Showing papers on "Pyruvate dehydrogenase kinase published in 2011"

Regulation of Metabolism by Hypoxia-Inducible Factor 1

Abstract: The maintenance of oxygen homeostasis is critical for survival, and the master regulator of this process in metazoan species is hypoxia-inducible factor 1 (HIF-1), which controls both O(2) delivery and utilization. Under conditions of reduced O(2) availability, HIF-1 activates the transcription of genes, whose protein products mediate a switch from oxidative to glycolytic metabolism. HIF-1 is activated in cancer cells as a result of intratumoral hypoxia and/or genetic alterations. In cancer cells, metabolism is reprogrammed to favor glycolysis even under aerobic conditions. Pyruvate kinase M2 (PKM2) has been implicated in cancer growth and metabolism, although the mechanism by which it exerts these effects is unclear. Recent studies indicate that PKM2 interacts with HIF-1α physically and functionally to stimulate the binding of HIF-1 at target genes, the recruitment of coactivators, histone acetylation, and gene transcription. Interaction with HIF-1α is facilitated by hydroxylation of PKM2 at proline-403 and -408 by PHD3. Knockdown of PHD3 decreases glucose transporter 1, lactate dehydrogenase A, and pyruvate dehydrogenase kinase 1 expression; decreases glucose uptake and lactate production; and increases O(2) consumption. The effect of PKM2/PHD3 is not limited to genes encoding metabolic enzymes because VEGF is similarly regulated. These results provide a mechanism by which PKM2 promotes metabolic reprogramming and suggest that it plays a broader role in cancer progression than has previously been appreciated.

Erk regulation of pyruvate dehydrogenase flux through PDK4 modulates cell proliferation

Abstract: Loss of extracellular matrix (ECM) attachment leads to metabolic impairments that limit cellular energy production. Characterization of the metabolic alterations induced by ECM detachment revealed a dramatic decrease in uptake of glucose, glutamine, and pyruvate, and a consequent decrease in flux through glycolysis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle. However, flux through pyruvate dehydrogenase (PDH) is disproportionally decreased, concomitant with increased expression of the PDH inhibitory kinase, PDH kinase 4 (PDK4), and increased carbon secretion. Overexpression of ErbB2 maintains PDH flux by suppressing PDK4 expression in an Erk-dependent manner, and Erk signaling also regulates PDH flux in ECM-attached cells. Additionally, epidermal growth factor (EGF), a potent inducer of Erk, positively regulates PDH flux through decreased PDK4 expression. Furthermore, overexpression of PDK4 in ECM-detached cells suppresses the ErbB2-mediated rescue of ATP levels, and in attached cells, PDK4 overexpression decreases PDH flux, de novo lipogenesis, and cell proliferation. Mining of microarray data from human tumor data sets revealed that PDK4 mRNA is commonly down-regulated in tumors compared with their tissues of origin. These results identify a novel mechanism by which ECM attachment, growth factors, and oncogenes modulate the metabolic fate of glucose by controlling PDK4 expression and PDH flux to influence proliferation.

Flux through hepatic pyruvate carboxylase and phosphoenolpyruvate carboxykinase detected by hyperpolarized 13C magnetic resonance

Abstract: In the heart, detection of hyperpolarized [13C]bicarbonate and 13CO2 by magnetic resonance (MR) after administration of hyperpolarized [1-13C]pyruvate is caused exclusively by oxidative decarboxylation of pyruvate via the pyruvate dehydrogenase complex (PDH). However, liver mitochondria possess alternative anabolic pathways accessible by [1-13C]pyruvate, which may allow a wider diagnostic range for hyperpolarized MR compared with other tissue. Metabolism of hyperpolarized [1-13C]pyruvate in the tricarboxylic acid (TCA) cycle was monitored in the isolated perfused liver from fed and fasted mice. Hyperpolarized [1-13C]pyruvate was rapidly converted to [1-13C]lactate, [1-13C]alanine, [1-13C]malate, [4-13C]malate, [1-13C]aspartate, [4-13C]aspartate, and [13C]bicarbonate. Livers from fasted animals had increased lactate:alanine, consistent with elevated NADH:NAD+. The appearance of asymmetrically enriched malate and aspartate indicated high rates of anaplerotic pyruvate carboxylase activity and incomplete equilibration with fumarate. Hyperpolarized [13C]bicarbonate was also detected, consistent with multiple mechanisms, including cataplerotic decarboxylation of [4-13C]oxaloacetate via phosphoenolpyruvate carboxykinase (PEPCK), forward TCA cycle flux of [4-13C]oxaloacetate to generate 13CO2 at isocitrate dehydrogenase, or decarboxylation of [1-13C]pyruvate by PDH. Isotopomer analysis of liver glutamate confirmed that anaplerosis was sevenfold greater than flux through PDH. In addition, signal from [4-13C]malate and [4-13C]aspartate was markedly blunted and signal from [13C]bicarbonate was completely abolished in livers from PEPCK KO mice, indicating that the major pathway for entry of hyperpolarized [1-13C]pyruvate into the hepatic TCA cycle is via pyruvate carboxylase, and that cataplerotic flux through PEPCK is the primary source of [13C]bicarbonate. We conclude that MR detection of hyperpolarized TCA intermediates and bicarbonate is diagnostic of pyruvate carboxylase and PEPCK flux in the liver.

Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa

Abstract: background: There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. methods: Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of 13 C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. results: The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed 13 C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. conclusions: Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energydemanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD + through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

Butyrate elicits a metabolic switch in human colon cancer cells by targeting the pyruvate dehydrogenase complex.

Abstract: Butyrate, a short-chain fatty acid produced by the colonic bacterial fermentation is able to induce cell growth inhibition and differentiation in colon cancer cells at least partially through its capacity to inhibit histone deacetylases. Since butyrate is expected to impact cellular metabolic pathways in colon cancer cells, we hypothesize that it could exert its antiproliferative properties by altering cellular metabolism. We show that although Caco2 colon cancer cells oxidized both butyrate and glucose into CO(2) , they displayed a higher oxidation rate with butyrate as substrate than with glucose. Furthermore, butyrate pretreatment led to an increase cell capacity to oxidize butyrate and a decreased capacity to oxidize glucose, suggesting that colon cancer cells, which are initially highly glycolytic, can switch to a butyrate utilizing phenotype, and preferentially oxidize butyrate instead of glucose as energy source to produce acetyl coA. Butyrate pretreated cells displayed a modulation of glutamine metabolism characterized by an increased incorporation of carbons derived from glutamine into lipids and a reduced lactate production. The butyrate-stimulated glutamine utilization is linked to pyruvate dehydrogenase complex since dichloroacetate reverses this effect. Furthermore, butyrate positively regulates gene expression of pyruvate dehydrogenase kinases and this effect involves a hyperacetylation of histones at PDK4 gene promoter level. Our data suggest that butyrate exerts two distinct effects to ensure the regulation of glutamine metabolism: it provides acetyl coA needed for fatty acid synthesis, and it also plays a role in the control of the expression of genes involved in glucose utilization leading to the inactivation of PDC.

Overexpression of Pyruvate Dehydrogenase Kinase 3 Increases Drug Resistance and Early Recurrence in Colon Cancer

Abstract: The switch of cellular metabolism from mitochondrial respiration to glycolysis is the hallmark of cancer cells and is associated with tumor malignancy. Pyruvate dehydrogenase kinase-1 (PDK1) and PDK3 participate in the metabolic switch of cancer cells; however, the medical significance of PDK1 and PDK3 in cancer progression is not known. Here, we assessed the expression profiles of PDK1 and PDK3 in colorectal cancer. Western blot analysis (n = 74) demonstrated that PDK3 was markedly increased in colon cancer compared to that in adjacent normal tissues, whereas PDK1 was decreased in cancer cells. In addition, PDK3 expression was positively correlated with that of hypoxia inducible factor-1α (HIF-1α) in cancer cells. Further analysis using immunohistochemical staining revealed that PDK3 levels were positively associated with severity of cancer and negatively associated with disease-free survival. In vitro studies using several colon cancer cell lines showed that PDK3 expression was controlled by HIF-1α and contributed to hypoxia-induced increased drug resistance, perhaps explaining why patients with PDK3 overexpression have a greater incidence of treatment failure. Taken together, our findings suggest that PDK3 plays an important role in the metabolic switch and drug resistance of colon cancer and is potentially a novel target for cancer therapy.

Role of Pyruvate Dehydrogenase Inhibition in the Development of Hypertrophy in the Hyperthyroid Rat Heart A Combined Magnetic Resonance Imaging and Hyperpolarized Magnetic Resonance Spectroscopy Study

Abstract: Background—Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy. Methods and Results—Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg · kg−1 · d−1). In vivo pyruvat...

Amyloid beta resistance in nerve cell lines is mediated by the Warburg effect.

Abstract: Amyloid beta (Aβ) peptide accumulation in the brains of patients with Alzheimer's disease (AD) is closely associated with increased nerve cell death. However, many cells survive and it is important to understand the mechanisms involved in this survival response. Recent studies have shown that an anti-apoptotic mechanism in cancer cells is mediated by aerobic glycolysis, also known as the Warburg effect. One of the major regulators of aerobic glycolysis is pyruvate dehydrogenase kinase (PDK), an enzyme which represses mitochondrial respiration and forces the cell to rely heavily on glycolysis, even in the presence of oxygen. Recent neuroimaging studies have shown that the spatial distribution of aerobic glycolysis in the brains of AD patients strongly correlates with Aβ deposition. Interestingly, clonal nerve cell lines selected for resistance to Aβ exhibit increased glycolysis as a result of activation of the transcription factor hypoxia inducible factor 1. Here we show that Aβ resistant nerve cell lines upregulate Warburg effect enzymes in a manner reminiscent of cancer cells. In particular, Aβ resistant nerve cell lines showed elevated PDK1 expression in addition to an increase in lactate dehydrogenase A (LDHA) activity and lactate production when compared to control cells. In addition, mitochondrial derived reactive oxygen species (ROS) were markedly diminished in resistant but not sensitive cells. Chemically or genetically inhibiting LDHA or PDK1 re-sensitized resistant cells to Aβ toxicity. These findings suggest that the Warburg effect may contribute to apoptotic-resistance mechanisms in the surviving neurons of the AD brain. Loss of the adaptive advantage afforded by aerobic glycolysis may exacerbate the pathophysiological processes associated with AD.

Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility.

Abstract: Under conditions of reduced tissue oxygenation, hypoxia-inducible factor (HIF) controls many processes, including angiogenesis and cellular metabolism, and also influences cell proliferation and survival decisions. HIF is centrally involved in tumour growth in inherited diseases that give rise to renal cell carcinoma (RCC), such as Von Hippel–Lindau syndrome and tuberous sclerosis complex. In this study, we examined whether HIF is involved in tumour formation of RCC in Birt–Hogg–Dube syndrome. For this, we analysed a Birt–Hogg–Dube patient-derived renal tumour cell line (UOK257) that is devoid of the Birt–Hogg–Dube protein (BHD) and observed high levels of HIF activity. Knockdown of BHD expression also caused a threefold activation of HIF, which was not as a consequence of more HIF1α or HIF2α protein. Transcription of HIF target genes VEGF, BNIP3 and CCND1 was also increased. We found nuclear localization of HIF1α and increased expression of VEGF, BNIP3 and GLUT1 in a chromophobe carcinoma from a Birt–Hogg–Dube patient. Our data also reveal that UOK257 cells have high lactate dehydrogenase, pyruvate kinase and 3-hydroxyacyl-CoA dehydrogenase activity. We observed increased expression of pyruvate dehydrogenase kinase 1 (a HIF gene target), which in turn leads to increased phosphorylation and inhibition of pyruvate dehydrogenase. Together with increased protein levels of GLUT1, our data reveal that UOK257 cells favour glycolytic rather than lipid metabolism (a cancer phenomenon termed the ‘Warburg effect’). UOK257 cells also possessed a higher expression level of the L-lactate influx monocarboxylate transporter 1 and consequently utilized L-lactate as a metabolic fuel. As a result of their higher dependency on glycolysis, we were able to selectively inhibit the growth of these UOK257 cells by treatment with 2-deoxyglucose. This work suggests that targeting glycolytic metabolism may be used therapeutically to treat Birt–Hogg–Dube-associated renal lesions.

Role of SLC5A8, a plasma membrane transporter and a tumor suppressor, in the antitumor activity of dichloroacetate

Abstract: There has been growing interest among the public and scientists in dichloroacetate (DCA) as a potential anticancer drug. Credible evidence exists for the antitumor activity of this compound, but high concentrations are needed for significant therapeutic effect. Unfortunately, these high concentrations produce detrimental side effects involving the nervous system, thereby precluding its use for cancer treatment. The mechanistic basis of the compound's antitumor activity is its ability to activate the pyruvate dehydrogenase complex through inhibition of pyruvate dehydrogenase kinase. As the compound inhibits the kinase at micromolar concentrations, it is not known why therapeutically prohibitive high doses are needed for suppression of tumor growth. We hypothesized that lack of effective mechanisms for the entry of DCA into tumor cells may underlie this phenomenon. Here we show that SLC5A8 transports DCA very effectively with high affinity. This transporter is expressed in normal cells, but expression is silenced in tumor cells by epigenetic mechanisms. The lack of the transporter makes tumor cells resistant to the antitumor activity of DCA. However, if the transporter is expressed in tumor cells ectopically, the cells become sensitive to the drug at low concentrations. This is evident in breast cancer cells, colon cancer cells and prostate cancer cells. Normal cells, which constitutively express the transporter, are however not affected by the compound, indicating tumor cell-selective therapeutic activity. The mechanism of the compound's antitumor activity still remains its ability to inhibit pyruvate dehydrogenase kinase and force mitochondrial oxidation of pyruvate. As silencing of SLC5A8 in tumors involves DNA methylation and its expression can be induced by treatment with DNA methylation inhibitors, our findings suggest that combining DCA with a DNA methylation inhibitor would offer a means to reduce the doses of DCA to avoid detrimental effects associated with high doses but without compromising antitumor activity.

Hypermethylation of Hepatic Glucokinase and L-Type Pyruvate Kinase Promoters in High-Fat Diet–Induced Obese Rats

Abstract: Obesity-dependent insulin resistance and type 2 diabetes mellitus are closely associated with decreased glucose utilization and down-regulation of hepatic glycolytic enzymes expression Previously, we showed that DNA hypermethylation is involved in age-dependent susceptibility to hepatic insulin resistance and diabetes However, what we cannot distinguish is whether the age-related obesity contributes to DNA hypermethylation in those natural aging rats In the present study, we hypothesize that DNA methylation plays a crucial role in the regulation of glycolytic enzymes in the high-fat diet-induced obesity Here, we report that DNA hypermethylation is correlated with a decline in hepatic glucokinase (Gck) and L-type pyruvate kinase (LPK) expression in high-fat diet-induced obese rats as compared with the control diet group Down-regulation of Gck and LPK expression are reversed by the 5-aza-2'-deoxycytidine in the cell model of steatosis These novel observations indicate that DNA methylation is involved in the development of metabolic diseases, such as obesity, insulin resistance, type 2 diabetes mellitus, and nonalcoholic steatohepatitis, suggesting that the hypermethylation level of Gck and LPK promoters may be a useful parameter for the evaluation of obesity-induced insulin resistance and fatty liver

Pyruvate dehydrogenase deficiency and epilepsy.

Abstract: The pyruvate dehydrogenase complex (PDHc) is a mitochondrial matrix multienzyme complex that provides the link between glycolysis and the tricarboxylic acid (TCA) cycle by catalyzing the conversion of pyruvate into acetyl-CoA. PDHc deficiency is one of the commoner metabolic disorders of lactic acidosis presenting with neurological phenotypes that vary with age and gender. In this mini-review, we postulate mechanisms of epilepsy in the setting of PDHc deficiency using two illustrative cases (one with pyruvate dehydrogenase complex E1-alpha polypeptide (PDHA1) deficiency and the second one with pyruvate dehydrogenase complex E1-beta subunit (PDHB) deficiency (a rare subtype of PDHc deficiency)) and a selected review of published case series. PDHc plays a critical role in the pathway of carbohydrate metabolism and energy production. In severe deficiency states the resulting energy deficit impacts on brain development in utero resulting in structural brain anomalies and epilepsy. Milder deficiency states present with variable manifestations that include cognitive delay, ataxia, and seizures. Epileptogenesis in PDHc deficiency is linked to energy failure, development of structural brain anomalies and abnormal neurotransmitter metabolism. The use of the ketogenic diet bypasses the metabolic block, by providing a direct source of acetyl-CoA, leading to amelioration of some symptoms. Genetic counseling is essential as PDHA1 deficiency (commonest defect) is X-linked although females can be affected due to unfavorable lyonization, while PDHB and PDH phosphatase (PDP) deficiencies (much rarer defects) are of autosomal recessive inheritance. Research is in progress for looking into animal models to better understand pathogenesis and management of this challenging disorder.

The pyruvate carboxylase-pyruvate dehydrogenase axis in islet pyruvate metabolism: Going round in circles?

Abstract: Pyruvate is the major product of glycolysis in pancreatic β-cells, and its ultimate metabolic fate depends on the relative activities of two enzymes. The first, pyruvate carboxylase (PC) replenishes oxaloacetate withdrawn from the tricarboxylic acid (TCA) cycle via the carboxylation of pyruvate to form oxaloacetate. Flux via PC is also involved in the formation of NADPH, one of several important coupling factors for insulin secretion. In most tissues, PC activity is enhanced by increased acetyl-CoA. The alternative fate of pyruvate is its oxidative decarboxylation to form acetyl-CoA via the pyruvate dehydrogenase complex (PDC). The ultimate fate of acetyl-CoA carbon is oxidation to CO2 via the TCA cycle, and so the PDC reaction results of the irreversible loss of glucose-derived carbon. Thus, PDC activity is stringently regulated. The mechanisms controlling PDC activity include end-product inhibition by increased acetyl-CoA, NADH and ATP, and its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDHKs 1-4). Here we review new developments in the regulation of the activities and expression of PC, PDC and the PDHKs in the pancreatic islet in relation to islet pyruvate disposition and glucose-stimulated insulin secretion (GSIS).

Mitochondrion as a novel site of dichloroacetate biotransformation by glutathione transferase ζ1

Abstract: Dichloroacetate (DCA) is a potential environmental hazard and an investigational drug. Repeated doses of DCA result in reduced drug clearance, probably through inhibition of glutathione transferase ζ1 (GSTZ1), a cytosolic enzyme that converts DCA to glyoxylate. DCA is known to be taken up by mitochondria, where it inhibits pyruvate dehydrogenase kinase, its major pharmacodynamic target. We tested the hypothesis that the mitochondrion was also a site of DCA biotransformation. Immunoreactive GSTZ1 was detected in liver mitochondria from humans and rats, and its identity was confirmed by liquid chromatography/tandem mass spectrometry analysis of the tryptic peptides. Study of rat submitochondrial fractions revealed GSTZ1 to be localized in the mitochondrial matrix. The specific activity of GSTZ1-catalyzed dechlorination of DCA was 2.5- to 3-fold higher in cytosol than in whole mitochondria and was directly proportional to GSTZ1 protein expression in the two compartments. Rat mitochondrial GSTZ1 had a 2.5-fold higher AppKm for glutathione than cytosolic GSTZ1, whereas the AppKm values for DCA were identical. Rats administered DCA at a dose of 500 mg/kg/day for 8 weeks showed reduced hepatic GSTZ1 activity and expression of ∼10% of control levels in both cytosol and mitochondria. We conclude that the mitochondrion is a novel site of DCA biotransformation catalyzed by GSTZ1, an enzyme colocalized in cytosol and mitochondrial matrix.

Pyruvate promotes tumor angiogenesis through HIF-1-dependent PAI-1 expression

Abstract: Cancer cells usually obtain energy from a high rate of glycolysis rather than oxidative phosphorylation under normoxia as well as hypoxia. Under these circumstances, pyruvate, the end-product of glycolysis, accumulates in cancer cells. We have previously reported that pyruvate activates endothelial cells and induces angiogenesis. Here, we examined the angiogenic activity of pyruvate in tumor cells. Plasminogen activator inhibitor-1 (PAI-1), the gene most upregulated by pyruvate, showed a pro-angiogenic activity, which was abolished by a PAI-1 neutralizing antibody. Moreover, stabilization of hypoxia-inducible factor-1α (HIF-1α) by pyruvate was required for induction of PAI-1 transcription through direct binding to hypoxia response element-2 (HRE-2) on the promoter. These results suggest that pyruvate can activate the angiogenic activity of cancer cells under normoxia and that PAI-1 may act as a pro-angiogenic factor in pyruvate-induced angiogenesis.

The oxygen sensor PHD3 limits glycolysis under hypoxia via direct binding to pyruvate kinase.

Abstract: The oxygen sensor PHD3 limits glycolysis under hypoxia via direct binding to pyruvate kinase

Decreasing lactate level and increasing polypeptide production by downregulating the expression of lactate dehydrogenase and pyruvate dehydrogenase kinase

Abstract: The present invention provides methods and compositions for reducing lactate production and increasing polypeptide production in cultured cells. In one aspect, the invention provides a method comprising culturing cells expressing a) a small interfering RNA (siRNA) specific for a lactate dehydrogenase (LDH) and b) an siRNA specific for a pyruvate dehydrogenase kinase (PDHK). In another aspect, the invention provides cultured cells or vectors comprising an siRNA specific for a LDH and an siRNA specific for a PDHK.

Determining the in vivo regulation of cardiac pyruvate dehydrogenase based on label flux from hyperpolarised [1‐13C]pyruvate

Abstract: Pyruvate dehydrogenase (PDH) is a key regulator of cardiac substrate selection and is regulated by both pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation and feedback inhibition. The extent to which chronic upregulation of PDK protein levels, acutely increased PDK activity and acute feedback inhibition limit PDH flux remains unclear because existing in vitro assessment methods inherently disrupt the regulation of the enzyme complex. We have demonstrated previously that hyperpolarised 13C-labelled metabolic tracers coupled with MRS can monitor flux through PDH in vivo. The aim of this study was to determine the relative contributions of acute and chronic changes in PDK and PDH activities to in vivo myocardial PDH flux. We examined both fed and fasted rats with either hyperpolarised [1-13C]pyruvate alone or hyperpolarised [1-13C]pyruvate co-infused with malate [to modulate mitochondrial nicotinamide adenine dinucleotide (NADH/NAD+) and acetyl-coenzyme A (acetyl-CoA)/CoA ratios, which alter both PDH activity and flux]. To confirm the metabolic fate of infused malate, we performed in vitro1H NMR spectroscopy on cardiac tissue extracts. We observed that, in fed rats, where PDH activity was high, the presence of malate increased PDH flux by 27%, whereas, in the fasted state, malate infusion had no effect on PDH flux. These observations suggest that pyruvate oxidation is limited by feedback inhibition from acetyl-CoA only when PDH activity is high. Therefore, in the case of PDH, and potentially other enzymes, hyperpolarised 13C MRI can be used to assess noninvasively enzymatic regulation. Copyright © 2011 John Wiley & Sons, Ltd.

PDH activation during in vitro muscle contractions in PDH kinase 2 knockout mice: effect of PDH kinase 1 compensation

Abstract: Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate oxidation in skeletal muscle. PDH is deactivated by a set of PDH kinases (PDK1, PDK2, PDK3, PDK4), with PDK2 and PDK4...