Pyruvate Dehydrogenase Enzyme Complex Research Articles

The Role of Structural Order in the Mechanism of Charge Transport across Tunnel Junctions with Various Iron‐Storing Proteins

In biomolecular electronics, the role of structural order in charge transport (CT) is poorly understood. It has been reported that the metal oxide cores of protein cages (e.g., iron oxide and ferrihydrite nanoparticles (NPs) present in ferritin and E2-LFtn, which is E2 protein engineered with an iron-binding sequence) play an important role in the mechanism of CT. At the same time, the NP core also plays a major role in the structural integrity of the proteins. This paper describes the role of structural order in CT across tunnel junctions by comparing three iron-storing proteins. They are (1) DNA binding protein from starved cells (Dps, diameter (∅) = 9 nm); (2) engineered archaeal ferritin (AfFtn-AA, ∅= 12 nm); and (3) engineered E2 of pyruvate dehydrogenase enzyme complex (E2-LFtn, ∅= 25 nm). Both holo-Dps and apo-Dps proteins undergo CT by coherent tunneling because their globular architecture and relative structural stability provide a coherent conduction pathway. In contrast, apo-AfFtn-AA forms a disordered structure across which charges have to tunnel incoherently, but holo-AfFtn-AA retains its globular structure and supports coherent tunneling. The large E2-LFtn always forms disordered structures across which charges incoherently tunnel regardless of the presence of the NP core. These findings highlight the importance of structural order in the mechanism of CT across biomolecular tunnel junctions.

Abstract PS17-50: Nuclear expression of acetyl-CoA producing enzymes and their roles in epigenetic reprogramming in breast cancer cells

Abstract Metabolic and epigenetic reprogramming are two key hallmarks of cancer. Rapidly proliferating cancer cells often exhibit a higher and differentially reprogrammed transcriptional activity than normal cells of the same tissue. To support transcriptional reprogramming and increased transcriptional load, sustained chromatin acetylation is required to loosen the chromatin and enable RNA transcription. Histone acetylation also requires a constant supply of acetyl-CoA, a labile, membrane impermeable metabolite that is the obligatory donor of the acetyl groups for histone acetylation. However, the pathways responsible for increased synthesis of acetyl-CoA inside the nucleus remain largely elusive. Using estrogen-responsive (MCF7) and their matched endocrine therapy resistant breast cancer cells (LCC9 and MCF7:5C), we show that blocking p300-mediated acetyltransferase activity is crucial for proliferation of endocrine therapy-resistant breast cancer cells. Furthermore, we found nuclear expression of two acetyl-CoA producing enzymes, i.e., pyruvate dehydrogenase complex (PDC), and ATP citrate lyase (ACLY), which implies their functional role inside the nucleus. PDC is a multimeric protein complex enzyme made up of five different subunits. Canonically, PDC is found in mitochondria and is responsible for the conversion of pyruvate to acetyl-CoA, linking glycolysis to the tricarboxylic acid (TCA) cycle. Our results show that multiple PDC enzyme subunits, e.g., pyruvate dehydrogenase (PDH) E1 alpha (E1 alpha), dihydrolipoal dehydrogenase (DLD), and PDHX (also known as E3BP) are expressed in the nucleus of MCF7, LCC9 and MCF7:5C cells. Moreover, expression of nuclear DLD and PDHX subunit is higher in endocrine resistant cells (LCC9 and MCF7:5C) than in their parental MCF7 cells, strongly suggesting a possible role in endocrine resistance. Notably, high expression of PDHX is correlated with poor relapse-free survival in estrogen-receptor positive breast cancer patients in four independent publicly available datasets. ACLY is a tetramer of identical subunits catalyzes conversion of citric acid and co-enzyme A to acetyl-CoA. ACLY is also overexpressed in the nucleus of endocrine-resistant breast cancer cells (LCC9 and MCF7:5C) when compared with their endocrine sensitive MCF7 controls. This study investigates the role of nuclear PDC and ACLY mediated acetyl-CoA synthesis and its impact on histone acetylation enabling reprogrammed transcriptional activity supporting proliferation of endocrine therapy-resistant breast cancer cells. Citation Format: Surojeet Sengupta, Shuait Nair, Lu Jin, Catherine M Sevigny, Brandon Jones, Robert Clarke. Nuclear expression of acetyl-CoA producing enzymes and their roles in epigenetic reprogramming in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-50.

