phosphorylation of glycogen synthase kinase-3β (p-GSK3β). In PAC mice, overexpression of Pdk4 blocked the inhibitory effect of LCZ696 on RV remodeling, whereas conditional knockout of Pdk4 attenuated PAC-induced RV remodeling. ConclusionsPdk4 is a common therapeutic target for pressure overload-induced left ventricular and RV remodeling, and LCZ696 attenuates RV remodeling by downregulating Pdk4 and inhibiting PDK4/p-GSK3β signal.

Remifentanil reduces multiple organ and energy metabolism disturbances in a rat sepsis model.

The aim of this study was to observe the effects of remifentanil on organ damage and energy metabolism in lipopolysaccharide (LPS)-induced septic rats. A total of 45 clean-grade male Wistar rats (weight 270-320 g) were randomly divided into three groups: a control group, an LPS group, and an LPS with remifentanil treatment (LPS+REM) group. After 6 hours of modeling, the levels of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in lung and kidney tissues of rats in each group were detected by ELISA. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in lung and kidney tissues were determined, and the content of lactic acid, pyruvate and epinephrine in heart and kidney tissues were detected. Reverse transcription polymerase chain reaction and the Western blot test were used to detect the expression of pyruvate dehydrogenase kinase 4 (PDK4) in the myocardial tissue. We found that remifentanil treatment inhibited the levels of IL-6, TNF-α, and MDA in the lung and kidneys 6 h after the administration of LPS and increased the level of SOD activity. Treatment with remifentanil reduced the expression of lactic acid, pyruvate, and epinephrine in the heart and kidney tissues and attenuated the expression of PDK4 messenger RNA and PDK4 protein in the myocardial tissue. We concluded that remifentanil might inhibit the release of tissue inflammatory factors, regulate the body's energy metabolism, and ultimately protect the sepsis tissue damage caused by LPS.

MuRF1 deficiency prevents age-related fat weight gain, possibly through accumulation of PDK4 in skeletal muscle mitochondria in older mice.

Recent studies show that muscle mass and metabolic function are interlinked. Muscle RING finger 1 (MuRF1) is a critical muscle-specific ubiquitin ligase associated with muscle atrophy. Yet, the molecular target of MuRF1 in atrophy and aging remains unclear. We examined the role of MuRF1 in aging, using MuRF1-deficient (MuRF1-/- ) mice in vivo, and MuRF1-overexpressing cell in vitro. MuRF1 deficiency partially prevents age-induced skeletal muscle loss in mice. Interestingly, body weight and fat mass of more than 7-month-old MuRF1-/- mice were lower than in MuRF1+/+ mice. Serum and muscle metabolic parameters and results of indirect calorimetry suggest significantly higher energy expenditure and enhanced lipid metabolism in 3-month-old MuRF1-/- mice than in MuRF1+/+ mice, resulting in suppressed adipose tissue gain during aging. Pyruvate dehydrogenase kinase 4 (PDK4) is crucial for a switch from glucose to lipid metabolism, and the interaction between MuRF1 and PDK4 was examined. PDK4 protein levels were elevated in mitochondria from the skeletal muscle in MuRF1-/- mice. In vitro, MuRF1 interacted with PDK4 but did not induce degradation through ubiquitination. Instead, SUMO posttranscriptional modification (SUMOylation) of PDK4 was detected in MuRF1-overexpressing cells, in contrast to cells without the RING domain of MuRF1. MuRF1 deficiency enhances lipid metabolism possibly by upregulating PDK4 localization into mitochondrial through prevention of SUMOylation. Inhibition of MuRF1-mediated PDK4 SUMOylation is a potential therapeutic target for age-related dysfunction of lipid metabolism and muscle atrophy.

Loss of PDK4 expression promotes proliferation, tumorigenicity, motility and invasion of hepatocellular carcinoma cells.

