Search

Background Dihydrolipoamide dehydrogenase deficiency (DLDD) is a rare metabolic disorder inherited in an autosomal recessive manner. This heterogeneous disease has a variable clinical presentation, onset, and biochemical markers. Materials and methods We retrospectively reviewed the clinical and molecular diagnosis of eight cases with DLDD from four referral centers in Saudi Arabia. Results Remarkably, we found hepatic involvement ranging from acute hepatic failure to chronic hepatitis in five patients. In addition, neurological disorders in the form of seizures, developmental delay, ataxia, hypotonia and psychomotor symptoms were found in five patients, two of them with a combination of hepatic and neurological symptoms. In addition, only one patient had recurrent episodes of hypoglycemia. While most patients had the hepatic form of homozygous variant c.685G > T in the DLD gene, one patient was found to have a novel variant c.623C > T that had neurological and hepatic symptoms. Conclusions We describe the largest reported DLDD cohort in the Saudi population. Clinical, biochemical, radiological, and molecular characterization was reviewed and no clear genotype-phenotype correlation was found in this cohort.

Summary: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor and bi-functional lipid and protein phosphatase. We report that the metabolic regulator pyruvate dehydrogenase kinase1 (PDHK1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The PTEN protein phosphatase dephosphorylates nuclear factor κB (NF-κB)-activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NF-κB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to induce aerobic glycolysis and PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, a biomarker of decreased patient survival. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers. : The tumor suppressor PTEN is widely inactivated in cancers and tumor syndromes. Currently, there is no effective therapeutic strategy in the clinic for PTEN-deficient cancers. Chatterjee et al. found that PTEN-deficient cells and cancers are uniquely sensitive to PDHK1 inhibition and propose PDHK1 as a potential therapeutic target in PTEN-deficient cancers. Keywords: PTEN, protein phosphatase, PDHK1, NKAP, NF-κB, synthetic lethality, metabolism, signaling, cancer

SUMMARY The impact of glucose metabolism on muscle regeneration remains unresolved. We identify glucose metabolism as a crucial driver of histone acetylation and myogenic cell fate. We use single-cell mass cytometry (CyTOF) and flow cytometry to characterize the histone acetylation and metabolic states of quiescent, activated, and differentiating muscle stem cells (MuSCs). We find glucose is dispensable for mitochondrial respiration in proliferating MuSCs, so that glucose becomes available for maintaining high histone acetylation via acetyl-CoA. Conversely, quiescent and differentiating MuSCs increase glucose utilization for respiration and have consequently reduced acetylation. Pyruvate dehydrogenase (PDH) activity serves as a rheostat for histone acetylation and must be controlled for muscle regeneration. Increased PDH activity in proliferation increases histone acetylation and chromatin accessibility at genes that must be silenced for differentiation to proceed, and thus promotes self-renewal. These results highlight metabolism as a determinant of MuSC histone acetylation, fate, and function during muscle regeneration., Graphical Abstract, In Brief Yucel et al. identify a link between stem cells’ metabolism and their fate and function. Mitochondrial glucose utilization determines remodeling of the histone acetylation landscape of muscle stem cells during tissue regeneration. Pyruvate dehydrogenase (PDH) is a pivotal control point for this and determines the differentiation potential of myogenic progenitors.

© 2020 The Author(s) Background & aims: This post hoc study aimed to determine whether major elective abdominal surgery had any acute impact on mitochondrial pyruvate dehydrogenase complex (PDC) activity and maximal mitochondrial ATP production rates (MAPR) in a large muscle group (vastus lateralis -VL) distant to the site of surgical trauma. Methods: Fifteen patients undergoing major elective open abdominal surgery were studied. Muscle biopsies were obtained after the induction of anesthesia from the VL immediately before and after surgery for the determination of PDC and maximal MAPR (utilizing a variety of energy substrates). Results: Muscle PDC activity was reduced by >50% at the end of surgery compared with pre-surgery (p < 0.05). Muscle MAPR were comprehensively suppressed by surgery for the substrate combinations: glutamate + succinate; glutamate + malate; palmitoylcarnitine + malate; and pyruvate + malate (all p < 0.05), and could not be explained by a lower mitochondrial yield. Conclusions: PDC activity and mitochondrial ATP production capacity were acutely impaired in muscle distant to the site of surgical trauma. In keeping with the limited data available, we surmise these events resulted from the general anesthesia procedures employed and the surgery related trauma. These findings further the understanding of the acute dysregulation of mitochondrial function in muscle distant to the site of major surgical trauma in patients, and point to the combination of general anesthesia and trauma related inflammation as being drivers of muscle metabolic insult that warrants further investigation. Clinical trial registration: Registered at (NCT01134809).

Objective Insulin resistance and altered hepatic mitochondrial function are central features of type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD), but the etiological role of these processes in disease progression remains unclear. Here we investigated the molecular links between insulin resistance, mitochondrial remodeling, and hepatic lipid accumulation. Methods Hepatic insulin sensitivity, endogenous glucose production, and mitochondrial metabolic fluxes were determined in wild-type, obese (ob/ob) and pioglitazone-treatment obese mice using a combination of radiolabeled tracer and stable isotope NMR approaches. Mechanistic studies of pioglitazone action were performed in isolated primary hepatocytes, whilst molecular hepatic lipid species were profiled using shotgun lipidomics. Results Livers from obese, insulin-resistant mice displayed augmented mitochondrial content and increased tricarboxylic acid cycle (TCA) cycle and pyruvate dehydrogenase (PDH) activities. Insulin sensitization with pioglitazone mitigated pyruvate-driven TCA cycle activity and PDH activation via both allosteric (intracellular pyruvate availability) and covalent (PDK4 and PDP2) mechanisms that were dependent on PPARγ activity in isolated primary hepatocytes. Improved mitochondrial function following pioglitazone treatment was entirely dissociated from changes in hepatic triglycerides, diacylglycerides, or fatty acids. Instead, we highlight a role for the mitochondrial phospholipid cardiolipin, which underwent pathological remodeling in livers from obese mice that was reversed by insulin sensitization. Conclusion Our findings identify targetable mitochondrial features of T2D and NAFLD and highlight the benefit of insulin sensitization in managing the clinical burden of obesity-associated disease., Highlights • Hepatic pyruvate dehydrogenase is hyperactivated in insulin-resistant mice. • Pioglitazone lowers hepatic PDH through covalent and allosteric actions. • Induction of hepatocyte PDK4 by pioglitazone requires PPARγ. • Improved mitochondrial function with pioglitazone precedes changes in liver fat. • Mitochondrial lipid remodeling is a modifiable feature of liver insulin resistance.