Regulation of pyruvate dehydrogenase complex related to lactate switch in CHO cells.

The metabolism of Chinese hamster ovary (CHO) cell lines is typically characterized by high rates of aerobic glycolysis with increased lactate formation, known as the ”Warburg” effect. Although this metabolic state can switch to lactate consumption, the involved regulations of the central metabolism have only been partially studied so far. An important reaction transferring the lactate precursor, pyruvate, into the tricarboxylic acid cycle is the decarboxylation reaction catalyzed by the pyruvate dehydrogenase enzyme complex (PDC). Among other mechanisms, PDC is mainly regulated by phosphorylation–dephosphorylation at the three sites Ser232, Ser293, and Ser300. In this work, the PDC phosphorylation in antibody‐producing CHO DP‐12 cell culture is investigated during the lactate switch. Batch cultivations were carried out with frequent sampling (every 6 h) during the transition from lactate formation to lactate uptake, and the PDC phosphorylation levels were quantified using a novel indirect flow cytometry protocol. Contrary to the expected activation of PDC (i.e., reduced PDC phosphorylation) during lactate consumption, Ser293 and Ser300 phosphorylation levels were 33% higher compared to the phase of glucose excess. At the same time, the relative phosphorylation level of Ser232 increased steadily throughout the cultivation (66% increase overall). The intracellular pyruvate was found to accumulate only during the period of high lactate production, while acetyl‐CoA showed nearly no accumulation. These results indicate a deactivation of PDC and reduced oxidative metabolism during lactate switch even though the cells undergo a metabolic transition to lactate‐based cell growth and metabolism. Overall, this study provides a unique view on the regulation of PDC during the lactate switch, which contributes to an improved understanding of PDC and its interaction with the bioprocess.

Emerging regulatory roles of mitochondrial sirtuins on pyruvate dehydrogenase complex and the related metabolic diseases: Review

The pyruvate dehydrogenase complex (PDC) is a multi-enzyme complex of the mitochondria that provides a link between glycolysis and the Krebs cycle. PDC plays an essential role in producing acetyl-CoA from glucose and the regulation of fuel consumption. In general, PDC enzyme is regulated in two different ways, end-product inhibition and posttranslational modifications (more extensive phosphorylation and dephosphorylation subunit E1). Posttranslational modifications of this enzyme are regulated by various factors. Sirtuins are the class III of histone deacylatases that catalyze protein posttranslational modifications, including deacetylation, adenosine diphosphate ribosylation, and deacylation. Sirt3, Sirt4, and Sirt5 are mitochondrial sirtuins that control the posttranslational modifications of mitochondrial protein. Considering the comprehensive role of sirtuins in post-translational modifications and regulation of metabolic processes, the aim of this review is to explore the role of mitochondrial sirtuins in the regulation of the PDC. PDC deficiency is a common metabolic disorder that causes pyruvate to be converted to lactate and alanine rather than to acetyl-CoA. because this enzyme is in the gateway of complete oxidation, glucose products entering the Krebs cycle and resulting in physiological and structural changes in the organs. Metabolic blockage such as ketogenic diet broken up by b -oxidation and producing acetyl-CoA can improve the patients. Sirtuins play a role in the production of acetyl-CoA through oxidation of fatty acids and other pathways. Thus, we hypothesize that the targets and bioactive compounds targeting mitochondrial sirtuins can be involved in the treatment of PDC deficiency. In general, this review discusses the present knowledge on how mitochondrial sirtuins are involved in the regulation of PDC as well as their possible roles in the treatment of PDC deficiency.

Toward Multiscale Modeling of Proteins and Bioagglomerates: An Orientation-Sensitive Diffusion Model for the Integration of Molecular Dynamics and the Discrete Element Method.