Although the roles and underlying mechanisms of other PDK family members (i.e., PDK1, PDK2 and PDK3) in tumor progression have been extensively investigated and are well understood, the functions and underlying molecular mechanisms of pyruvate dehydrogenase kinase 4 (PDK4) in the tumorigenesis and progression of various cancers [including hepatocellular carcinoma (HCC)] remain largely unknown. In this study, we examined the expression profile of PDK4 in HCC clinical tissue specimens and the roles of PDK4 in the proliferation, tumorigenicity, motility and invasion of HCC cells. The immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) results revealed that PDK4 was significantly downregulated in the cohort of HCC clinical specimens. Additionally, PDK4 protein was found in both the nucleus and cytoplasm of HCC cells based on an immunofluorescence (ICC) assay, and PDK4 protein was also found in the nucleus and cytoplasm of cancer cells contained in HCC clinical specimens based on IHC. The CCK-8 assay and cell colony formation assay demonstrated that stable depletion of endogenous PDK4 by lentivirus-mediated RNA interference (RNAi) markedly promoted the proliferation of HCC cell lines (i.e., BEL-7402 and BEL-7404 cells) in vitro, while PDK4 silencing significantly enhanced the tumorigenic ability of BEL-7404 cells in vivo. In addition to enhance proliferation and tumorigenesis induced by PDK4 silencing, additional studies demonstrated that knockdown of PDK4 led to increase migration and invasion of BEL-7402 and BEL-7404 cells in vitro. Taken together, these findings suggest that the loss of PDK4 expression contributes to HCC malignant progression.

P731High-density lipoproteins rescue diabetes-impaired angiogenesis by restoring cellular metabolic reprogramming responses to hypoxia

Abstract Background We have previously identified that high-density lipoproteins (HDL) rescue hypoxia-induced angiogenesis in diabetes, however the underlying mechanisms remain unknown. A central component of hypoxia-induced angiogenesis is the reprogramming of endothelial cell (EC) metabolism to improve tolerance of hypoxia at the site of injury/repair, including following myocardial infarction. Pyruvate dehydrogenase kinase 4 (PDK4) suppresses mitochondrial respiration in hypoxia to decrease oxygen consumption and preserve cell survival. Diabetes impairs this adaptation to hypoxia, causing cellular dysfunction and may underlie delayed angiogenic responses to ischaemia in hyperglycaemia. Purpose To determine the role of metabolic reprogramming in angiogenesis and diabetes and the effect of reconstituted HDL (rHDL). Methods and results Using an in vitro functional angiogenesis assay, siRNA-mediated knockdown of PDK4 impaired human coronary artery endothelial cell (HCAEC) tubulogenesis in hypoxia by 82% versus siScrambled controls (P<0.0001). HCAECs were treated with rHDL (20μM, apolipoproteinA-I + phosphatidylcholine) or PBS (vehicle) and exposed to glucose (5 or 25 mM, 72 h), then normoxia or hypoxia (1.2% O2, 6 h). PDK4 expression was increased by 65% in hypoxia versus the normoxia control (P<0.05). By contrast, in high glucose PDK4 expression failed to increase in response to hypoxia. Incubation with rHDL rescued this impairment and elevated PDK4 expression by 40% in hypoxia and in high glucose (P<0.01), versus the PBS control. In parallel, rHDL rescued high glucose-impaired tubulogenesis in hypoxia by 64% versus the PBS/normoxia control (P<0.001). We next used a murine model of diabetic wound healing that tracks angiogenesis in the wound bed. We found daily topical application of rHDL (50μg/wound/day) increased wound angiogenesis by 10% (as assessed by Laser Doppler perfusion imaging) and promoted CD31+ neovessel formation by 46% in diabetic wounds, supporting an increased rate of wound closure in diabetic animals of 30% (P<0.05). rHDL treated wounds from diabetic mice had a striking increase in PDK4 gene (180%) and protein expression (350%) in the early-mid stages (24 h and 3-day time points, P<0.05) post-wounding, when the angiogenic response to wound ischaemia is most important. Conclusions We have demonstrated that the key protein controlling the metabolic reprogramming response to hypoxia, PDK4, plays an important role in endothelial cell angiogenesis. We also show that the PDK4 and angiogenesis responses to hypoxia are impaired in high glucose and can be rescued by rHDL. In vivo we find that topical rHDL increases wound angiogenesis and PDK4 levels, explaining the enhanced rate of wound closure in diabetic mice and has implications for improving cardiovascular outcomes for diabetic patients following myocardial infarction.