Inborn errors of carbohydrate metabolism covered in this chapter include disaccharidase deficiencies, disorders of monosaccharide metabolism, glycogen storage diseases, and gluconeogenic disorders. This chapter focuses mainly on clinical aspects, genetics and current treatments for these disorders. The defective digestion of dietary disaccharides starch, lactose and sucrose is due to deficiencies of congenital lactase, adult-type lactase, or sucrase-isomaltase. Disorders of monosaccharide metabolism include defects in transport (glucose-galactose malabsorption), enzymes of galactose metabolism (galactokinase, galactose-1-phosphate uridyl transferase, and uridine diphosphate galactose-4-epimerase deficiency), enzymes of fructose metabolism (liver fructose-1-phosphate aldolase), and dehydrogenases of pentose metabolism (fructose-6-phosphate and glucose-6-phosphate). To date, there are over 12 glycogenoses, or glycogen metabolism disorders, that have been cataloged. Glycogen storage diseases (GSD), a major category of glycogenoses, are categorized by the type of tissue involved: liver, muscle, and/or cardiac. Gluconeogenic disorders entail deficiencies of fructose-1,6-diphosphatase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate dehydrogenase complex.

A series of 1-(substituted phenoxyacetoxy)-1-(furan-2-yl)methylphosphonates as potential inhibitor of pyruvate dehydrogenase complex (PDHc) was synthesized and studied. Some of them were found to be a kind of effective plant PDHc inhibitor with higher inhibitory potency against broadleaf plant PDHc. The most effective compound II-17 showed good herbicidal activity against broadleaf weeds at 100–300 g ai/ha and it was very safe for rice, maize, and wheat even at the rate of 800–1200 g ai/ha. The recent field trials at different regions in China further showed that II-17 (O,O-dimethyl 1-(2,4-dichlorophenoxyacetoxy)-1-(furan-2-yl)methylphosphonate, HWS) could be applied to control a broad spectrum of broad-leaved weeds at the rate of 135–450 g ai/ha for postemergence in maize fields by using the formulation of 30% EC (Emulsifiable concentrate) or 30% powder. This compound as dicot PDHc inhibitor displayed the good utility as a selective postemergence herbicide.

There is increasing evidence showing that oxidative glucose metabolism in mitochondria is impaired in epilepsy and the involved metabolic mechanisms have been further characterized by recent work. Glucose is the main energy source in brain in people who eat a conventional mixed diet with carbohydrates, fats, and proteins. In the cytoplasm, glucose is metabolized to pyruvate by glycolysis, which produces small amounts of energy. Entry of pyruvate into mitochondria and subsequent metabolism via the tricarboxylic acid cycle generates large amounts of adenosine triphosphate as well as precursors for cellular biosynthesis of lipids and amino acids. Sufficient energy is important for the brain to be able to signal normally and to keep the sodium and potassium gradients stable across cellular membranes. In contrast, insufficient energy can contribute to surges in extracellular potassium levels, which destabilizes membrane potentials and signaling and can result in seizure generation in the chronic epileptic brain. The known biochemical mechanisms leading to mitochondrial impairments of glucose carbon metabolism in epilepsy are summarized in this chapter, including reduced glucose utilization, decreases in enzyme activities, such as pyruvate dehydrogenase, as well as increased anaplerotic demand. Based on this current knowledge, auxiliary brain fuels would be useful to provide extra energy to the epileptic brain. This includes ketones, tricarboxylic acid cycle intermediates, and precursors as well as even medium chain fatty acids and triheptanoin.

Sestrin2 (Sesn2) is a stress-inducible protein that declines with aging in the heart. We reported that rescue Sesn2 levels in aged mouse hearts through gene therapy improves the resistance of aged hearts to ischemia and reperfusion (I/R) insults. We hypothesize that Sesn2 as a scaffold protein maintains mitochondrial integrity to protect heart from ischemic injury during I/R. Young C57BL/6 J (3–6 months), aged C57BL/6 J (24–26 months), and young Sesn2 KO (3–6 months, C57BL/6 J background) mice were subjected to in vivo regional ischemia and reperfusion. The left ventricle was collected for transcriptomics, proteomics and metabolomics analysis. The results demonstrated that Sesn2 deficiency leads to aging-like cardiac diastolic dysfunction and intolerance to ischemia reperfusion stress. Seahorse analysis demonstrated that Sesn2 deficiency in aged and young Sesn2 KO versus young hearts lead to impaired mitochondrial respiration rate with defects in Complex I and Complex II activity. The Sesn2 targeted proteomics analysis revealed that Sesn2 plays a critical role in maintaining mitochondrial functional integrity through modulating mitochondria biosynthesis and assembling of oxidative phosphorylation (OXPHOS) complexes. The RNA-Seq data showed that alterations in the expression of mitochondrial compositional and functional genes and substrate metabolism related genes in young Sesn2 KO and aged versus young hearts. Further immunofluorescence and immunoprecipitation analysis demonstrated that Sesn2 is translocated into mitochondria and interacts with OXPHOS components to maintain mitochondrial integrity in response to I/R stress. Biochemical analysis revealed that Sesn2 is associated with citrate cycle components to modulate pyruvate dehydrogenase and isocitrate dehydrogenase activities during I/R stress. Thus, Sesn2 serves as a scaffold protein interacting with OXPHOS components to maintain mitochondrial integrity under I/R stress. Age-related downregulation of cardiac Sesn2 fragilizes mitochondrial functional integrity in response to ischemic stress., Graphical abstract Image 1, Highlights • Ischemia reperfusion stress triggers Sesn2 accumulation in mitochondria. • Sesn2 interacts with OXPHOS complexes to modulate the adaptive substrate metabolism. • Age-related Sesn2 maintains mitochondrial functional integrity under stress conditions.