Most processes involved in biological and biotechnological systems spread over many scales in space and time. For example, the interaction of multiple enzymes in heterogeneous enzymatic agglomerates or clusters, necessary for efficient enzymatic conversion, is of high interest for research and enzyme engineering. In order to understand and predict their overall behavior and performance, it is important to describe these scales as completely as possible, known as multiscale modeling. While many different approaches have been presented in recent years, knowledge about protein formation and bioagglomeration at the micro scale is still very limited. In an attempt to address such systems, we propose a bottom- up multiscale modeling methodology, bridging the gaps between molecular dynamics (MD) with an explicit solvent and the larger scale discrete element method (DEM) using an implicit solvent and abstracting macromolecules (e.g., proteins) as objects with anisotropic properties. We term this approach the molecular discrete element method (MDEM). For this, we present an orientation-sensitive diffusion model for DEM, which describes the dynamics of anisotropic translational and rotational diffusion, while implicitly considering solvent molecules and enforcing a canonical ensemble. A general-purpose model and parametrization approach is presented, which can be used to simulate any process involving diffusion of discrete particles. Effects of temperature and viscosity changes can be considered, and guidance is provided concerning time step selection. This model is generally applicable and serves as a precondition to enforce the proper dynamics (i.e., diffusion characteristics and canonical ensemble, similar to a thermostat in MD) for the proposed multiscale modeling methodology with anisotropic properties. Thereby, it presents a first step toward modeling at the micro scale and is integral to enforcing dynamics of such systems and therefore extensively validated. As a next step, interaction models are to be defined and added to the presented model. In comparison to atomistic and coarse-grained (CG) MD, a speedup of 5-7 orders of magnitude can be achieved. The approach is demonstrated on multiple components of the pyruvate dehydrogenase enzyme complex, a multienzymatic machinery that involves very different types of enzymes and is of high value to further elucidate the mechanisms of bioagglomeration and metabolic channeling.

MiR‑21‑5p targets PDHA1 to regulate glycolysis and cancer progression in gastric cancer

Pyruvate dehydrogenaseA1 (PDHA1) is a component of the pyruvate dehydrogenase enzyme complex, which links glycolysis and the tricarboxylic acid cycle, and is important for cancer metabolism shift. PDHA1 downregulation has been revealed in several types of cancer to enhance glycolysis. However, the role of PDHA1 in gastric cancer remains largely unknown. In the present study, we found that PDHA1 was significantly downregulated in gastric cancer, and associated with poor prognosis. PDHA1 downregulation promoted gastric cancer glycolysis and cancer progression. miR‑21‑5p directly targeted PDHA1 to suppress PDHA1 expression, and promote glycolysis as well as cell proliferation in gastric cancer. Moreover, miR‑21‑5p was significantly upregulated in gastric cancer and negatively associated with PDHA1 expression in gastric cancer samples. Our results indicated that miR‑21‑5p targeted PDHA1 to regulate a metabolic switch and cancer progression in gastric cancer, and reveal the potential role of the miR‑21‑5p/PDHA1 axis in gastric cancer treatment.

PO-197 A possible link between the mitochondrial gatekeeper pyruvate dehydrogenase enzyme complex and EMT

Introduction In recent years, mitochondria have attracted much attention in metastatic cancer research. The first finding of the importance of mitochondria in cancer was reported by Otto Warburg in 1920. In the following years,it was found that cancer is closely related to mitochondrial characteristics such as glucose metabolism and chemoresistance. In this context,epithelial mesenchymal transition (EMT), due to its potential of being a molecular marker for tumour metastasis, clarification of this process is essential. Although increments have been made in metastatic cancer research,there are no studies showing the relationship between pyruvate dehydrogenase enzyme complex (PDH) and EMT. Material and methods To inhibit PDH activity, pharmacological (PDH inhibitor, Cpi-613) and genomic approaches (stable cell lines were established by lentivirus) were used in A549 cells. The antiproliferative effect of Cpi-613 was investigated by xCELLigence System, SRB and ATP viability assays. After determining anti-proliferative doses of Cpi-613 and/or getting monoclonal cell lines (shCtrl and shPDHA1); wound healing, invasion and drug sensitivity tests were also applied. To demonstrate the EMT phenotype in cells, EMT-related protein expressions were analysed via western blotting in cell fractionations. Genes that regulate the activity of PDH, were also checked by qRt-PCR. Cell cycle specifity of Cpi-613 was also investigated. Glycolysis and/or survival-related proteins and cancer stem cell characteristics were also determined.SB431542 (TGF-βRI inhibitor) was also used to determine Cpi-613 induced EMT depends on TGF-β signalling. Results and discussions Inhibition of PDH by Cpi-613 and PDHA1 knockdown induced morphological changes which are characteristics of EMT. A more rapid wound healing, increased invasive potential and cemoresistance were also shown. The effect of Cpi-613 was non-specific for cell cycle. The possible inactivating kinase of PDH was found to be PDK4. SB431542 treatment also reversed the EMT phenotype. In shPDHA1 cells the expressions of GLUT1; MDR1 and MRP1; AKT, PI3K, MAPK; and OCT-4 proteins found to be increased. The proportion of CD133 + cells were also higher in shPDHA1 cells than those of shCtrl cells. Conclusion Knockdown of PDHA1 expression or inhibition of PDH activity induced the EMT phenotype and more importantly resulted in resistance to anticancer drugs. The induction of PDH enzyme activity could be a novel approach for the development of anti-metastatic cancer drugs.

