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The Warburg effect describes the circumstance that tumor cells preferentially use glycolysis rather than oxidative phosphorylation for energy production. It has been reported that this metabolic reconfiguration originates from a switch in the expression of alternative splice forms (PKM1 and PKM2) of the glycolytic enzyme pyruvate kinase (PK), which is also important for malignant transformation. However, analytical evidence for this assumption was still lacking. Using mass spectrometry, we performed an absolute quantification of PKM1 and PKM2 splice isoforms in 25 human malignant cancers, 6 benign oncocytomas, tissue matched controls, and several cell lines. PKM2 was the prominent isoform in all analyzed cancer samples and cell lines. However, this PKM2 dominance was not a result of a change in isoform expression, since PKM2 was also the predominant PKM isoform in matched control tissues. In unaffected kidney, lung, liver, and thyroid, PKM2 accounted for a minimum of 93% of total PKM, for 80% - 96% of PKM in colon, and 55% - 61% of PKM in bladder. Similar results were obtained for a panel of tumor and non-transformed cell lines, where PKM2 was the predominant form. Thus, our results reveal that an exchange in PKM1 to PKM2 isoform expression during cancer formation is not occurring, nor do these results support conclusions that PKM2 is specific for proliferating, and PKM1 for non-proliferating tissue.

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

Lipoate is a covalently bound cofactor essential for five redox reactions in humans: in four 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). Two enzymes are from the energy metabolism, α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; and three are from the amino acid metabolism, branched-chain ketoacid dehydrogenase, 2-oxoadipate dehydrogenase, and the GCS. All these enzymes consist of multiple subunits and share a similar architecture. Lipoate synthesis in mitochondria involves mitochondrial fatty acid synthesis up to octanoyl-acyl-carrier protein; and three lipoate-specific steps, including octanoic acid transfer to glycine cleavage H protein by lipoyl(octanoyl) transferase 2 (putative) (LIPT2), lipoate synthesis by lipoic acid synthetase (LIAS), and lipoate transfer by lipoyltransferase 1 (LIPT1), which is necessary to lipoylate the E2 subunits of the 2-oxoacid dehydrogenases. The reduced form dihydrolipoate is reactivated by dihydrolipoyl dehydrogenase (DLD). Mutations in LIAS have been identified that result in a variant form of nonketotic hyperglycinemia with early-onset convulsions combined with a defect in mitochondrial energy metabolism with encephalopathy and cardiomyopathy. LIPT1 deficiency spares the GCS, and resulted in a combined 2-oxoacid dehydrogenase deficiency and early death in one patient and in a less severely affected individual with a Leigh-like phenotype. As LIAS is an iron-sulphur-cluster-dependent enzyme, a number of recently identified defects in mitochondrial iron-sulphur cluster synthesis, including NFU1, BOLA3, IBA57, GLRX5 presented with deficiency of LIAS and a LIAS-like phenotype. As in DLD deficiency, a broader clinical spectrum can be anticipated for lipoate synthesis defects depending on which of the affected enzymes is most rate limiting.

Purpose: Many solid tumors exhibit abnormal aerobic metabolism characterized by increased glycolytic capacity and decreased cellular respiration. Recently, mutations in the nuclear encoded mitochondrial enzymes fumarate hydratase and succinate dehydrogenase have been identified in certain tumor types, thus demonstrating a direct link between mitochondrial energy metabolism and tumorigenesis. Although mutations in the mitochondrial genome (mitochondrial DNA, mtDNA) also can affect aerobic metabolism and mtDNA alterations are frequently observed in tumor cells, evidence linking respiratory chain deficiency in a specific tumor type to a specific mtDNA mutation has been lacking. Experimental Design: To identify mitochondrial alterations in oncocytomas, we investigated the activities of respiratory chain enzymes and sequenced mtDNA in 15 renal oncocytoma tissues.Results: Here, we show that loss of respiratory chain complex I (NADH/ubiquinone oxidoreductase) is associated with renal oncocytoma. Enzymatic activity of complex I was undetectable or greatly reduced in the tumor samples (n = 15). Blue Native gel electrophoresis of the multisubunit enzyme complex revealed a lack of assembled complex I. Mutation analysis of the mtDNA showed frame-shift mutations in the genes of either subunit ND1, ND4, or ND5 of complex I in 9 of the 15 tumors. Conclusion: Our data indicate that isolated loss of complex I is a specific feature of renal oncocytoma and that this deficiency is frequently caused by somatic mtDNA mutations.A shift in cellular energy production from aerobic oxidation in mitochondria to anaerobic glycolysis is a fundamental property of cancer cells, also called the Warburg effect (1, 2). Otto Warburg postulated that damage of the aerobic energy metabolism is a primary and irreversible event in tumor formation (2). Recently, this hypothesis has been supported by the demonstration that mutations of single enzymes of the mitochondrial energy metabolism are associated with tumorigenesis (3, 4). Germline mutations in distinct subunits of succinate dehydrogenase predispose to hereditary paragangliomas and pheochromocytomas (4, 5). Germline mutations in the fumarate hydratase gene can either cause leiomyoma, leiomyosarcoma, and renal cell carcinoma (3) or Leydig cell tumor (6). Therefore, succinate dehydrogenase and fumarate hydratase are regarded as mitochondrial tumor suppressors for these types of tumors (5).Mitochondrial DNA (mtDNA), the small genome of the mitochondrion, is essential for aerobic energy metabolism and encodes some of the subunits of respiratory chain complexes I, III, and IV, as well as the F 1 F 0 -ATP synthase. Complex I consists of 46 different subunits, with a molecular mass totaling 980 kDa. Seven of these subunits (ND1 -ND6 and ND4L) are encoded by mtDNA; the other complex I genes are located on nuclear chromosomes.Because of its essential role in energy metabolism, the mitochondrial genome has long been suspected of contributing to metabolic alterations in tumors. Such investigations d...

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Our findings establish MFF loss of function as a cause of disturbed mitochondrial and peroxisomal dynamics associated with early-onset Leigh-like basal ganglia disease. We suggest that, even if laboratory findings are not indicative of mitochondrial or peroxisomal dysfunction, the co-occurrence of optic and/or peripheral neuropathy with seizures warrants genetic testing for MFF mutations.

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals’ samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp−/− mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp−/− MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia.