Pyruvate Dehydrogenase Kinase Isoenzyme 4 Research Articles

Reversing tozasertib resistance in glioma through inhibition of pyruvate dehydrogenase kinases.

Acquired resistance to conventional chemotherapeutic agents limits their effectiveness and can cause cancer treatment to fail. Because enzymes in the aurora kinase family are vital regulators of several mitotic events, we reasoned that targeting these kinases with tozasertib, a pan‐aurora kinase inhibitor, would not only cause cytokinesis defects, but also induce cell death in high‐grade pediatric and adult glioma cell lines. We found that tozasertib induced cell cycle arrest, increased mitochondrial permeability and reactive oxygen species generation, inhibited cell growth and migration, and promoted cellular senescence and pro‐apoptotic activity. However, sustained exposure to tozasertib at clinically relevant concentrations conferred resistance, which led us to examine the mechanistic basis for the emergence of drug resistance. RNA‐sequence analysis revealed a significant upregulation of the gene encoding pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), a pyruvate dehydrogenase (PDH) inhibitory kinase that plays a crucial role in the control of metabolic flexibility under various physiological conditions. Upregulation of PDK1, PDK2, PDK3, or PDK4 protein levels was positively correlated with tozasertib‐induced resistance through inhibition of PDH activity. Tozasertib‐resistant cells exhibited increased mitochondrial mass as measured by 10‐N‐nonyl‐Acridine Orange. Inhibition of PDK with dichloroacetate resulted in increased mitochondrial permeability and cell death in tozasertib‐resistant glioma cell lines. Based on these results, we believe that PDK is a selective target for the tozasertib resistance phenotype and should be considered for further preclinical evaluations.

High hepatic expression of PDK4 improves survival upon multimodal treatment of colorectal liver metastases

BackgroundPatients with borderline resectable colorectal liver metastases (CRLM) frequently receive neoadjuvant chemotherapy (NC) to reduce tumour burden, thus making surgical resection feasible. Even though NC can induce severe liver injury, most studies investigating tissue-based prognostic markers focus on tumour tissue. Here, we assessed the prognostic significance of pyruvate-dehydrogenase-kinase isoenzyme 4 (PDK4) within liver tissue of patients undergoing surgical resection due to CRLM.MethodsTranscript levels of hypoxia-adaptive genes (such as PDK isoenzymes) were assessed in the tissue of healthy liver, corresponding CRLM, healthy colon mucosa and corresponding tumour. Uni- and multivariate analyses were performed. Responses to chemotherapy upon up- or down-regulation of PDK4 were studied in vitro.ResultsPDK4 expression within healthy liver tissue was associated with increased overall survival and liver function following surgical resection of CRLM. This association was enhanced in patients with NC. PDK4 expression in CRLM tissue did not correlate with overall survival. Up-regulation of PDK4 increased the resistance of hepatocytes and colon cancer cells against chemotherapy-induced toxicity, whereas knockdown of PDK4 enhanced chemotherapy-associated cell damage.ConclusionOur findings suggest that up-regulated PDK4 expression reduces hepatic chemotherapy-induced oxidative stress and is associated with improved postoperative liver function in patients undergoing multimodal treatment and resection of CRLM.

Transcriptomics in the wild: Hibernation physiology in free-ranging dwarf lemurs.

Hibernation is an adaptive strategy some mammals use to survive highly seasonal or unpredictable environments. We present the first investigation on the transcriptomics of hibernation in a natural population of primate hibernators: Crossley's dwarf lemurs (Cheirogaleus crossleyi). Using capture-mark-recapture techniques to track the same animals over a period of 7months in Madagascar, we used RNA-seq to compare gene expression profiles in white adipose tissue (WAT) during three distinct physiological states. We focus on pathway analysis to assess the biological significance of transcriptional changes in dwarf lemur WAT and, by comparing and contrasting what is known in other model hibernating species, contribute to a broader understanding of genomic contributions of hibernation across Mammalia. The hibernation signature is characterized by a suppression of lipid biosynthesis, pyruvate metabolism and mitochondrial-associated functions, and an accumulation of transcripts encoding ribosomal components and iron-storage proteins. The data support a key role of pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) in regulating the shift in fuel economy during periods of severe food deprivation. This pattern of PDK4 holds true across representative hibernating species from disparate mammalian groups, suggesting that the genetic underpinnings of hibernation may be ancestral to mammals.

Pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes to the pathogenesis of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive disease and poses a high risk of severe liver damage. However, the pathogenesis of NASH is still unclear. Accumulation of lipid droplets and insulin resistance is the hallmark of NASH. Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) plays key role in glucose metabolism via regulating the activity of pyruvate dehydrogenase complex (PDC). Here, we demonstrated a novel of PDK4 in NASH by regulating hepatic steatosis and insulin signaling pathway in methionine and choline deficient (MCD) diet induced NASH model. Hepatic PDK4 levels were highly induced in human patients with NASH and MCD diet fed mice, as well as in hepatocytes treated with oleic acid. The glucose and lipid metabolism were impaired in Pdk4−/− mice. Pdk4 deficiency ameliorated the hepatic steatosis significantly in NASH mice. Pdk4−/−-MCD mice had reduced liver weights and triglyceride (TG) levels. And Pdk4 deficiency dramatically reduced the expression of genes related to fatty acid uptake, synthesis and gluconeogenesis. In addition, elevated phosphorylated AMPK (p-AMPK), p-SAPK/JNK and diminished p-ERK, p-P38, p-Akt and p-mTOR/p-4EBP1 proteins were observed. In conclusion, our data indicated that PDK4 potentially contributes to the hepatic steatosis in NASH via regulating several signaling pathway and PDK4 may be a new therapeutic strategy against NAFLD.

Effects of neonatal dexamethasone administration on cardiac recovery ability under ischemia-reperfusion in 24-wk-old rats.

Evaluations of stress-induced cardiac functional alterations in adults after neonatal glucocorticoid (GC) treatment have been limited. In the present study, we evaluated adult cardiac functional recovery during postischemic reperfusion and measured cardiac gene expression involved energy metabolism in rats neonatally treated with dexamethasone (DEX). Male Wistar rats were injected DEX in first 3 d after birth and controls were received saline (SAL). At 24 wk of age, insulin tolerance tests were performed, plasma lipid levels were measured, and left ventricular function and myocardial infarct size were evaluated. Expressions of genes involved in cardiac energy metabolism were measured by quantitative real-time polymerase chain reaction (PCR) and western blot. In 24-wk-old rats, neonatal DEX administration caused dyslipidemia, impaired cardiac recovery function and increased size of infarction, decreased cardiac expression of glucose transporter 4(GLUT4), peroxisome proliferative-activated receptor gamma coactivator 1α (PGC-1α) and ratios of phospho-forkhead box O1/forkhead box O1 (p-FoxO1/FoxO1) and phospho AMP-activated protein kinase/AMP-activated protein kinase (p-AMPK/AMPK) but increased pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) expression compared with controls. Neonatal DEX administration impairs cardiac functional recovery during reperfusion following ischemia in 24-wk-old rats. Reduced cardiac glucose utilization may contribute to the long-term detrimental effects caused by neonatal DEX treatment.

Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high saturated fat diet

Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) may prove beneficial in the treatment of type 2 diabetes or diet-induced obesity, it may have detrimental effects by inhibiting fatty acid oxidation. Peroxisome proliferator-activated receptor α (PPARα) agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment using a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of a PDK4 inhibitor because PPARα agonists induce PDK4 expression. In the present study, the effects of clofibric acid, a PPARα agonist, on blood and liver lipids were determined in wild-type and PDK4 knockout mice fed a high-fat diet. As expected, treatment of wild-type mice with clofibric acid resulted in less body weight gain, smaller epididymal fat pads, greater insulin sensitivity, and lower levels of serum and liver triacylglycerol. Surprisingly, rather than decreasing the effectiveness of clofibric acid, PDK4 deficiency enhanced the beneficial effects of clofibric acid on hepatic steatosis, reduced blood glucose levels, and did not prevent the positive effects of clofibric acid on serum triacylglycerols and free fatty acids. The metabolic effects of clofibric acid are therefore independent of the induction of PDK4 expression. The additive beneficial effects on hepatic steatosis may be due to induction of increased capacity for fatty acid oxidation and partial uncoupling of oxidative phosphorylation by clofibric acid, and a reduction in the capacity for fatty acid synthesis as a result of PDK4 deficiency.