Enzymatic testing sensitivity, variability and practical diagnostic algorithm for pyruvate dehydrogenase complex (PDC) deficiency.

Pyruvate dehydrogenase complex (PDC) deficiency is a major cause of primary lactic acidemia in children. Prompt and correct diagnosis of PDC deficiency and differentiating between specific vs generalized, or secondary deficiencies has important implications for clinical management and therapeutic interventions. Both genetic and enzymatic testing approaches are being used in the diagnosis of PDC deficiency. However, the diagnostic efficacy of such testing approaches for individuals affected with PDC deficiency has not been systematically investigated in this disorder. We sought to evaluate the diagnostic sensitivity and variability of the various PDC enzyme assays in females and males at the Center for Inherited Disorders of Energy Metabolism (CIDEM). CIDEM data were filtered by lactic acidosis and functional PDC deficiency in at least one cell/tissue type (blood lymphocytes, cultured fibroblasts or skeletal muscle) identifying 186 subjects (51% male and 49% female), about half were genetically resolved with 78% of those determined to have a pathogenic PDHA1 mutation. Assaying PDC in cultured fibroblasts in cases where the underlying genetic etiology is PDHA1, was highly sensitive irrespective of gender; 97% (95% confidence interval [CI]: 90%-100%) and 91% (95% CI: 82%-100%) in females and males, respectively. In contrast to the fibroblast-based testing, the lymphocyte- and muscle-based testing were not sensitive (36% [95% CI: 11%-61%, p=0.0003] and 58% [95% CI: 30%-86%, p=0.014], respectively) for identifying known PDC deficient females with pathogenic PDHA1 mutations. In males with a known PDHA1 mutation, the sensitivity of the various cell/tissue assays (75% lymphocyte, 91% fibroblast and 88% muscle) were not statistically different, and the discordance frequency due to the specific cell/tissue used for assaying PDC was 0.15±0.11. Based on this data, a practical diagnostic algorithm is proposed accounting for current molecular approaches, enzyme testing sensitivity, and variability due to gender, cell/tissue type used for testing, and successive repeat testing.

Metabolic engineering of Escherichia coli for 1,3-butanediol biosynthesis through the inverted fatty acid β-oxidation cycle

The feasibility of 1,3-butanediol biosynthesis through the inverted cycle of fatty acid β-oxidation in Escherichia coli cells was investigated by the rational metabolic engineering approach. CoA-dependent aldehyde dehydrogenase MhpF and alcohol dehydrogenases FucO and YqhD were used as terminal enzymes catalyzing conversion of 3-hydroxybutyryl-CoA to 1,3-butanediol. Constitutive expression of the corresponding genes in E. coli strains, which are deficient in mixed acid fermentation pathways and expressing fad regulon genes under control of P trc-ideal-4 promoter, did not lead to the synthesis of 1,3-butanediol during anaerobic glucose utilization. Additional inactivation of fadE and ydiO genes, encoding acyl-CoA dehydrogenases, also did not cause synthesis of the target product. Constitutive expression of aceEF-lpdA operon genes encoding enzymes of pyruvate dehydrogenase complex led to an increase in anaerobic synthesis of ethanol. Synthesis of 1,3-butanediol was observed with the overexpression of acetyl-CoA C-acetyltransferase AtoB. Constitutive expression of atoB gene in a strain with a basal expression of alcohol/aldehyde dehydrogenase leads to synthesis of 0.3 mM of 1,3-butanediol.