PDK4‐Deficiency Switches the Pro‐Survival NF‐κB/TNF Signaling to Pro‐Apoptosis in Hepatocytes

ObjectiveAlthough pyruvate dehydrogenase kinase 4 (PDK4) is a well‐stablished regulator of glucose oxidation in the mitochondria, the non‐metabolic function of PDK4 and how it coordinates cellular signaling events remain unexplored. This study delineates a novel cytoplasmic function of PDK4 in hepatocyte apoptosis via its control of the NF‐κB/TNF signaling.MethodsWe used primary hepatocytes, and hepatocellular carcinoma (HCC) cell lines with PDK4 knockdown using shRNAs or overexpression. Mouse models: wild‐type (WT), Pdk4−/− and PDK4 overexpression mice treated with anti‐mouse CD95, or D‐galactosamine and lipopolysaccharide (D‐GalN/LPS) to induce liver failure. TNF and NF‐κB signaling pathways were inhibited by shRNAs against TNF receptor (TNFR1) and NF‐κB/p65, respectively. Apoptosis, mitochondria function, cell death genes, TNF levels, NF‐κB activity, ROS, GSH, JNK activity and protein interaction were evaluated.ResultsIn HCC cells, knockdown of PDK4 (shPDK4) remarkably decreased cell viability and triggered apoptosis. Transmission electronic microscopy revealed increased abnormal mitochondria by shPDK4. Seahorse identified marked elevation in mitochondria respiration accompanied with enhanced ATP and ROS production. qPCR array identified a common upregulation of TNF, BIRC3, and CCDC103 in shPDK4 Huh7 and Hep3B cells; the three genes were demonstrated to be NF‐κB/p65 targets. Cytoplasmic PDK4 protein bound to p65 and sequestered it from nuclear translocation. Hydrogen peroxide, nutrient deprivation, or shPDK4 disrupted p65 and PDK4 interaction, allowing p65 to translocate to the nucleus and bind to the TNF promoter, resulting in TNF activation and production, TNFR1 signaling and cell apoptosis. In vivo, Pdk4−/− livers developed more severe apoptosis than WT mice after administration of the anti‐mouse CD95 or GalN/LPS, which was accompanied by increased production of ROS, sustained activation of JNK, and lower levels of GSH. In contrast, PDK4 overexpression in WT mice prevented NF‐κB activation and TNF production, attenuated ROS, reduced JNK activation and GSH depletion, and thereof protected livers against apoptosis; the liver injury was reduced through impairing NF‐κB/TNF signaling in Pdk4−/− mice. Notably, TNF treatment sensitized apoptosis in shPDK4 cells, and induced apoptosis in Pdk4−/− primary hepatocytes but not WT cells. PDK4‐deficiency changed intracellular environment, leading to enhanced ROS production, sustained JNK and GSH depletion in HCC cells and primary hepatocytes. In this scenario, TNF treatment could not effectively activate anti‐apoptotic NF‐κB targets TRAF2, A20, c‐IAPs, BCL2 and FLIP in shPDK4 cells and Pdk4−/− primary hepatocytes, in contrary to control cells. Therefore, the pro‐survival activity of TNF was switched to a pro‐apoptotic activity by PDK4‐deficiency.ConclusionsIntact PDK4 function is indispensable in TNF/NF‐κB‐mediated hepatocyte survival. In the absence of PDK4, the survival function of NF‐κB is switched to pro‐apoptosis and pro‐liver injury.Support or Funding InformationAll authors have nothing to disclose. Grant support: L.W. is supported by NIH R01ES025909, R01DK104656, R21AA022482, R21AA024935, R01AA026322, VA Merit Award 1I01BX002634.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

180. The Role of the Pyruvate Dehydrogenase Kinase 4 (PDK4) Gene Mutation in the Development of Dilated Cardiomyopathy in Dobermanpinschers and Potential Application of Cardiac Gene Therapy