Nucleotide-binding oligomerization domain-Like Receptor with a Pyrin domain 3 (NLRP3) inflammasome was emerged as a marker of metabolic dysregulation. We revealed that age-related Sirtuin-1 (SIRT1) modulates cardiac metabolism that medicated inflammatory response during ischemia and reperfusion (I/R) stress. We hypothesize that SIRT1 attenuates NLRP3 inflammasome-dependent inflammation and pyroptosis during myocardial I/R through metabolic modulation. C57BL/6J wild type (WT) mice, inducible cardiomyocyte specific SIRT1 knockout (icSIRT1 KO) and inducible cardiomyocyte specific PDH E1α knockout (icPDH E1α KO) mice were subjected to ligation and release of left anterior descending coronary artery for in vivo regional I/R models. The echocardiography measurement demonstrated that SIRT1 agonist SRT1720 (30 μg/g) improved cardiac systolic function during 45 min of ischemia and 6 h of reperfusion in C57BL/6J WT mice. The biochemical analysis showed that I/R triggered activation of cardiac pyruvate dehydrogenase (PDH), while SIRT1 agonist SRT1720 inhibited I/R-induced PDH activity and reduced production of reactive oxygen species (ROS) during myocardial I/R. Moreover, SRT1720 regulates PDH-related glucose oxidative metabolism to reduce NLRP3 inflammasome activation and pyroptosis in an Akt signaling dependent manner during I/R. Furthermore, an impaired Akt signaling was observed in icSIRT1 KO versus SIRT1fox/flox mice under I/R stress. Intriguingly, we observed lower levels of ROS generation, decreased NLRP3 levels and less pyroptosis occurred in the icPDH E1α KO versus PDH E1αflox/flox hearts during I/R. Taken together, the results indicate that SIRT1 agonism can inhibit activation of NLRP3 inflammasome via Akt-dependent metabolic regulation during ischemic insults by I/R., Graphical abstract Image 1, Highlights • Reactive oxygen species (ROS) mediate cardiac NLRP3 inflammasome activation during ischemia and reperfusion. • SIRT1 agonist modulates cardiac metabolic homeostasis in response to ischemic insults. • Pyruvate dehydrogenase plays a role in SIRT1 regulating NLRP3 inflammasome activation.

Malignant cells frequently demonstrate an oncogenic-driven reliance on glycolytic metabolism to support their highly proliferative nature. Overexpression of pyruvate dehydrogenase kinase (PDK) may promote this unique metabolic signature of tumor cells by inhibiting mitochondrial function. PDKs function to phosphorylate and inhibit pyruvate dehydrogenase (PDH) activity. Silencing of PDK expression has previously been shown to restore mitochondrial function and reduce tumor cell proliferation. High dose Vitamin B1, or thiamine, possesses antitumor properties related to its capacity to reduce PDH phosphorylation and promote its enzymatic activity, presumably through PDK inhibition. Though a promising nutraceutical approach for cancer therapy, thiamine's low bioavailability may limit clinical effectiveness. Here, we have demonstrated exploiting the commercially available lipophilic thiamine analogs sulbutiamine and benfotiamine increases thiamine's anti-cancer effect in vitro. Determined by crystal violet proliferation assays, both sulbutiamine and benfotiamine reduced thiamine's millimolar IC50 value to micromolar equivalents. HPLC analysis revealed that sulbutiamine and benfotiamine significantly increased intracellular thiamine and TPP concentrations in vitro, corresponding with reduced levels of PDH phosphorylation. Through an ex vitro kinase screen, thiamine's activated cofactor form thiamine pyrophosphate (TPP) was found to inhibit the function of multiple PDK isoforms. Attempts to maximize intracellular TPP by exploiting thiamine homeostasis gene expression resulted in enhanced apoptosis in tumor cells. Based on our in vitro evaluations, we conclude that TPP serves as the active species mediating thiamine's inhibitory effect on tumor cell proliferation. Pharmacologic administration of benfotiamine, but not sulbutiamine, reduced tumor growth in a subcutaneous xenograft mouse model. It remains unclear if benfotiamine's effects in vivo are associated with PDK inhibition or through an alternative mechanism of action. Future work will aim to define the action of lipophilic thiamine mimetics in vivo in order to translate their clinical usefulness as anticancer strategies.

The Jordan group's interest center on: E. coli (ec) and human (h) pyruvate dehydrogenase (PDHc), 2-oxoglutarate dehydrogenase (OGDHc) and 2-oxoadipate dehydrogenase (OADHc) complexes; and on 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). The most important findings were: (1) the first components of OGDHc and OADHc and ecDXPS aerobically generated the ThDP-enamine radical species in “off-pathway” mechanism. (2) The pre-steady-state rate constants for conversion of pyruvate to all intermediates were determined on the entire E. coli PDHc. (3) The hydrogen–deuterium exchange MS (HDX-MS) method provided a snapshot of (i) the conformational dynamics of the full-length E. coli DXPS; (ii) interaction loci of the ecE1p-ecE2p subcomplex; (iii) the unique inter-component interaction sites in the hE1o-hE2o subcomplex, also complemented by chemical cross-linking MS. (4) Protein engineering using site saturation mutagenesis was applied to the first and second components of the ecOGDHc and led to formation of acyl-Coenzyme A with a variety acyl groups.

This chapter focuses on the methods to measure unique metabolites, specific enzymes, and metabolic flux in fatty acid β-oxidation, and on biochemical assays of tricarboxylic acid (TCA) cycle enzymes and the pyruvate dehydrogenase complex. These assays play an important role in the diagnosis of genetic diseases, newborn screening, and in cancer and metabolism research. The rationale, protocol, pros and cons, and alternative methods are discussed. Nevertheless, each laboratory should adapt the preferred method optimizing sample preparation and assay conditions for linearity and a low signal-to-noise ratio. The reader is also referred to the additional literature citing methods and clinical descriptions of the various diseases.

Vitamin B1 (thiamine) is converted in living cells into biologically active forms, among which thiamine diphosphate (ThDP) predominates. ThDP is a coenzyme for several key enzymes of carbohydrate metabolism including pyruvate dehydrogenase complex (PDC) which is involved in pyruvate decarboxylation with the acetyl-CoA formation. ThDP-dependent pyruvate dehydrogenase, the first enzyme in the complex, is regulated by phosphorylation–dephosphorylation performed by specific regulatory enzymes. It was shown that ThDP and thiamine triphosphate can participate in this PDC regulation as effectors modulating the activity of the regulatory enzymes. Numerous studies indicate that PDC plays a central role in the maintenance of glucose homeostasis in the body and the disturbance of its regulation can initiate the development of metabolic and neurodegenerative diseases. The search for effectors of the PDC regulatory enzymes, including, among others, thiamine derivatives, is a promising approach to the treatment of the above diseases.

