Can a single subunit yeast NADH dehydrogenase (Ndi1) remedy diseases caused by respiratory complex I defects?

The proton-translocating NADH-quinone oxidoreductase (complex I) is one of five enzyme complexes in the oxidative phosphorylation system in mammalian mitochondria. Complex I is composed of 46 different subunits, 7 of which are encoded by mitochondrial DNA. Defects of complex I are involved in many human mitochondrial diseases; therefore, the authors proposed to use the NDI1 gene encoding a single subunit NADH dehydrogenase of Saccharomyces cerevisiae for repair of respiratory activity. The yeast NDI1 gene was successfully introduced into 10 mammalian cell lines (two of which were complex I-deficient mutants). The expressed Ndi1 protein was correctly targeted to the matrix side of the inner mitochondrial membranes, was fully functional, and restored the NADH oxidase activity to the complex I-deficient cells. The NDI1-transduced cells were more resistant to complex I inhibitors and diminished production of reactive oxygen species. It was further shown that the Ndi1 protein can be functionally expressed in tissues such as skeletal muscles and brain of rodents. The Ndi1 expression scarcely induced an inflammatory response as assessed by hematoxylin and eosin (H&E) staining. The Ndi1 protein expressed in the substantia nigra (SN) elicited protective effects against neurodegeneration caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The Ndi1 protein has a great potential as a molecular remedy for complex I deficiencies.     

Expression of the yeast NADH dehydrogenase Ndi1 in Drosophila confers increased lifespan independently of dietary restriction

Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling.     

Restoration of electron transport without proton pumping in mammalian mitochondria

We have restored the CoQ oxidative capacity of mouse mtDNA-less cells (ρ° cells) by transforming them with the alternative oxidase Aox of Emericella nidulans. Cotransforming ρ° cells with the NADH dehydrogenase of Saccharomyces cerevisiae, Ndi1 and Aox recovered the NADH DH/CoQ reductase and the CoQ oxidase activities. CoQ oxidation by AOX reduces the dependence of ρ° cells on pyruvate and uridine. Coexpression of AOX and NDI1 further improves the recycling of NAD⁺. Therefore, 2 single-protein enzymes restore the electron transport in mammalian mitochondria substituting >80 nuclear DNA-encoded and 11 mtDNA-encoded proteins. Because those enzymes do not pump protons, we were able to split electron transport and proton pumping (ATP synthesis) and inquire which of the metabolic deficiencies associated with the loss of oxidative phosphorylation should be attributed to each of the 2 processes.     

The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates

Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I–V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD⁺- and ubiquinone- (UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1–NAD⁺ and Ndi1–UQ2 complexes show overlapping binding sites for the NAD⁺ and quinone substrates.     

The pleiotropic effects of decanoic acid treatment on mitochondrial function in fibroblasts from patients with complex I deficient Leigh syndrome

There is growing interest in the use of the ketogenic diet (KD) to treat inherited metabolic diseases including mitochondrial disorders. However, neither the mechanism whereby the diet may be working, nor if it could benefit all patients with mitochondrial disease, is known. This study focusses on decanoic acid (C10), a component of the medium chain triglyceride KD, and a ligand for the nuclear receptor PPAR-γ known to be involved in mitochondrial biogenesis. The effects of C10 were investigated in primary fibroblasts from a cohort of patients with Leigh syndrome (LS) caused by nuclear-encoded defects of respiratory chain complex I, using mitochondrial respiratory chain enzyme assays, gene expression microarray, qPCR and flow cytometry. Treatment with C10 increased citrate synthase activity, a marker of cellular mitochondrial content, in 50 % of fibroblasts obtained from individuals diagnosed with LS in a PPAR-γ-mediated manner. Gene expression analysis and qPCR studies suggested that treating cells with C10 supports fatty acid metabolism, through increasing ACADVL and CPT1 expression, whilst downregulating genes involved in glucose metabolism (PDK3, PDK4). PCK2, involved in blocking glucose metabolism, was upregulated, as was CAT, encoding catalase. Moreover, treatment with C10 also decreased oxidative stress in complex I deficient (rotenone treated) cells. However, since not all cells from subjects with LS appeared to respond to C10, prior cellular testing in vitro could be employed as a means for selecting individuals for subsequent clinical studies involving C10 preparations.     