Adaptive gene regulation of pyruvate dehydrogenase kinase isoenzyme 4 in hepatotoxic chemical-induced liver injury and its stimulatory potential for DNA repair and cell proliferation

The processes involved in the adaptation of animals to environmental factors remain unclear. We examined the mechanisms underlying the adaptive potential of the mouse against hepatotoxic chemical-induced injury. Microarray analysis revealed that ethylbenzene, a hepatotoxic chemical, upregulated PDK4 (encoding pyruvate dehydrogenase kinase isoenzyme 4) in mouse livers and that the upregulation was enhanced by previous exposure to the chemical. Although PDK4 is an energy resource regulator induced by starvation, expression of other fasting-inducible genes was unaffected. PDK4 induced by chemical stress developed hepatic accumulation of sirtuin 1 by regulating pyruvate concentration and activated the Nbn and ATM, which are critical for DNA repair and checkpoint activation. PDK4 overexpression on carbon tetrachloride (CCl4)-induced liver injury resulted in delayed necrotic tissue recovery with cell cycle arrest and decreased γH2AX foci and micronucleus formation. PDK4 silencing on CCl4-induced liver injury accelerated necrotic tissue recovery and increased γH2AX foci and micronucleus formation, indicating the essential role of PDK4 in DNA repair and checkpoint activation. PDK4 overexpression induced pancreas-specific transcription factor 1a (Ptf1a) upregulation and transcriptional activation of several pancreatic genes in the liver. Ptf1a overexpression by adenoviral gene delivery resulted in accelerated tissue recovery on CCl4-induced liver injury. Our data identified PDK4 as a novel pivotal factor in adaptation to chemical stress.

Cardioprotective Effects of a Selective B2 Receptor Agonist of Bradykinin Post-Acute Myocardial Infarct

The cardioprotective benefits of bradykinin are attributable to activation of its B(2) receptor (B(2)R)-mediated actions and abolished by B(2)R antagonists. The current experiments evaluated the cardioprotective potential of a potent, long-acting B(2)R-selective agonist peptide analogue of bradykinin, the compound NG291. We compared the extent of cardiac tissue damage and remodeling and expression pattern of selected genes in mice submitted to acute myocardial infarct (MI) and treated for 1 week with either NG291 [Hyp(3),Thi(5),(N)Chg(7),Thi(8)]-bradykinin or with saline delivered via osmotic minipump. Active treatment resulted in better ejection fraction (EF) 69 +/- 1% vs. 61 +/- 3.1% (P = 0.01), (vs. 85 +/- 1.3% in sham-operated controls), fractional shortening (FS) 38 +/- 4% vs. 32 +/- 8% (NS) (vs. 53 +/- 1.2 in sham-operated controls), and fewer markers of myocyte apoptosis (TUNEL-positive nuclei 4.9 +/- 1.1% vs. 9.7 +/- 0.03%, P = 0.03). Systolic blood pressure (SBP) at end point was normal at 110 +/- 4.2 in actively treated mice, but tended to be lower at 104 +/- 4.7 mm Hg in saline controls with decreased cardiac systolic capacity. Expression patterns of selected genes to factors related to tissue injury, inflammation, and metabolism (i.e., the B(1)R, B(2)R, endothelial nitric oxide synthase (eNOS), TNF-alpha, cardiomyopathy-associated 3 (Cmya3), and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4)) showed that acute MI induced significant upregulation of these genes, and active treatment prevented or attenuated this upregulation, whereas the B(2)R agonist itself produced no difference in the myocardium of sham-operated mice. Treatment with a selective B(2)R agonist initiated at the time of induction of acute MI in mice had a beneficial effect on cardiac function, tissue remodeling, and inflammation-related tissue gene expression, which may explain its structural and functional benefits.

Pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) deficiency attenuates the long-term negative effects of a high-saturated fat diet

The hypothesis that PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) has potential as a target for the treatment of type 2 diabetes was tested by feeding wild-type and PDHK4 knockout mice a high saturated fat diet that induces hyperglycemia, hyperinsulinaemia, glucose intolerance, hepatic steatosis and obesity. Previous studies have shown that PDHK4 deficiency lowers blood glucose by limiting the supply of three carbon gluconeogenic substrates to the liver. There is concern, however, that the increase in glucose oxidation caused by less inhibition of the pyruvate dehydrogenase complex by phosphorylation will inhibit fatty acid oxidation, promote ectopic fat accumulation and worsen insulin sensitivity. This was examined by feeding wild-type and PDHK4 knockout mice a high saturated fat diet for 8 months. Fasting blood glucose levels increased gradually in both groups but remained significantly lower in the PDHK4 knockout mice. Hyperinsulinaemia developed in both groups, but glucose tolerance was better and body weight was lower in the PDHK4 knockout mice. At termination, less fat was present in the liver and skeletal muscle of the PDHK4 knockout mice. Higher amounts of PGC-1alpha [PPARgamma (peroxisome proliferator-activated receptor gamma) coactivator 1alpha] and PPARalpha and lower amounts of fatty acid synthase and acetyl-CoA carboxylase isoenzyme 1 were present in the liver of the PDHK4 knockout mice. These findings suggest PDHK4 deficiency creates conditions that alter upstream signalling components involved in the regulation of lipid metabolism. The findings support the hypothesis that PDHK4 is a viable target for the treatment of type 2 diabetes.

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

Pharmacodynamic/Pharmacogenomic Modeling of Insulin Resistance Genes in Rat Muscle after Methylprednisolone Treatment: Exploring Regulatory Signaling Cascades

Corticosteroids (CS) effects on insulin resistance related genes in rat skeletal muscle were studied. In our acute study, adrenalectomized (ADX) rats were given single doses of 50 mg/kg methylprednisolone (MPL) intravenously. In our chronic study, ADX rats were implanted with Alzet mini-pumps giving zero-order release rates of 0.3 mg/kg/h MPL and sacrificed at various times up to 7 days. Total RNA was extracted from gastrocnemius muscles and hybridized to Affymetrix GeneChips. Data mining and literature searches identified 6 insulin resistance related genes which exhibited complex regulatory pathways. Insulin receptor substrate-1 (IRS-1), uncoupling protein 3 (UCP3), pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), fatty acid translocase (FAT) and glycerol-3-phosphate acyltransferase (GPAT) dynamic profiles were modeled with mutual effects by calculated nuclear drug-receptor complex (DR(N)) and transcription factors. The oscillatory feature of endothelin-1 (ET-1) expression was depicted by a negative feedback loop. These integrated models provide testable quantitative hypotheses for these regulatory cascades.

Cloning and expression of PDK4, FOXO1A and DYRK1A from the hibernating greater horseshoe bat ( Rhinolophus ferrumequinum)

Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) cDNA was cloned from the brain of greater horseshoe bat ( Rhinolophus ferrumequinum). The deduced amino acid sequence shares strong homology with these PDK4 of other mammals. Moreover, we partially cloned homologues of dual-specificity tyrosine-phosphorylated and regulated protein kinase 1A (DYRK1A), and forkhead box protein O1A (FOXO1A) from greater horseshoe bat. Among five different tissues tested, PDK4 mRNA was highly expressed in the heart, white adipose tissue and muscle, but weakly expressed in the brain and liver, while DYRK1A and FOXO1A were expressed in all five tissues. Moreover, the transcript levels of PDK4, DYRK1A, and FOXO1A were measured in the heart, white adipose tissue and muscle of hibernating and arousal greater horseshoe bats by Northern blot and real time PCR. The results showed that transcript level of PDK4 was significantly higher in white adipose tissue. Expression level of DYRK1A was significantly higher in hibernating state in white adipose tissue, and expression level of FOXO1A was significantly higher in muscle in aroused state. These results suggest that up-regulation of the transcript levels of PDK4 during hibernation were not regulated via DYRK1A and FOXO1A in white adipose tissue and muscle, and the possible presence of another isoenzyme of PDK which is responsible for the tissue-specific regulation of Pyruvate dehydrogenase complex (PDC) activity in the bat heart during hibernation.