Dilated cardiomyopathy (DCM) is estimated to be the third most common inherited type of heart disease in humans reported to affect 35.6 and 0.57 in 100,000 adults and children, respectively. The Doberman Pinscher (DP) is a canine breed affected with idiopathic, non-ischemic DCM that closely resembles the human counterpart acting as a clinically relevant human model. Recently, a 16 base pair deletion in the 5’ donor splice site of intron 10 of the pyruvate dehydrogenase kinase 4 (PDK4) gene, encoding for a mitochondrial protein involved in energy metabolism, has been identified but the relationship between the PDK4 mutation, mitochondrial function and phenotypic development of DCM is largely unknown. Our research has focused on understanding the relationship between the consequences of the PDK4 mutation on mitochondrial function and phenotypic development of DCM as well as the potential of cardiac gene therapy to correct the phenotype. A total of 64 DP were tested for the PDK4 mutation and screened for DCM. Healthy dogs and dogs diagnosed with DCM were further divided into 3 groups based on the presence of the genetic mutation: Wild-type (PDK4wt/wt), Heterozygous (PDK4wt/del), and Homozygous (PDK4del/del). Preliminary analyses using isolated mitochondria from skin fibroblasts of these dogs demonstrated a lack of detectable PDK4 protein expression in PDK4del/del, and a significant decrease in PDK4wt/del (48% of normal). Phase contrast microscopy and immunofluorescence studies revealed significantly different morpho-phenotypic characteristics and reduced capacity to adapt to unfavorable metabolic conditions as compared to healthy dogs. Assessment of mitochondrial metabolic potential and function (oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)) were performed using an extracellular flux analyzer. Cells were stressed using serum glucose starvation. Compared to healthy dogs, PDK4wt/del and PDK4del/del revealed a much lower basal respiration rate (OCR reduction of 46% and 68% of normal, respectively) and decreased metabolic potential to meet energy demands (85% and 72% of normal, respectively). Maximal mitochondrial respiration and spare respiratory capacity were all significantly lower in the affected dogs (PDK4wt/del 42% and 14%, and PDK4del/del 40% and 1.4% of normal OCR, respectively). Our results indicate that the mitochondria of affected dogs have a lower metabolic capacity and thus exhaust the two major energy-producing pathways, aerobic and glycolytic metabolism, at a much faster rate when under stress compared to healthy control dogs. Although we detected a significant decrease in PDK4 protein levels, PDK4 transcription levels have yet to be determined in these samples. Our immediate future goals include 1) quantitative PCR to compare PDK4 transcript levels, 2) PCR and sequencing to identify abnormalities in PDK4 mRNA splicing in affected dogs, 3) assessments of mitochondria ATP production and PDK4 protein kinase function. Our long-term goal is to perform in vitro experiments to assess if mitochondrial abnormalities can be improved with gene therapy. To this end, we have cloned canine healthy PDK4 into an adeno-associated virus construct. Ultimately, our efforts will lead us to a cardiac gene therapy clinical trial for Doberman PDK4-DCM.

Inflammation increases pyruvate dehydrogenase kinase 4 (PDK4) expression via the Jun N-Terminal Kinase (JNK) pathway in C2C12 cells

Chronic inflammation augments the deleterious effects of several diseases, particularly diabetes, cancer, and sepsis. It is also involved in the process of metabolic shift from glucose oxidation to lactate production. Although several studies suggest that the change in activity of the pyruvate dehydrogenase complex (PDC) is a major factor causing this metabolic change, the exact mechanism of the inflammatory state remains unclear. In this study, we investigated the effect of lipopolysaccharide (LPS) on the expression of pyruvate dehydrogenase kinase 4 (PDK4), which is strongly associated with inactivation of the PDC in C2C12 myoblasts.In C2C12 myoblasts, LPS exposure led to increased PDK4 mRNA and protein expression levels as well as lactate production in culture medium. However, the expression levels of other PDK isoenzymes (PDK1 – 3) remained unchanged. Additionally, we observed that LPS treatment induced phosphorylation of Jun N-Terminal Kinases (JNK). To confirm the role of JNK, we inhibited the JNK pathway and observed that PDK4 expression and lactate production were decreased, but p38 and ERK were not significantly changed.Taken together, our results suggest that LPS induces PDK4 expression and alters glucose metabolism via the JNK pathway.

5'-AMP activated protein kinase α2 controls substrate metabolism during post-exercise recovery via regulation of pyruvate dehydrogenase kinase4.