Immunometabolic profiling of T cells from patients with relapsing-remitting multiple sclerosis reveals an impairment in glycolysis and mitochondrial respiration

BackgroundMetabolic reprogramming is shaped to support specific cell functions since cellular metabolism controls the final outcome of immune response. Multiple sclerosis (MS) is an autoimmune disease resulting from loss of immune tolerance against central nervous system (CNS) myelin. Metabolic alterations of T cells occurring during MS are not yet well understood and their studies could have relevance in the comprehension of the pathogenetic events leading to loss of immune tolerance to self and to develop novel therapeutic strategies aimed at limiting MS progression.Methods and ResultsIn this report, we observed that extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), indicators of glycolysis and oxidative phosphorylation, respectively, were impaired during T cell activation in naïve-to-treatment relapsing remitting (RR)MS patients when compared with healthy controls. These results were also corroborated at biochemical level by a reduced expression of the glycolitic enzymes aldolase, enolase 1, hexokinase I, and by reduction of Krebs cycle enzymes dihydrolipoamide-S-acetyl transferase (DLAT) and dihydrolipoamide-S-succinyl transferase (DLST). Treatment of RRMS patients with interferon beta-1a (IFN beta-1a) was able to restore T cell glycolysis and mitochondrial respiration as well as the amount of the metabolic enzymes to a level comparable to that of healthy controls. These changes associated with an up-regulation of the glucose transporter-1 (GLUT-1), a key element in intracellular transport of glucose.ConclusionsOur data suggest that T cells from RRMS patients display a reduced engagement of glycolysis and mitochondrial respiration, reversible upon IFN beta-1a treatment, thus suggesting an involvement of an altered metabolism in the pathogenesis of MS.     

Host-derived glucose and its transporter in the obligate intracellular pathogen Toxoplasma gondii are dispensable by glutaminolysis

Toxoplasma gondii, as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgST1–3), of which TgST2 is expressed at its surface, whereas TgST1 and TgST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike Δtgst2, the modest impairment in Δtggt1 and Δtggt1/Δtgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The Δtggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1-complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in Δtggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of Δtggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells.     

Studies of foetal mouse hearts in organ culture: metabolic requirements for prolonged function in vitro and the influence of cardiac maturation on substrate utilization

Isolated, intact hearts from foetal mice of 14 to 21 days' gestational age survive and beat in organ culture for several days when the sole external substrate available for energy production is pyruvate, acetoacetate, β-hydroxybutyrate, octanoate, or glucose. Survival with glucose is significantly longer than with the other substrates. Inhibition of oxidative phosphorylation or of glycolysis causes early death of the hearts. Utilization of glucose is greater in hearts of younger (14 to 17 day) foetuses than in those of older foetuses near term (> 19 days' gestation). Release of lactate is also greater in younger foetuses, both in absolute terms and as a proportion of the glucose consumed. The results suggest that long-term function in vitro of intact hearts of late-foetal mice requires the utilization of glucose as well as the oxidation of substrates through the tricarboxylic acid cycle. Glycolytic capacity assumes relatively less importance in cardiac metabolism as the foetus approaches term, but the availability of glucose remains essential for prolonged function.     

Carbohydrate Catabolism and the Enhancement of Uptake of Galactose in Hamster Cells Transformed by Polyoma Virus

Untransformed as well as polyoma virus-transformed hamster cells can be grown equally well on a slow catabolite like galactose as on a rapid catabolite like glucose. The rate of uptake of galactose is greatly enhanced in the transformed cells as compared with untransformed cells, and this enhancement of entry was as markedly expressed in galactose-grown cultures as in glucose-grown cultures. Since the transformed cultures grown in glucose medium consume practically all of their carbohydrate, contrary to the galactose-grown cultures, problems dealing with regulation of transport by substrate concentrations have to be dealt with also. The galactose captured by the cells accumulates initially as galactose, α-galactose-1-phosphate, and UDP-galactose. However, after a 24-hr growth on a galactose growth medium, the product accumulated was almost exclusively galactitol. In spite of the enhancement of entry of galactose into the transformed cells, the metabolic pathway becomes stalled even before it has reached the stage of glucose-1-phosphate, largely due to a choke of the enzyme UDP-galactose-4-epimerase (EC 5.1.3.2). Among the sparse amounts of catabolic products generated by the transformed cells from galactose, carbon dioxide (allebeit no D-xylose) and lactic acid were found, both of them in much smaller amounts than seen if glucose is the carbohydrate source. Also, growth of transformed cells on a galactose medium gradually tends to become oriented, a phenomenon that could be called “contact promotion.” Subsequent addition of glucose disturbs the oriented growth and interferes with contact promotion.     

Inactivation of chemokine (C-C motif) receptor 1 (CCR1) suppresses colon cancer liver metastasis by blocking accumulation of immature myeloid cells in a mouse model

Recent reports have suggested critical roles of myeloid cells in tumor invasion and metastasis, although these findings have not led to therapeutics. Using a mouse model for liver dissemination, we show that mouse and human colon cancer cells secrete CC-chemokine ligands CCL9 and CCL15, respectively, and recruit CD34⁺ Gr-1⁻ immature myeloid cells (iMCs). They express CCL9/15 receptor CCR1 and produce matrix metalloproteinases MMP2 and MMP9. Lack of the Ccr1, Mmp2, or Mmp9 gene in the host dramatically suppresses outgrowths of disseminated tumors in the liver. Importantly, CCR1 antagonist BL5923 blocks the iMC accumulation and metastatic colonization and significantly prolongs the survival of tumor-bearing mice. These results suggest that CCR1 antagonists can provide antimetastatic therapies for patients with disseminated colon cancer in the liver.     