Role of pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during starvation

The PDC (pyruvate dehydrogenase complex) is strongly inhibited by phosphorylation during starvation to conserve substrates for gluconeogenesis. The role of PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) in regulation of PDC by this mechanism was investigated with PDHK4-/- mice (homozygous PDHK4 knockout mice). Starvation lowers blood glucose more in mice lacking PDHK4 than in wild-type mice. The activity state of PDC (percentage dephosphorylated and active) is greater in kidney, gastrocnemius muscle, diaphragm and heart but not in the liver of starved PDHK4-/- mice. Intermediates of the gluconeogenic pathway are lower in concentration in the liver of starved PDHK4-/- mice, consistent with a lower rate of gluconeogenesis due to a substrate supply limitation. The concentration of gluconeogenic substrates is lower in the blood of starved PDHK4-/- mice, consistent with reduced formation in peripheral tissues. Isolated diaphragms from starved PDHK4-/- mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis. BCAAs (branched chain amino acids) are higher in the blood in starved PDHK4-/- mice, consistent with lower blood alanine levels and the importance of BCAAs as a source of amino groups for alanine formation. Non-esterified fatty acids are also elevated more in the blood of starved PDHK4-/- mice, consistent with lower rates of fatty acid oxidation due to increased rates of glucose and pyruvate oxidation due to greater PDC activity. Up-regulation of PDHK4 in tissues other than the liver is clearly important during starvation for regulation of PDC activity and glucose homoeostasis.

Coordinate expression of the PDK4 gene: a means of regulating fuel selection in a hibernating mammal.

Hibernation in mammals requires a metabolic shift away from the oxidation of carbohydrates and toward the combustion of stored fatty acids as the primary source of energy during torpor. A key element involved in this fuel selection is pyruvate dehydrogenase kinase isoenzyme 4 (PDK4). PDK4 inhibits pyruvate dehydrogenase and thus minimizes carbohydrate oxidation by preventing the flow of glycolytic products into the tricarboxylic acid cycle. This paper examines expression of the PDK4 gene during hibernation in heart, skeletal muscle, and white adipose tissue (WAT) of the 13-lined ground squirrel, Spermophilus tridecemlineatus. During hibernation PDK4 mRNA levels increase 5-fold in skeletal muscle and 15-fold in WAT compared with summer-active levels. Similarly, PDK4 protein is increased threefold in heart, fivefold in skeletal muscle, and eightfold in WAT. High levels of serum insulin, likely to have an inhibitory effect on PDK4 gene expression, are seen during fall when PDK4 mRNA levels are low. Coordinate upregulation of PDK4 in three distinct tissues suggests a common signal that regulates PDK4 expression and fuel selection during hibernation.

Starvation Increases the Amount of Pyruvate Dehydrogenase Kinase in Several Mammalian Tissues

Covalent modification of the pyruvate dehydrogenase complex provides an important regulatory mechanism for controlling the disposal of glucose and other compounds metabolized to pyruvate. Regulation of the complex by this mechanism is achieved in part by tissue-specific expression of the genes encoding isoenzymes of pyruvate dehydrogenase kinase (PDK). Starvation is known from our previous work to increase PDK activity of heart and skeletal muscle by increasing the amount of PDK isoenzyme 4 (PDK4) present in these tissues. This study demonstrates that increased expression of both PDK4 and PDK2 occurs in rat liver, kidney, and lactating mammary gland in response to starvation. PDK4 and PDK2 message levels were also increased by starvation in the two tissues examined (liver and kidney), suggesting enhancement of gene transcription. Changes in PDK2 message and protein were of similar magnitude, but changes in PDK4 message were greater than those in PDK4 protein, suggesting regulation at the level of translation. In contrast to these tissues, starvation had little or no effect on PDK2 and PDK4 protein in brain, white adipose tissue, and brown adipose tissue. Nevertheless, PDK4 message levels were significantly increased in brain and white adipose tissue by starvation. The findings of this study indicate that increased expression of PDK isoenzymes is an important mechanism for bringing about inactivation of the pyruvate dehydrogenase complex during starvation in many but not all tissues of the body. The absence of this mechanism preserves the capacity of neuronal tissue to utilize glucose for energy during starvation.