It is well known that exercise has a major impact on substrate metabolism for many hours after exercise. However, the regulatory mechanisms increasing lipid oxidation and facilitating glycogen resynthesis in the post-exercise period are unknown. To address this, substrate oxidation was measured after prolonged exercise and during the following 6 h post-exercise in 5´-AMP activated protein kinase (AMPK) α2 and α1 knock-out (KO) and wild-type (WT) mice with free access to food. Substrate oxidation was similar during exercise at the same relative intensity between genotypes. During post-exercise recovery, a lower lipid oxidation (P < 0.05) and higher glucose oxidation were observed in AMPKα2 KO (respiratory exchange ratio (RER) = 0.84 ± 0.02) than in WT and AMPKα1 KO (average RER = 0.80 ± 0.01) without genotype differences in muscle malonyl-CoA or free-carnitine concentrations. A similar increase in muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in WT and AMPKα2 KO was observed following exercise, which is consistent with AMPKα2 deficiency not affecting the exercise-induced activation of the PDK4 transcriptional regulators HDAC4 and SIRT1. Interestingly, PDK4 protein content increased (63%, P < 0.001) in WT but remained unchanged in AMPKα2 KO. In accordance with the lack of increase in PDK4 protein content, lower (P < 0.01) inhibitory pyruvate dehydrogenase (PDH)-E1α Ser(293) phosphorylation was observed in AMPKα2 KO muscle compared to WT. These findings indicate that AMPKα2 regulates muscle metabolism post-exercise through inhibition of the PDH complex and hence glucose oxidation, subsequently creating conditions for increased fatty acid oxidation.

Pharmacological activation of the pyruvate dehydrogenase complex reduces statin‐mediated upregulation of FOXO gene targets and protects against statin myopathy in rodents

We previously reported that statin myopathy is associated with impaired carbohydrate (CHO) oxidation in fast-twitch rodent skeletal muscle, which we hypothesised occurred as a result of forkhead box protein O1 (FOXO1) mediated upregulation of pyruvate dehydrogenase kinase-4 (PDK4) gene transcription. Upregulation of FOXO gene targets known to regulate proteasomal and lysosomal muscle protein breakdown was also evident. We hypothesised that increasing CHO oxidation in vivo, using the pyruvate dehydrogenase complex (PDC) activator, dichloroacetate (DCA), would blunt activation of FOXO gene targets and reduce statin myopathy. Female Wistar Hanover rats were dosed daily for 12 days (oral gavage) with either vehicle (control, 0.5% w/v hydroxypropyl-methylcellulose 0.1% w/v polysorbate-80; n = 9), 88 mg( )kg(-1) day(-1) simvastatin (n = 8), 88 mg( )kg(-1) day(-1) simvastatin + 30 mg kg(-1) day(-1) DCA (n = 9) or 88 mg kg(-1) day(-1) simvastatin + 40 mg kg(-1) day(-1) DCA (n = 9). Compared with control, simvastatin reduced body mass gain and food intake, increased muscle fibre necrosis, plasma creatine kinase levels, muscle PDK4, muscle atrophy F-box (MAFbx) and cathepsin-L mRNA expression, increased PDK4 protein expression, and proteasome and cathepsin-L activity, and reduced muscle PDC activity. Simvastatin with DCA maintained body mass gain and food intake, abrogated the myopathy, decreased muscle PDK4 mRNA and protein, MAFbx and cathepsin-L mRNA, increased activity of PDC and reduced proteasome activity compared with simvastatin. PDC activation abolished statin myopathy in rodent skeletal muscle, which occurred at least in part via inhibition of FOXO-mediated transcription of genes regulating muscle CHO utilisation and protein breakdown.

Hypoxia Induces PDK4 Gene Expression through Induction of the Orphan Nuclear Receptor ERRγ