Mitochondria supply ~90% of the ATP required for contractile function in cardiac cells. While adult cardiomyocytes preferentially utilize fatty acids as a fuel source for oxidative phosphorylation, cardiac mitochondria can switch to other substrates when required. This change is driven in part by a combination of extracellular and intracellular signal transduction pathways that alter mitochondrial gene expression and enzymatic activity. The mechanisms by which extracellular metabolic information is conveyed to cardiac mitochondria are not currently well defined. Recent work has shown that adropin – a liver-secreted peptide hormone – can induce changes in mitochondrial fuel substrate utilization in skeletal muscle, leading to increased glucose use. In this study, we examined whether adropin could regulate mitochondrial glucose utilization pathways in cardiac cells. We show that stimulation of cultured cardiac cells with adropin leads to decreased expression of the pyruvate dehydrogenase (PDH) negative regulator PDK4, which reduces inhibitory PDH phosphorylation. The downregulation of PDK4 expression by adropin is lost when GPR19 – a putative adropin receptor – is genetically depleted in H9c2 cells. Loss of GRP19 expression alone increased PDK4 expression, leading to a reduction in mitochondrial respiration. Finally, we show that adropin-mediated GPR19 signaling relies on the p44/42 MAPK pathway, and that pharmacological disruption of this pathway blocks the effects of adropin on PDK4 in cardiac cells. These findings suggest that adropin may be a key regulator of fuel substrate utilization in the heart, and implicates an orphan G-protein coupled receptor in a novel signaling pathway controlling mitochondrial fuel metabolism., Graphical abstract fx1

Objective Mitochondrial pyruvate dehydrogenase kinases 1–4 (PDKs1–4) negatively regulate activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation. PDKs play a pivotal role in maintaining energy homeostasis and contribute to metabolic flexibility by attenuating PDC activity in various mammalian tissues. Cumulative evidence has shown that the up-regulation of PDK4 expression is tightly associated with obesity and diabetes. In this investigation, we test the central hypothesis that PDKs1-4 are a pharmacological target for lowering glucose levels and restoring insulin sensitivity in obesity and type 2 diabetes (T2D). Methods Diet-induced obese (DIO) mice were treated with a liver-specific pan-PDK inhibitor 2-[(2,4-dihydroxyphenyl) sulfonyl]isoindoline-4,6-diol (PS10) for four weeks, and results compared with PDK2/PDK4 double knockout (DKO) mice on the same high fat diet (HFD). Results Both PS10-treated DIO mice and HFD-fed DKO mice showed significantly improved glucose, insulin and pyruvate tolerance, compared to DIO controls, with lower plasma insulin levels and increased insulin signaling in liver. In response to lower glucose levels, phosphorylated AMPK in PS10-treated DIO and HFD-fed DKO mice is upregulated, accompanied by decreased nuclear carbohydrate-responsive element binding protein (ChREBP). The reduced ChREBP signaling correlates with down-regulation of hepatic lipogenic enzymes (ACC1, FAS, and SCD1), leading to markedly diminished hepatic steatosis in both study groups, with lower circulating cholesterol and triacylglyceride levels as well as reduced fat mass. PS10-treated DIO as well as DKO mice showed predominant fatty acid over glucose oxidation. However, unlike systemic DKO mice, increased hepatic PDC activity alone in PS10-treated DIO mice does not raise the plasma total ketone body level. Conclusion Our findings establish that specific targeting of hepatic PDKs with the PDK inhibitor PS10 is an effective therapeutic approach to maintaining glucose and lipid homeostasis in obesity and T2D, without the harmful ketoacidosis associated with systemic inhibition of PDKs., Highlights • Diet-induced obese (DIO) mice were treated with a novel liver-specific pyruvate dehydrogenase kinase (PDK) inhibitor PS10. • PS10-treated DIO mice and PDK2/PDK4-DKO mice fed indentical HFD showed improved glucose, insulin and pyruvate tolerance. • PS10-treated DIO and untreated DKO mice manifest reduced hepatic steatosis and attenuation of ChREBP-mediated lipogenesis. • Unlike DKO mice, targeting hepatic PDK alone in DIO mice with PS10 does not increase total plasma ketone-body levels.

Graphical abstract, Highlights • Pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) translocate to the nucleus in injured liver cells. • Increased nuclear PDHC and LDH induce increased nuclear acetyl-CoA and lactate, histone hyper-acetylation, and damage response gene expression. • Inhibition of histone acetylation or inhibition of PDHC and LDH reduce damage and inflammation in injured livers. • Galloflavin inhibits both PDH and LDH and protects from damage and mortality induced by hepatotoxins., Background & Aims Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression. Methods Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure. Results Levels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. Conclusion PDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of acetyl-CoA and lactate. This results in histone H3 hyper-acetylation and expression of damage response genes. Inhibition of PDHC and LDH reduces liver damage and improves survival in mice with acute liver failure. Thus, PDHC and LDH are targets for therapy of acute liver failure. Lay summary Acute liver failure is a rapidly progressive deterioration of liver function resulting in high mortality. In experimental mouse models of acute liver failure, we found that two metabolic enzymes, namely pyruvate dehydrogenase complex and lactic dehydrogenase, translocate to the nucleus resulting in detrimental gene expression. Treatment with an inhibitor of these two enzymes was found to reduce liver damage and to improve survival.

Research over the past seventy years has established that mitochondrial-l-lactate dehydrogenase (m-L-LDH) is vital for mitochondrial bioenergetics. However, in recent report, Fulghum et al. concluded that lactate is a poor fuel for mitochondrial respiration [1]. In the present study, we have followed up on these findings and conducted an independent investigation to determine if lactate can support mitochondrial bioenergetics. We demonstrate herein that lactate can fuel the bioenergetics of heart, muscle, and liver mitochondria. Lactate was just as effective as pyruvate at stimulating mitochondrial coupling efficiency. Inclusion of LDH (sodium oxamate or GSK 2837808A) and pyruvate dehydrogenase (PDH; CPI-613) inhibitors abolished respiration in mitochondria energized with lactate. Lactate also fueled mitochondrial ROS generation and was just as effective as pyruvate at stimulating H2O2 production. Additionally, lactate-induced ROS production was inhibited by both LDH and PDH inhibitors. Enzyme activity measurements conducted on permeabilized mitochondria revealed that LDH is localized in mitochondria. In aggregate, we can conclude that mitochondrial LDH fuels bioenergetics in several tissues by oxidizing lactate., Graphical abstract Image 1, Highlights • Lactate can fuel mitochondrial respiration. • Lactate serves as a substrate for H2O2 production. • Mitochondria contain LDH.