Enhancing the [13C]bicarbonate signal in cardiac hyperpolarized [1‐13C]pyruvate MRS studies by infusion of glucose, insulin and potassium

A change in myocardial metabolism is a known effect of several diseases. MRS with hyperpolarized (13)C-labelled pyruvate is a technique capable of detecting changes in myocardial pyruvate metabolism, and has proven to be useful for the evaluation of myocardial ischaemia in vivo. However, during fasting, the myocardial glucose oxidation is low and the fatty acid oxidation (β-oxidation) is high, which complicates the interpretation of pyruvate metabolism with the technique. The aim of this study was to investigate whether the infusion of glucose, insulin and potassium (GIK) could increase the myocardial glucose oxidation in the citric acid cycle, reflected as an increase in the [(13)C]bicarbonate signal in cardiac hyperpolarized [1-(13)C]pyruvate MRS measurements in fasted rats. Two groups of rats were infused with two different doses of GIK and investigated by MRS after injection of hyperpolarized [1-(13)C]pyruvate. No [(13)C]bicarbonate signal could be detected in the fasted state. However, a significant increase in the [(13)C]bicarbonate signal was observed by the infusion of a high dose of GIK. This study demonstrates that a high [(13)C]bicarbonate signal can be achieved by GIK infusion in fasted rats. The increased [(13)C]bicarbonate signal indicates an increased flux of pyruvate through the pyruvate dehydrogenase enzyme complex and an increase in myocardial glucose oxidation through the citric acid cycle.

Phenylbutyrate Therapy for Pyruvate Dehydrogenase Complex Deficiency and Lactic Acidosis

Lactic acidosis is a buildup of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1α subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57BL/6 wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1α in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1α through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noa(m631) zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis.

Comparative13C Metabolic Flux Analysis of Pyruvate Dehydrogenase Complex-Deficient,l-Valine-Producing Corynebacterium glutamicum

L-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by (13)C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an L-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated for L-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP.

Redesigning Escherichia coli Metabolism for Anaerobic Production of Isobutanol

Fermentation enables the production of reduced metabolites, such as the biofuels ethanol and butanol, from fermentable sugars. This work demonstrates a general approach for designing and constructing a production host that uses a heterologous pathway as an obligately fermentative pathway to produce reduced metabolites, specifically, the biofuel isobutanol. Elementary mode analysis was applied to design an Escherichia coli strain optimized for isobutanol production under strictly anaerobic conditions. The central metabolism of E. coli was decomposed into 38,219 functional, unique, and elementary modes (EMs). The model predictions revealed that during anaerobic growth E. coli cannot produce isobutanol as the sole fermentative product. By deleting 7 chromosomal genes, the total 38,219 EMs were constrained to 12 EMs, 6 of which can produce high yields of isobutanol in a range from 0.29 to 0.41 g isobutanol/g glucose under anaerobic conditions. The remaining 6 EMs rely primarily on the pyruvate dehydrogenase enzyme complex (PDHC) and are typically inhibited under anaerobic conditions. The redesigned E. coli strain was constrained to employ the anaerobic isobutanol pathways through deletion of 7 chromosomal genes, addition of 2 heterologous genes, and overexpression of 5 genes. Here we present the design, construction, and characterization of an isobutanol-producing E. coli strain to illustrate the approach. The model predictions are evaluated in relation to experimental data and strategies proposed to improve anaerobic isobutanol production. We also show that the endogenous alcohol/aldehyde dehydrogenase AdhE is the key enzyme responsible for the production of isobutanol and ethanol under anaerobic conditions. The glycolytic flux can be controlled to regulate the ratio of isobutanol to ethanol production.

An overview of a cohort of South African patients with mitochondrial disorders

Mitochondrial disorders are frequently encountered inherited diseases characterized by unexplained multisystem involvement with a chronic, intermittent, or progressive nature. The objective of this paper is to describe the profile of patients with mitochondrial disorders in South Africa. Patients with possible mitochondrial disorders were accessed over 10 years. Analyses for respiratory chain and pyruvate dehydrogenase complex enzymes were performed on muscle. A diagnosis of a mitochondrial disorder was accepted only if an enzyme activity was deficient. Sixty-three patients were diagnosed with a mitochondrial disorder, including 40 African, 20 Caucasian, one mixed ancestry, and two Indian patients. The most important findings were the difference between African patients and other ethnicities: respiratory chain enzyme complexes CI+III or CII+III deficiencies were found in 52.5% of African patients, being of statistical significance (p value = 0.0061). They also presented predominantly with myopathy (p value = 0.0018); the male:female ratio was 1:1.2. Twenty-five (62.5%) African patients presented with varying degrees of a myopathy accompanied by a myopathic face, high palate, and scoliosis. Fourteen of these 25 also had ptosis and/or progressive external ophthalmoplegia. No patients of other ethnicities presented with this specific myopathic phenotype. Caucasian patients (16/20) presented predominantly with central nervous system involvement. Of the South African pediatric neurology patients, Africans are more likely to present with myopathy and CII+III deficiency, and Caucasian patients are more likely to present with encephalopathy or encephalomyopathy.

Plasmodium pyruvate dehydrogenase activity is only essential for the parasite's progression from liver infection to blood infection

Plasmodium parasites possess a single pyruvate dehydrogenase (PDH) enzyme complex that is localized to the plastid-like organelle known as the apicoplast. Unlike most eukaryotes, Plasmodium parasites lack a mitochondrial PDH. The PDH complex catalyses the conversion of pyruvate to acetyl-CoA, an important precursor for the tricarboxylic acid cycle and type II fatty acid synthesis (FAS II). In this study, using a rodent malaria model, we show that the PDH E1 alpha and E3 subunits colocalize with the FAS II enzyme FabI in the apicoplast of liver stages but are not significantly expressed in blood stages. Deletion of the E1 alpha or E3 subunit genes of Plasmodium yoelii PDH caused no defect in blood stage development, mosquito stage development or early liver stage development. However, the gene deletions completely blocked the ability of the e1 alpha(-) and e3(-) parasites to form exo-erythrocytic merozoites during late liver stage development, thus preventing the initiation of a blood stage infection. This phenotype is similar to that observed for deletions of genes involved in FAS II elongation. The data strongly support the hypothesis that the sole role of PDH is to provide acetyl-CoA for FAS II.

Potential Dysregulation of the Pyruvate Dehydrogenase Complex by Bacterial Toxins and Insulin

The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl CoA, effectively controlling the entrance of glycolysis products into aerobic metabolism. Because hyperlactatemia is one of the hallmarks of sepsis, we hyphothesized that gram-positive and negative bacterial toxin treatment will interfere with mRNA levels of regulatory enzymes of the PDC and overall enzyme activity in hepatocytes. HEP G2 hepatocarcinoma cells were incubated for 24 hours in the presence of lipopolysaccaride (LPS) or lipoteichoic acid. Total RNA was then isolated and message RNA levels for both pyruvate dehydrogense kinase 4 and phosphatase 2 were determined by RTPCR. Amplified DNA fragments were visualized by ethidium bromide in agarose gels and densitometry of the bands was performed. Data were then normalized to the housekeeping gene, GAPDH. Enzyme activity was then determined by capturing intact PDC on nitrocellulose membranes then determining PDC-dependent production of NADH. LPS treatment led to a time dependent increase in PDK4 message while decreasing PDP2 levels. Enzyme activity, in these cells, also significantly decreased 24 hours after exposure to LPS. Cells cultured in the presence of lipoteichoic acid and insulin exhibited differing message ratios and activity levels when evaluated at 4 hours, but at 24 hours shifted to mimic those observed in LPS treated cells. This data may indicate that exposure to bacterial cell wall components and insulin could create cellular environments that result in a build-up of lactate.

The effect of hyperpolarized tracer concentration on myocardial uptake and metabolism

Hyperpolarized (13)C-labeled substrates directly provide a source of magnetic resonance (MR) signal to observe the substrates' real-time uptake and enzymatic conversion. The aim of this study was to optimize the concentration of hyperpolarized [1-(13)C]pyruvate infused as a metabolic tracer, by observing the mitochondrial conversion of pyruvate to H(13)CO(3)(-) in heart tissue. Hyperpolarized pyruvate was infused into rats at concentrations between 20 mM and 80 mM and the relationships between [1-(13)C]lactate, [1-(13)C]alanine, and H(13)CO(3)(-) production and the infused pyruvate concentration were investigated. H(13)CO(3)(-) production reached saturation above 40 mM infused pyruvate concentration, indicating that hyperpolarized MR experiments performed at this concentration maximize the H(13)CO(3)(-) signal with minimal alterations to in vivo substrate composition. Additionally, the linear dependence of alanine production on pyruvate concentration confirmed that hyperpolarized MR methods in the heart reveal enzyme activity, rather than cellular uptake. H(13)CO(3)(-) production demonstrated evidence of sigmoidal enzyme kinetics, a reflection of the allosteric nature of the pyruvate dehydrogenase (PDH) enzyme complex. This protocol could be useful to optimize the infused concentration of other hyperpolarized metabolites in different organs, to ensure adequate MR signal with minimum metabolic perturbation.

In vivo assessment of pyruvate dehydrogenase flux in the heart using hyperpolarized carbon-13 magnetic resonance

The advent of hyperpolarized (13)C magnetic resonance (MR) has provided new potential for the real-time visualization of in vivo metabolic processes. The aim of this work was to use hyperpolarized [1-(13)C]pyruvate as a metabolic tracer to assess noninvasively the flux through the mitochondrial enzyme complex pyruvate dehydrogenase (PDH) in the rat heart, by measuring the production of bicarbonate (H(13)CO(3)(-)), a byproduct of the PDH-catalyzed conversion of [1-(13)C]pyruvate to acetyl-CoA. By noninvasively observing a 74% decrease in H(13)CO(3)(-) production in fasted rats compared with fed controls, we have demonstrated that hyperpolarized (13)C MR is sensitive to physiological perturbations in PDH flux. Further, we evaluated the ability of the hyperpolarized (13)C MR technique to monitor disease progression by examining PDH flux before and 5 days after streptozotocin induction of type 1 diabetes. We detected decreased H(13)CO(3)(-) production with the onset of diabetes that correlated with disease severity. These observations were supported by in vitro investigations of PDH activity as reported in the literature and provided evidence that flux through the PDH enzyme complex can be monitored noninvasively, in vivo, by using hyperpolarized (13)C MR.

Antibody binding and inhibition of pyruvate dehydrogenase (PDH) in sera from patients with primary biliary cirrhosis

Autoantibodies reactive against mitochondria which are present in sera from patients with primary biliary cirrhosis (PBC) have been shown to bind to the lipoamide acetyltransferase moiety (E2) of the pyruvate dehydrogenase enzyme complex (PDH). This newly described antigen has been shown to be identical to M2, a 70-kD antigen of the inner mitochondrial membrane. Sera from 10 patients with PBC, 11 patients with chronic active hepatitis (CAH), 20 healthy controls and patients with thyroiditis and systemic lupus erythematosus (SLE) were tested by ELISA for the presence of antibodies (IgG and IgM classes and IgG subclasses) reactive against PDH. The effect of serum and separated IgG and IgM on the PDH enzyme activity was measured spectrophotometrically. Nine out of 10 PBC sera were positive by immunofluorescence for mitochondria (M2 pattern). These nine sera reacted strongly with both IgG and IgM to PDH in the ELISA and also inhibited the enzyme activity by 80% (s.d. 25%). This was significantly different compared with the controls (4 +/- 6%; P less than 0.001). Enzyme inhibition was mainly caused by IgG. Of all control sera (from healthy and patient individuals) only one patient with CAH reacted significantly in the tests. This CAH patient had a high antibody titre against mitochondria as measured by immunofluorescence.

Ca2+-independent effects of spermine on pyruvate dehydrogenase complex activity in energized rat liver mitochondria incubated in the absence of exogenous Ca2+ and Mg2 +

In the absence of exogenous Ca(2+) and Mg(2+) and in the presence of EGTA, which favours the release of endogenous Ca(2+), the polyamine spermine is able to stimulate the activity of pyruvate dehydrogenase complex (PDC) of energized rat liver mitochondria (RLM). This stimulation exhibits a gradual concentration-dependent trend, which is maximum, about 140%, at 0.5 mM concentration, after 30 min of incubation. At concentrations higher than 0.5 mM, spermine still stimulates PDC, when compared with the control, but shows a slight dose-dependent decrease. Changes in PDC stimulation are very close to the phosphorylation level of the E(1alpha) subunit of PDC, which regulates the activity of the complex, but it is also the target of spermine. In other words, progressive dephosphorylation gradually enhances the stimulation of RLM and progressive phosphorylation slightly decreases it. These results provide the first evidence that, when transported in RLM, spermine can interact in various ways with PDC, showing dose-dependent behaviour. The interaction most probably takes place directly on a specific site for spermine on one of the regulatory enzymes of PDC, i.e. pyruvate dehydrogenase phosphatase (PDP). The interaction of spermine with PDC may also involve activation of another regulatory enzyme, pyruvate dehydrogenase kinase (PDK), resulting in an increase in E(1alpha) phosphorylation and consequently reduced stimulation of PDC at high polyamine concentrations. The different effects of spermine in RLM are discussed, considering the different activities of PDP and PDK isoenzymes. It is suggested that the polyamine at low concentrations stimulates the isoenzyme PDP(2) and at high concentrations it stimulates PDK(2).