Multiple cellular signaling pathways that control metabolism and survival are activated when cell are incubated under hypoxic conditions. Activation of the hypoxia inducible factor (HIF)-1 promotes expression of genes that increase the capacity to cope with the stress imposed by a reduced oxygen environment. Here we show that the orphan nuclear receptor estrogen related receptor γ (ERRγ) plays a critical role in hypoxia–mediated activation of pyruvate dehydrogenase kinase 4 (PDK4) gene expression. ERRγ mRNA and protein levels were increased by hypoxia or desferrioxamine (DFO) treatment in hepatoma cell lines. Co-expression of HIF-1α and β increased ERRγ promoter activity as well as mRNA expression, while knockdown of endogenous HIF-1α reduced the hypoxia-mediated induction of ERRγ. In addition, hypoxia also increased the promoter activity and mRNA level of PDK4 in HepG2 cells. Adenovirus mediated-overexpression of ERRγ specifically increased PDK4 gene expression, while ablation of endogenous ERRγ significantly decreased hypoxia-mediated induction of PDK4 gene expression. Finally, GSK5182, an inverse agonist of ERRγ, strongly inhibited the hypoxia-mediated induction of PDK4 protein and promoter activity. Regulation of the transcriptional activity of ERRγ may provide a therapeutic approach for the regulation of PDK4 gene expression under hypoxia.

Effects of high-fat diet and physical activity on pyruvate dehydrogenase kinase-4 in mouse skeletal muscle

BackgroundThe expression of PDK4 is elevated by diabetes, fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. It is previously shown that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a master regulator of energy metabolism, coactivates in cell lines pyruvate dehydrogenase kinase-4 (PDK4) gene expression via the estrogen-related receptor α (ERRα). We investigated the effects of long-term high-fat diet and physical activity on the expression of PDK4, PGC-1α and ERRα and the amount and function of mitochondria in skeletal muscle.MethodsInsulin resistance was induced by a high-fat (HF) diet for 19 weeks in C57BL/6 J mice, which were either sedentary or with access to running wheels. The skeletal muscle expression levels of PDK4, PGC-1α and ERRα were measured and the quality and quantity of mitochondrial function was assessed.ResultsThe HF mice were more insulin-resistant than the low-fat (LF) -fed mice. Upregulation of PDK4 and ERRα mRNA and protein levels were seen after the HF diet, and when combined with running even more profound effects on the mRNA expression levels were observed. Chronic HF feeding and voluntary running did not have significant effects on PGC-1α mRNA or protein levels. No remarkable difference was found in the amount or function of mitochondria.ConclusionsOur results support the view that insulin resistance is not mediated by the decreased qualitative or quantitative properties of mitochondria. Instead, the role of PDK4 should be contemplated as a possible contributor to high-fat diet-induced insulin resistance.

Induction of PDK4 in the heart muscle of JVS mice, an animal model of systemic carnitine deficiency, does not appear to reduce glucose utilization by the heart

Pyruvate dehydrogenase kinase 4 (PDK4) mRNA has been reported as an up-regulated gene in the heart and skeletal muscle of carnitine-deficient juvenile visceral steatosis (JVS) mice under fed conditions. PDK4 plays an important role in the inhibition of glucose oxidation via the phosphorylation of pyruvate dehydrogenase complex (PDC). This study evaluated the meaning of increased PDK4 mRNA in glucose metabolism by investigating PDK4 protein levels, PDC activity and glucose uptake by the heart and skeletal muscle of JVS mice. PDK4 protein levels in the heart and skeletal muscle of fed JVS mice were increased in accordance with mRNA levels, and protein was enriched in the mitochondria. PDK4 protein was co-fractionated with PDC in sucrose density gradient centrifugation, like PDK2 protein; however, the activities of the pyruvate dehydrogenase complex (PDC) active form in the heart and skeletal muscle of fed JVS mice were similar to those in fed control mice. Fed JVS mice showed significantly higher glucose uptake in the heart and similar uptake in the skeletal muscle compared with fed control mice. Thus, in carnitine deficiency under fed conditions, glucose was preferentially utilized in the heart as an energy source despite increased PDK4 protein levels in the mitochondria. The preferred glucose utilization may be involved in developing cardiac hypertrophy from carnitine deficiency in fatty acid oxidation abnormality.