Dichloroacetate (DCA) has been the focus of research by both environmental toxicologists and biomedical scientists for over 50 years. As a product of water chlorination and a metabolite of certain industrial chemicals, DCA is ubiquitous in our biosphere at low μg/kg body weight daily exposure levels without obvious adverse effects in humans. As an investigational drug for numerous congenital and acquired diseases, DCA is administered orally or parenterally, usually at doses of 10-50mg/kg per day. As a therapeutic, its principal mechanism of action is to inhibit pyruvate dehydrogenase kinase (PDK). In turn, PDK inhibits the key mitochondrial energy homeostat, pyruvate dehydrogenase complex (PDC), by reversible phosphorylation. By blocking PDK, DCA activates PDC and, consequently, the mitochondrial respiratory chain and ATP synthesis. A reversible sensory/motor peripheral neuropathy is the clinically limiting adverse effect of chronic DCA exposure and experimental data implicate the Schwann cell as a toxicological target. It has been postulated that stimulation of PDC and respiratory chain activity by DCA in normally glycolytic Schwann cells causes uncompensated oxidative stress from increased reactive oxygen species production. Additionally, the metabolism of DCA interferes with the catabolism of the amino acids phenylalanine and tyrosine and with heme synthesis, resulting in accumulation of reactive molecules capable of forming adducts with DNA and proteins and also resulting in oxidative stress. Preliminary evidence in rodent models of peripheral neuropathy suggest that DCA-induced neurotoxicity may be mitigated by naturally occurring antioxidants and by a specific class of muscarinic receptor antagonists. These findings generate a number of testable hypotheses regarding the etiology and treatment of DCA peripheral neuropathy.

Seeds play a central role in the life cycle of plants. Seed hardness in pomegranates is of economic relevance, yet scarcely studied and poorly understood in China. In this study, we compared the proteomic differences between Zhongnonghong (soft-seeded) and Sanbai (hard-seeded) pomegranates. A total of 892 protein spots from both varieties were detected on two-dimensional electrophoresis gels (2-DE); 76 spots showed greater than a 1.5-fold or less than a 0.66-fold difference (P

Mitochondrial DNA (mtDNA) resides in close proximity to metabolic reactions, and is maintained by the 8-oxoguanine DNA glycosylase (Ogg1) and other members of the base excision repair pathway. Here, we tested the hypothesis that changes in liver metabolism as under fasting/feeding conditions would be sensed by liver mtDNA, and that Ogg1 deficient mice might unravel a metabolic phenotype. Wild type (WT) and ogg1−/− mice were either fed ad libitum or subjected to fasting for 24 h, and the corresponding effects on liver gene expression, DNA damage, as well as serum values were analyzed. Ogg1 deficient mice fed ad libitum exhibited hyperglycemia, elevated insulin levels and higher liver glycogen content as well as increased accumulation of 8oxoG in mtDNA compared to age- and gender matched WT mice. Interestingly, these phenotypes were absent in ogg1−/− mice during fasting. Gene expression and functional analyses suggest that the diabetogenic phenotype in the ogg1−/− mice is due to a failure to suppress gluconeogensis in the fed state. The ogg1−/− mice exhibited reduced mitochondrial electron transport chain (ETC) capacity and a combined low activity of the pyruvate dehydrogenase (PDH), alluding to inefficient channeling of glycolytic products into the citric acid cycle. Our data demonstrate a physiological role of base excision repair that goes beyond DNA maintenance, and implies that DNA repair is involved in regulating metabolism. © 2017. This is the authors’ accepted and refereed manuscript to the article. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

Gray mouse lemurs (Microcebus murinus) from Madagascar present an excellent model for studies of torpor regulation in a primate species. In the present study, we analyzed the response of the insulin signaling pathway as well as controls on carbohydrate sparing in six different tissues of torpid versus aroused gray mouse lemurs. We found that the relative level of phospho-insulin receptor substrate (IRS-1) was significantly increased in muscle, whereas the level of phospho-insulin receptor (IR) was decreased in white adipose tissue (WAT) of torpid animals, both suggesting an inhibition of insulin/insulin-like growth factor-1 (IGF-1) signaling during torpor in these tissues. By contrast, the level of phospho-IR was increased in the liver. Interestingly, muscle, WAT, and liver occupy central roles in whole body homeostasis and each displays regulatory controls operating at the plasma membrane. Changes in other tissues included an increase in phospho-glycogen synthase kinase 3α (GSK3α) and decrease in phospho-ribosomal protein S6 (RPS6) in the heart, and a decrease in phospho-mammalian target of rapamycin (mTOR) in the kidney. Pyruvate dehydrogenase (PDH) that gates carbohydrate entry into mitochondria is inhibited via phosphorylation by pyruvate dehydrogenase kinase (e.g., PDK4). In the skeletal muscle, the protein expression of PDK4 and phosphorylated PDH at Ser 300 was increased, suggesting inhibition during torpor. In contrast, there were no changes in levels of PDH expression and phosphorylation in other tissues comparing torpid and aroused animals. Information gained from these studies highlight the molecular controls that help to regulate metabolic rate depression and balance energetics during primate torpor.

Objective Dysregulated muscle metabolism is a cardinal feature of human insulin resistance (IR) and associated diseases, including type 2 diabetes (T2D). However, specific reactions contributing to abnormal energetics and metabolic inflexibility in IR are unknown. Methods We utilize flux balance computational modeling to develop the first systems-level analysis of IR metabolism in fasted and fed states, and varying nutrient conditions. We systematically perturb the metabolic network to identify reactions that reproduce key features of IR-linked metabolism. Results While reduced glucose uptake is a major hallmark of IR, model-based reductions in either extracellular glucose availability or uptake do not alter metabolic flexibility, and thus are not sufficient to fully recapitulate IR-linked metabolism. Moreover, experimentally-reduced flux through single reactions does not reproduce key features of IR-linked metabolism. However, dual knockdowns of pyruvate dehydrogenase (PDH), in combination with reduced lipid uptake or lipid/amino acid oxidation (ETFDH), does reduce ATP synthesis, TCA cycle flux, and metabolic flexibility. Experimental validation demonstrates robust impact of dual knockdowns in PDH/ETFDH on cellular energetics and TCA cycle flux in cultured myocytes. Parallel analysis of transcriptomic and metabolomics data in humans with IR and T2D demonstrates downregulation of PDH subunits and upregulation of its inhibitory kinase PDK4, both of which would be predicted to decrease PDH flux, concordant with the model. Conclusions Our results indicate that complex interactions between multiple biochemical reactions contribute to metabolic perturbations observed in human IR, and that the PDH complex plays a key role in these metabolic phenotypes., Graphical abstract Schematic of targeted knockdowns that model phenotypes of insulin resistance. Knockdowns (KD; green) of fatty acid transporter (FAT), pyruvate dehydrogenase (PDH), and electron transfer flavoprotein (ETFDH) recapitulated the metabolic phenotypes of insulin resistance (red), defined as reduced ATP + P-Cr synthesis, TCA flux, and metabolic flexibility (RQ). Additional metabolic alterations identified are depicted by orange arrows.

This chapter begins with pyruvate conversion into acetyl CoA and CO 2 by the enzyme pyruvate dehydrogenase. It then describes the combination of acetyl CoA with oxaloacetate to form citric acid. It then follows all of the biochemical transformations that convert citric acid back to oxaloacetate and 2 CO 2 molecules, producing NADH and FADH 2 and GTP along the way. How NADH and FADH 2 lead to the synthesis of ATP is considered in the discussion of the electron transport chain and ATP synthetase. The energetic of the electrochemical potential of hydrogen ions across the inner mitochondrial membrane is discussed

Compounds of different origin and nature can produce common metabolites and products. Hexoses, glycerol, fatty acids, and amino acids render acetyl-CoA, which is oxidized in the citric acid cycle. The respiratory chain is the final common destination of electrons from different substrates. Carbohydrates generate fatty acids and triacylglycerols. Amino acids form α-ketoacids by transamination. Glucose also produces α-ketoacids. After deamination, amino acids can form carbohydrates (glucogenic amino acids) or ketone bodies (ketogenic amino acids). Some metabolites (glucose-6-P, pyruvate, acetyl-CoA) are “crossroads” compounds of several metabolic pathways. Metabolic regulation is achieved by targeting key enzymes on a pathway, either by modifying the activity of preexistent enzymes (changes in substrate level, allosteric effectors, covalent modification) or changing the amount of enzyme (synthesis or degradation). Glycogenolysis is regulated through the control of glycogen phosphorylase and phosphorylase kinase . Glycogenesis is regulated by modulating the activity of glycogen synthase . Glycolysis is controlled by targeting hexokinase and phosphofructokinase . Gluconeogenesis is modulated at the level of g lucose-6-P phosphatase , fructose-l,6-bisP phosphatase , and pyruvate carboxylase. Oxidative decarboxylation of pyruvate is modulated via the pyruvate dehydrogenase (PDH) multienzyme complex. The citric acid cycle is regulated at various levels, including citrate synthase , isocitrate dehydrogenase , α-ketoglutarate dehydrogenase , and glutamate dehydrogenase . The Pasteur effect describes a phenomenon consisting of the decrease in glucose consumption in the presence of oxygen. Allosteric regulation of phosphofructokinase is responsible for this effect. Triacylglycerols in adipose tissue are hydrolyzed by lipase , a hormone-regulated enzyme. Fatty acid biosynthesis is mainly regulated at the level of acetyl CoA carboxylase . Cholesterol biosynthesis is regulated by controlling 3-OH-3-methylglutaryl-CoA reductase . Metabolism of nitrogenous compounds, such as the synthesis of some amino acids, of purines, and pyrimidines is regulated by the final product. Cellular oxidations are adjusted by the content of nucleotides in the cell. The energy charge of the cell depends on the relative concentration of ATP, ADP, and AMP of cells. When the energy charge is high, energy-consuming metabolic pathways are stimulated, whereas those producing ATP are inhibited.

Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes.

Large numbers of genes are responsible for Leigh syndrome (LS), making genetic confirmation of LS difficult. We screened our patients with LS using a limited set of 21 primers encompassing the frequently reported gene for the respiratory chain complexes I (ND1–ND6, and ND4L), IV(SURF1), and V(ATP6) and the pyruvate dehydrogenase E1α-subunit. Of 18 LS patients, we identified mutations in 11 patients, including 7 in mDNA (two with ATP6), 4 in nuclear (three with SURF1). Overall, we identified mutations in 61% of LS patients (11/18 individuals) in this cohort. Sanger sequencing with our limited set of primers allowed us a rapid genetic confirmation of more than half of the LS patients and it appears to be efficient as a primary genetic screening in this cohort.

Dihydrolipoamide dehydrogenase deficiency, also known as maple syrup urine disease (MSUD) type III, is caused by the deficiency of the E3 subunit of branched chain alpha-ketoacid dehydrogenase (BCKDH), α-ketoglutarate dehydrogenase (αKGDH), and pyruvate dehydrogenase (PDH). DLD deficiency variably presents with either a severe neonatal encephalopathic phenotype or a primarily hepatic phenotype. As a variant form of MSUD, it is considered a core condition recommended for newborn screening. The detection of variant MSUD forms has proven difficult in the past with no asymptomatic DLD deficiency patients identified by current newborn screening strategies. Citrulline has recently been identified as an elevated dried blood spot (DBS) metabolite in symptomatic patients affected with DLD deficiency. Here we report the retrospective DBS analysis and second-tier allo-isoleucine testing of 2 DLD deficiency patients. We show that an elevated citrulline and an elevated allo-isoleucine on second-tier testing can be used to successfully detect DLD deficiency. We additionally recommend that DLD deficiency be included in the "citrullinemia/elevated citrulline" ACMG Act Sheet and Algorithm.