Inhibition of gluconeogenesis by tolbutamide in isolated rat hepatocytes: modulation of glucose-6-phosphate substrate cycle

In hepatocytes isolated from 24-hour fasted rats, the oral hypoglycemic agent tolbutamide (1 mmol/L) inhibited glucose formation from different concentrations (1 to 20 mmol/L) of galactose, dihydroxyacetone, glycerol, and a mixture of L-lactate:pyruvate (molar ratio, 10:1). Parallel to the reduction of gluconeogenesis, tolbutamide stimulated L-lactate formation when cells were incubated with either galactose, dihydroxyacetone, or glycerol. All these tolbutamide effects occurred without significant modification of hepatocyte fructose-2,6-bisphosphate (F-2,6-P2) levels. Only when glucose was included in the incubation medium was the inhibition of gluconeogenesis caused by the sulfonylurea accompanied by a significant increment of the cellular F-2,6-P2 concentration. Under these conditions, tolbutamide potentiated the effect of glucose in promoting the increase of this regulatory metabolite, as well as the stimulation of glycolysis; in addition, tolbutamide increased the cellular pool of hexose-6-phosphates and the rate of tritium release from (2-3H)glucose. These results support the hypothesis that tolbutamide regulates hepatic glucose metabolism, at least, by modulating the glucose-6-phosphate substrate cycle.     

Immune profile and mitotic index of metastatic melanoma lesions enhance clinical staging in predicting patient survival

Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. We used gene expression profiling, mitotic index (MI), and quantification of tumor infiltrating leukocytes (TILs) and CD3+ cells in metastatic lesions to search for a molecular basis for this observation and to develop improved methods for predicting patient survival. We identified a group of 266 genes associated with postrecurrence survival. Genes positively associated with survival were predominantly immune response related (e.g., ICOS, CD3d, ZAP70, TRAT1, TARP, GZMK, LCK, CD2, CXCL13, CCL19, CCR7, VCAM1) while genes negatively associated with survival were cell proliferation related (e.g., PDE4D, CDK2, GREF1, NUSAP1, SPC24). Furthermore, any of the 4 parameters (prevalidated gene expression signature, TILs, CD3, and in particular MI) improved the ability of Tumor, Node, Metastasis (TNM) staging to predict postrecurrence survival; MI was the most significant contributor (HR = 2.13, P = 0.0008). An immune response gene expression signature and presence of TILs and CD3+ cells signify immune surveillance as a mechanism for prolonged survival in these patients and indicate improved patient subcategorization beyond current TNM staging.     

An Alteration in Glucose Metabolism Associated with a Defect in Ketone Body Metabolism

Skin fibroblasts (CC-69) cultured from a patient with a unique syndrome of ketoacidosis associated with coenzyme A transferase (EC 2.8.3.5) deficiency showed an altered pattern of carbohydrate metabolism. These cells used glucose at a rate significantly less than controls (125 against 680 nmol/mg per hr). The oxidation of [6-¹⁴C]glucose to ¹⁴CO₂ by these cells was also significantly diminished (12 against 350 pmol/mg per hr), but [2-¹⁴C]pyruvate and [1,4-¹⁴C]succinate oxidation by these cells did not differ from that by control cells. Measurements of glycolytic intermediates showed a reduction of several intermediates in the CC-69 cells that confirmed an inhibition of glycolysis between fructose-1,6-bisphosphate and pyruvate. The apparent inhibition in these cells could be reversed by an extended incubation of the cells in a buffered glucose solution. After 18 hr of incubation in 2.5 mM glucose, glucose uptake by the CC-69 cells increased 20-fold to 2560 nmol/mg per hr, whereas the rate for control cells remained constant at 640 ± 90. Concomitant with this increase, [6-¹⁴C]glucose oxidation rose from 8 to 2261 pmol/mg per hr while controls remained constant at 428 ± 175. This change was not due to new enzyme formation because incubation with puromycin had no effect on the increased use of glucose. Mixing experiments demonstrated no transfer of a permeable inhibitor or activating substances. In view of the deficiency of coenzyme A transferase in these cells, the data suggest an indirect regulatory role for this enzyme in peripheral tissue glycolysis.     