PGC-1α increases PDH content but does not change acute PDH regulation in mouse skeletal muscle

The aim of this study was to test whether the transcriptional coactivator peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC)1α regulates the content of pyruvate dehydrogenase (PDH)-E1α and influences PDH activity through regulation of pyruvate dehydrogenase kinase-4 (PDK4) expression and subsequently PDH phosphorylation. PGC-1α whole body knockout (KO), muscle-specific PGC-1α overexpressing mice (MCK PGC-1α), and littermate wild-type (WT) mice underwent two interventions known to affect PDH. Quadriceps muscles were removed from fed and 24-h fasted mice as well as at 6 h of recovery after 1-h running and from mice that did not run acutely. PDH-E1α protein content and PDH-E1α phosphorylation were lower in PGC-1α KO and higher in MCK PGC-1α mice at rest, but, while MCK PGC-1α had higher PDK4 protein content, KO of PGC-1α had no effect on PDK4 protein content. The differences in phosphorylation partly vanished when expressing phosphorylation relative to the PDH-E1α content with only a maintained elevated phosphorylation in MCK PGC-1α mice. Fasting upregulated PDK4 protein in PGC-1α KO, MCK PGC-1α and WT mice, but this was not consistently associated with increased PDH-E1α phosphorylation. Downregulation of the activity of PDH in the active form (PDHa) at 6-h recovery from exercise in both the PGC-1α KO and MCK PGC-1α mice and the association between PDH-E1α phosphorylation and PDHa activity in PGC-1α KO mice indicate that PGC-1α is not required for these responses. In conclusion, PGC-1α regulates PDH-E1α protein content in parallel with mitochondrial oxidative proteins, but does not seem to influence PDH regulation in mouse skeletal muscle in response to fasting and in recovery from exercise.

The Increase in Cardiac Pyruvate Dehydrogenase Kinase-4 after Short-Term Dexamethasone Is Controlled by an Akt-p38-Forkhead Box Other Factor-1 Signaling Axis

Glucocorticoids increase pyruvate dehydrogenase kinase-4 (PDK4) mRNA and protein expression, which phosphorylates pyruvate dehydrogenase, thereby preventing the formed pyruvate from undergoing mitochondrial oxidation. This increase in PDK4 expression is mediated by the mandatory presence of Forkhead box other factors (FoxOs) in the nucleus. In the current study, we examined the importance of the nongenomic effects of dexamethasone (Dx) in determining the compartmentalization of FoxO and hence its transcriptional activity. Rat cardiomyocytes exposed to Dx produced a robust decrease in glucose oxidation. Measurement of FoxO compartmentalization demonstrated increase in nuclear but resultant decrease in cytosolic content of FoxO1 with no change in the total content. The increase in nuclear content of FoxO1 correlated to an increase in nuclear phospho-p38 MAPK together with a robust association between this transcription factor and kinase. Dx also promoted nuclear retention of FoxO1 through a decrease in phosphorylation of Akt, an effect mediated by heat shock proteins binding to Akt. Measurement of the nuclear and total expression of sirtuin-1 protein showed no change after Dx. Instead, Dx increased the association of sirtuin-1 with FoxO1, thereby causing a decrease in FoxO acetylation. Manipulation of FoxO1 through agents that interfere with its nuclear shuttling or acetylation were effective in reducing Dx-induced increase in PDK4 protein expression. Our data suggest that FoxO1 has a major PDK4-regulating function. In addition, given the recent suggestions that altering glucose use can set the stage for heart failure, manipulating FoxO could assist in devising new therapeutic strategies to optimize cardiac metabolism and prevent PDK4 induced cardiac complications.

PDC deletion: the way to a man's heart disease

the heart has continuous high-energy demands required to sustain efficient contraction. This is met by the metabolism of major circulating substrates (e.g., glucose, lactate, or lipids), according to availability, since the heart has a limited capacity for nutrient storage (reviewed in Ref. [16][1

Dysregulated pyruvate dehydrogenase complex in Zucker diabetic fatty rats

The mitochondrial pyruvate dehydrogenase complex (PDC) is inactivated in many tissues during starvation and diabetes. We investigated carbohydrate oxidation (CHO) and the regulation of the PDC in lean and obese Zucker diabetic fatty (ZDF) rats during fed and starved conditions as well as during an oral glucose load without and with pharmacologically reduced levels of free fatty acids (FFA) to estimate the relative contribution of FFA on glucose tolerance, CHO, and PDC activity. The increase in total PDC activity (20-45%) was paralleled by increased protein levels ( approximately 2-fold) of PDC subunits in liver and muscle of obese ZDF rats. Pyruvate dehydrogenase kinase-4 (PDK4) protein levels were higher in obese rats, and consequently PDC activity was reduced. Although PDK4 protein levels were rapidly downregulated (57-62%) in both lean and obese animals within 2 h after glucose challenge, CHO over 3 h as well as the peak of PDC activity (1 h after glucose load) in liver and muscle were significantly lower in obese rats compared with lean rats. Similar differences were obtained with pharmacologically suppressed FFA by nicotinic acid, but with significantly improved glucose tolerance in obese rats, as well as increased CHO and delta increases in PDC activity (0-60 min) both in muscle and liver. These results demonstrated the suppressive role of FFA acids on the measured parameters. Furthermore, the results clearly demonstrate a rapid reactivation of PDC in liver and muscle of lean and obese rats after a glucose load and show that PDC activity is significantly lower in obese ZDF rats.

PGC-1α Coactivates PDK4 Gene Expression via the Orphan Nuclear Receptor ERRα: a Mechanism for Transcriptional Control of Muscle Glucose Metabolism

The transcriptional coactivator PGC-1alpha is a key regulator of energy metabolism, yet little is known about its role in control of substrate selection. We found that physiological stimuli known to induce PGC-1alpha expression in skeletal muscle coordinately upregulate the expression of pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of glucose oxidation. Forced expression of PGC-1alpha in C(2)C(12) myotubes induced PDK4 mRNA and protein expression. PGC-1alpha-mediated activation of PDK4 expression was shown to occur at the transcriptional level and was mapped to a putative nuclear receptor binding site. Gel shift assays demonstrated that the PGC-1alpha-responsive element bound the estrogen-related receptor alpha (ERRalpha), a recently identified component of the PGC-1alpha signaling pathway. In addition, PGC-1alpha was shown to activate ERRalpha expression. Chromatin immunoprecipitation assays confirmed that PGC-1alpha and ERRalpha occupied the mPDK4 promoter in C(2)C(12) myotubes. Additionally, transfection studies using ERRalpha-null primary fibroblasts demonstrated that ERRalpha is required for PGC-1alpha-mediated activation of the mPDK4 promoter. As predicted by the effects of PGC-1alpha on PDK4 gene transcription, overexpression of PGC-1alpha in C(2)C(12) myotubes decreased glucose oxidation rates. These results identify the PDK4 gene as a new PGC-1alpha/ERRalpha target and suggest a mechanism whereby PGC-1alpha exerts reciprocal inhibitory influences on glucose catabolism while increasing alternate mitochondrial oxidative pathways in skeletal muscle.

PPAR alpha ligand protects during cisplatin-induced acute renal failure by preventing inhibition of renal FAO and PDC activity.

Previous studies demonstrated that during cisplatin-induced acute renal failure, there is a significant reduction in proximal tubule fatty acid oxidation. We now report on the effects of peroxisome proliferator-activated receptor-alpha (PPAR alpha) ligand Wy-14643 (WY) on the abnormalities of medium chain fatty acid oxidation and pyruvate dehydrogenase complex (PDC) activity in kidney tissue of cisplatin-treated mice. Cisplatin causes a significant reduction in mRNA levels and enzyme activity of mitochondrial medium chain acyl-CoA dehydrogenase (MCAD). PPAR alpha ligand WY ameliorated cisplatin-induced acute renal failure and prevented cisplatin-induced reduction of mRNA levels and enzyme activity of MCAD. In contrast, in cisplatin-treated PPAR alpha null mice, WY did not protect kidney function and did not reverse cisplatin-induced decreased expression of MCAD. Cisplatin inhibited renal PDC activity before the development of acute tubular necrosis, and PDC inhibition was reversed by pretreatment with PPAR alpha agonist WY. Cisplatin also induced increased mRNA and protein levels of pyruvate dehydrogenase kinase-4 (PDK4), and PPAR alpha ligand WY prevented cisplatin-induced increased expression of PDK4 protein levels in wild-type mice. We conclude that PPAR alpha agonists have therapeutic potential for cisplatin-induced acute renal failure. Use of PPAR alpha ligands prevents acute tubular necrosis by ameliorating cisplatin-induced inhibition of two distinct metabolic processes, MCAD-mediated fatty acid oxidation and PDC activity.