Alzheimer disease (AD) is an age-related neurodegenerative disease characterized by the presence of three pathological hallmarks: synapse loss, extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NFTs). The major component of SP is amyloid β-peptide (Aβ), which has been shown to induce oxidative stress. The AD brain shows increased levels of lipid peroxidation products, including 4-hydroxy-2-nonenal (HNE). HNE can react covalently with Cys, His, or Lys residues on proteins, altering structure and function of the latter. In the present study we measured the levels of the HNE-modified lipoic acid in brain of subjects with AD and age-matched controls. Lipoic acid is a key co-factor for a number of proteins including pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, key complexes for cellular energetics. We observed a significant decrease in the levels of HNE-lipoic acid in the AD brain compared to that of age-matched controls. To investigate this phenomenon further, the levels and activity of lipoamide dehydrogenase (LADH) were measured in AD and control brains. Additionally, LADH activities were measured after in-vitro HNE-treatment to mice brains. Both LADH levels and activities were found to be significantly reduced in AD brain compared to age-matched control. HNE-treatment also reduced the LADH activity in mice brain. These data are consistent with a two-hit hypothesis of AD: oxidative stress leads to lipid peroxidation that, in turn, causes oxidative dysfunction of key energy-related complexes in mitochondria, triggering neurodegeneration. This study is consonant with the notion that lipoic acid supplementation could be a potential treatment for the observed loss of cellular energetics in AD and potentiate the antioxidant defense system to prevent or delay the oxidative stress in and progression of this devastating dementing disorder., Graphical abstract The NADH-dependent oxido-reductase enzyme lipoamide dehydrogenase (LADH) is an important member of the mitochondrial energy generation complex. Alteration of the structure and activity of LADH by elevated reactive oxygen species (ROS) may hamper energy metabolism and ATP production. Lipoic acid (LA) must be in the reduced form as part of its co-factor function for mitochondrial TCA complexes such as α-ketoglutarate dehydrogenase. However, oxidized LADH is unable to reduce LA to DHLA, and therefore HNE is unable to bind to DHLA efficiently. Consequently, in this study, decreased LA-HNE binding was observed in Alzheimer disease brain. Severe effects on learning, memory, and higher executive functioning, all significantly lost in AD patients, would be expected. Supplementation of LA conceivably may protect LADH from ROS or end products of ROS (e.g., HNE) by self-sacrifice mechanism, potentially providing protection against dementia or slowing the rate of progression of AD. Highlights ► HNE-bound lipoic acid (HNE-LA) levels were decreased in the IPL region of AD brain. ► The TCA enzyme LADH converts inactive lipoic acid to its active-antioxidant form. ► LADH levels and activity were deceased in AD in IPL brain region. ► In-vitro HNE-treatment to mouse brain reduced LADH activity. ► The results fit the two-hit hypothesis of AD.

Pyruvate dehydrogenase complex (PDC) deficiencies are inborn errors of carbohydrate metabolism that can profoundly impact the nervous system. Though the presentation can range from fatal neonatal lactic acidosis to ataxia and peripheral neuropathy in otherwise healthy school-age children, global developmental delay, hypotonia, microcephaly, and seizures are common. These nonspecific features can mimic other disorders. Both congenital brain malformations and destructive lesions can be seen. Ventriculomegaly and abnormalities of the corpus callosum are frequently observed, and Leigh syndrome may occur. The biochemical hallmarks are lactic acidemia, elevated pyruvate with a normal lactate-to-pyruvate ratio, and hyperalaninemia. Nearly all cases can be confirmed with combined assaying of PDC activity (in lymphocytes or cultured skin fibroblasts) and PDHA1 sequencing; additional genetic testing may identify the cause of cases due to mutations in other genes. It is increasingly recognized that mutations in genes that do not encode PDC proteins but are related to this pathway can also be implicated in PDC deficiency. The X-linked PDHA1 gene is responsible for the majority of cases, and this inheritance pattern likely accounts for the observation that females tend to have better survival than males, but often at the cost of poorer neurological and cognitive outcomes. PDC is a multiple enzyme complex that catalyzes the production of acetyl-CoA from pyruvate produced by glycolysis. PDC contains three catalytic enzymes, two regulatory enzymes, and a binding protein. It also requires the cofactors thiamine pyrophosphate (TPP), lipoic acid, and flavin adenine dinucleotide. While there is no proven effective treatment for PDC deficiency, the ketogenic diet is frequently employed to bypass the block in carbohydrate metabolism. Thiamine (vitamin B1), from which the cofactor TPP is derived, is typically given. Other potential treatments, such as dichloroacetate or phenylbutyrate, are candidates for further study.

Pyruvate occupies a unique position at a major crossroad of carbohydrate, protein, and lipid metabolism. Heart muscle removes lactate from the circulation during exercise. Primary functions for alanine are incorporation into protein and participation in transamination. Cytoplasmic malate and malic enzyme are a source of NADPH for lipogenesis, as well as a source of pyruvate. Acetyl-CoA, and increases in mitochondrial ATP/ADP and NADH/NAD+ ratios inhibit pyruvate dehydrogenase. Acetyl-CoA is an allosteric activator of pyruvate carboxylase. A dietary deficiency of thiamin or niacin will reduce pyruvate dehydrogenase activity, and a biotin or Zn++ deficiency will reduce pyruvate carboxylase activity.

Considered collectively, energy metabolism disorders spare no organ or tissue and therefore can mimic diseases routinely encountered by primary and specialty care clinicians. In addition to the well-established role of many energy metabolism enzymes and transporters in the formation and dissolution of high-energy molecular bonds and in the supply of fuels to cells, some also serve dual or, possibly, multiple or multifaceted roles. For example, some pyruvate metabolism enzyme mutations impair axonal migration or alter craniofacial configuration; many are linked to neuronal necrosis and apoptosis and to edema (spongiosis) of the cerebral white matter and, paradoxically, can manifest in the same individual with both low basal cerebral activity and intermittent hyperexcitation (seizures), which is associated with interneuron synaptic failure and resultant disinhibition. Even Krebs cycle enzyme variants are associated with specific cancers and may confer significant biological aggressivity to glioma, a phenomenon diagnostically exploited via magnetic resonance imaging spectroscopy. These a priori unexpected neurological manifestations probably stem from the fact that flux through energy metabolism reactions sustains the synthesis and recycling of neurotransmitters and other signaling molecules by neural cells and circuits. Consequently, the brain often bears the full burden of these diseases, in conjunction with cardiac and skeletal muscle, liver, and kidney. Metabolic therapies continue to be the mainstay approach to treatment, which assumes that the long-range metabolic and nonmetabolic consequences of enzyme and transport reactions are reversible, although this has not been proven because most such consequences are not well understood.

This chapter focuses on glycolysis and the tricarboxylic acid cycle (TCA). Glucose transports to the cells are mediated by a family of transmembrane facilitative glucose transporters (GLUTs). In the gastrointestinal (GI) tract and renal tubule cells glucose is actively transported against the concentration gradient along with Na + mediated by sodium-dependent transporter (SGLT). The major glucose transporter (GLUT4) of muscle and adipose tissue cells is regulated by insulin. The chapter discusses the ten steps involved in glycolytic pathways. Three allosteric enzymes hexokinase, phosphofructokinase and pyruvate kinase mediated by positive and negative effectors regulate glycolysis. Insulin promotes glycolysis and glucagon has opposite effects. AMP activated protein kinase (AMPK) promotes glycolysis. It is noted that under anaerobic and limited oxygen supply the end product of glycolysis is lactate. In aerobic cell the end product is pyruvate, which is oxidized in the mitochondria. TCA cycle consists of series biochemical reactions in which two carbon atoms of acetyl-CoA is oxidized to CO 2 and the energy stored in the two carbon atoms is transferred to NAD + and FAD, yielding NADH and FADH 2 .

Pyruvate dehydrogenase and pyruvate carboxylase deficiency are the most common disorders in pyruvate metabolism. Diagnosis is made by enzymatic and DNA analysis after basic biochemical tests in plasma, urine, and CSF. Pyruvate dehydrogenase has three main subunits, an additional E3-binding protein and two complex regulatory enzymes. Most frequent are deficiencies in PDH-E1α. There is a spectrum of clinical presentations in E1α deficiency, ranging in boys from severe neonatal lactic acidosis, Leigh encephalopathy, to later onset of neurological disease such as intermittent ataxia or dystonia. Females tend to have a more uniform presentation resembling nonprogressive cerebral palsy. Neuroradiological abnormalities such as corpus callosum agenesis are seen more frequently in girls, basal ganglia and midbrain disturbances in boys. Deficiencies in the other subunits have also been described, but in a smaller number of patients. Pyruvate carboxylase deficiency has three clinical phenotypes. The infantile type is characterized mainly by severe developmental delay, failure to thrive, and seizures. The second type is characterized by neonatal onset of severe lactic acidosis with rigidity and hypokinesia. A third form is rarer with intermittent episodes of lactic acidosis and ketoacidosis. Neuroradiological findings such as cystic periventricular leukomalacia have been described.

Lipoic acid is a relatively small dithiol-containing metabolite that serves as a key cofactor in several multienzyme complexes that function in energy metabolism or the degradation of several amino acids. In its cofactor form, it is tethered in an amide linkage to the e-amino group of conserved lysyl residues that reside on designated lipoyl carrier proteins (LCPs). Although not absolutely ubiquitous, it is found in Eukarya, Archaea, and Bacteria. Its cellular importance is underscored by the observation that many organisms can synthesize it de novo as well as scavenge it from nutrient sources. Lipoic acid is also gaining momentum as a broad-spectrum antioxidant, and in some countries has been prescribed since the 1960s for the treatment of a number of diseases associated with oxidative stress. The participation of lipoic acid in its role as a cofactor has been studied in detail, particularly with respect to the pyruvate dehydrogenase complex (PDC), the multienzyme complex that serves as the prototype for enzymes that utilize it as a cofactor. By contrast, the pathways by which the cofactor is formed have only fairly recently been illuminated. This chapter will describe these pathways, most clearly understood in Escherichia coli, and highlight the enzymology associated with the participant enzymes.

Publisher Summary This chapter provides significant information on mitochondrial diseases of the kidney. Many chronic renal progressive diseases result from nephron degeneration. Focal segmental glomerosclerosis could represent the final common pathway for this degenerative disorder. The initial changes are believed to occur in glomerular epithelial cells. The A3243G point mutation in the tRNALeu is considered to be the most common mtDNA defect. The A3243G mutation is shown to cause type II diabetes with deafness, cardiomyopathy, or progressive external ophthalmoplegia. Oncocytomas are benign tumors predominantly occurring in the kidney. Histologically, they reveal epithelial cells with granular eosinophilic cytoplasm. These cells have an increased number of mitochondria and the mitochondria are dysmorphic. While patients with Fanconi’s syndrome show many electrolyte abnormalities such as hypophosphatemia, hypokalemia, and hypomagnesemia, there are a large number of patients with electrolyte abnormalities and mitochondrial mutations who lack aminoaciduria, acidosis, and glucosuria. There is no effective treatment for diseases of the mitochondria. Treatment is primarily symptomatic. In addition, patients should avoid respiratory chain toxic medications. Patients with complex III deficiency may benefit from vitamin K-3 or coenzyme Q10. Patients with complex I deficiency may be treated with riboflavin. Vitamin C has been prescribed to prevent reactive oxidative species (ROS) damage. Dichloroacetate, an activator of pyruvate dehydrogenase complex, accelerates the oxidation of glucose, lactate, and pyruvate to acetylcoenzyme A. This product has been used at 25-200 mg/kg and improvement has been observed in patients with lactic acidosis.

Publisher Summary This chapter focuses on the biochemical assays for mitochondrial activity, which is assay of tricarboxylic acid (TCA) cycle enzymes and pyruvate dehydrogenase complex (PDHc). Defects in one of the counterparts is likely compatible with life but present clinically in a manner that seems far from a TCA cycle defect, as in mitochondrial DNA (mtDNA) replication defects or tumor formation. With the advancement of linkage analysis techniques, it is expected that molecular defects of most TCA components will be identified. Enzymatic methodologies will, therefore, be indispensable for confirming pathogenicity and the study of disease mechanisms. The assays may be performed on tissue homogenates or in isolated mitochondria. Congenital defects of TCA enzymes were previously thought to be rare, overshadowed by the more common defects in PDHc and the mitochondrial respiratory chain (MRC) disruption of the sample before assay is a crucial step. As opposed to the significant proportion of MRC disorders, which are maternally inherited, all PDHc and TCA defects are inherited as Mendelian traits.The most suitable disruption method depends on the enzyme and the material to be assayed. Several methods are employed, including treatment with various detergents such as Triton X-100, cholic acid, or dodecyl maltoside. A number of assays have been the subject of modifications by many distinguished laboratories. Thus, each laboratory should adapt the preferred method, optimizing sample preparation, disruption, and assay conditions to aim for a linear reaction with increased signal—background ratio.