The synergism of MCL1 and glycolysis on pediatric acute lymphoblastic leukemia cell survival and prednisolone resistance

In vitro and in vivo resistance to prednisolone are predictive for an adverse prognosis in pediatric precursor B-acute lymphoblastic leukemia. Causes of resistance are still poorly understood. In this study, we observed that prednisolone exposure of prednisolone-sensitive patients’ leukemic cells decreased anti-apoptotic MCL1 protein levels by 2.9-fold, while MCL1 protein expression in prednisolone-resistant leukemic patients’ cells was unaffected (P<0.01). Locked nucleic acid oligonucleotides directed against MCL1 reduced MCL1 protein levels by 82±16% (P<0.05) in leukemic cells, decreased proliferation by 9-fold and sensitized to prednisolone up to 80.8-fold, compared to a non-silencing-control locked nucleic acid (P<0.05). Remarkably, we discovered that MCL1-silencing up-regulated the glucose consumption of leukemic cells by 2.5-fold (P<0.05), suggesting a potential rescue mechanism mediated by glycolysis. Targeting glycolysis by 2-deoxyglucose synergistically inhibited leukemic survival by 23.2-fold in MCL1-silenced cells (P<0.05). Moreover, 2-deoxyglucose and MCL1 locked nucleic acid concomitantly sensitized leukemic cells to prednisolone compared to MCL1 locked nucleic acid or 2-deoxyglucose alone (P<0.05). In conclusion, these results indicate the need to target both MCL1 and glycolysis simultaneously to inhibit leukemic survival and sensitize acute leukemia patients towards prednisolone.     

The loss of Sirt1 in mouse pancreatic beta cells impairs insulin secretion by disrupting glucose sensing

Aims/hypothesis

Sirtuin 1 (SIRT1) has emerged as a key metabolic regulator of glucose homeostasis and insulin secretion. Enhanced SIRT1 activity has been shown to be protective against diabetes, although the mechanisms remain largely unknown. The aim of this study was to determine how SIRT1 regulates insulin secretion in the pancreatic beta cell.

Methods

Pancreatic beta cell-specific Sirt1 deletion was induced by tamoxifen injection in 9-week-old Pdx1CreER:floxSirt1 mice (Sirt1BKO). Controls were injected with vehicle. Mice were assessed metabolically via glucose challenge, insulin tolerance tests and physical variables. In parallel, Sirt1 short interfering RNA-treated MIN6 cells (SIRT1KD) and isolated Sirt1BKO islets were used to investigate the effect of SIRT1 inactivation on insulin secretion and gene expression.

Results

OGTTs showed impaired glucose disposal in Sirt1BKO mice due to insufficient insulin secretion. Isolated Sirt1BKO islets and SIRT1KD MIN6 cells also exhibited impaired glucose-stimulated insulin secretion. Subsequent analyses revealed impaired α-ketoisocaproic acid-induced insulin secretion and attenuated glucose-induced Ca²⁺ influx, but normal insulin granule exocytosis in Sirt1BKO beta cells. Microarray studies revealed a large cluster of mitochondria-related genes, the expression of which was dysregulated in SIRT1KD MIN6 cells. Upon further analysis, we demonstrated an explicit defect in mitochondrial function: the inability to couple nutrient metabolism to mitochondrial membrane hyperpolarisation and reduced oxygen consumption rates.

Conclusions/interpretation

Taken together, these findings indicate that in beta cells the deacetylase SIRT1 regulates the expression of specific mitochondria-related genes that control metabolic coupling, and that a decrease in beta cell Sirt1 expression impairs glucose sensing and insulin secretion.     

Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase activity that is a functional analog of the Fpg protein from Escherichia coli and excises 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged DNA. The repair of this ubiquitous kind of oxidative damage is essential to prevent mutations both in bacteria and in yeast. A human cDNA clone carrying an ORF displaying homology to the yeast protein was identified. The predicted protein has 345 amino acids and a molecular mass of 39 kDa. This protein shares a 38% sequence identity with the yeast Ogg1 protein, adding this novel human gene product to the growing family of enzymes that the repair of oxidatively damaged bases and are related to the E. coli endonuclease III. Northern blot analysis indicates that this gene, localized to chromosome 3p25, is ubiquitously expressed in human tissues. The cloned coding sequence was expressed in an E. coli strain that carried a disrupted fpg gene, the bacterial functional analog of OGG1. Cell-free extracts from these cultures displayed a specific lyase activity on duplex DNA that carried an 8-oxoG/C base pair. The products of the reaction are consistent with an enzymatic activity like the one displayed by the yeast Ogg1. Analysis of the substrate specificity reveals a very strong preference for DNA fragments harboring 8-oxoG/C base pairs. The pattern of specificity correlates well with the one found for the yeast enzyme. Moreover, when the human coding sequence was expressed in a yeast strain mutant in OGG1 it was able to complement the spontaneous mutator phenotype. These results make this novel gene (hOGG1) a strong candidate for the human homolog of the yeast OGG1 and suggest an important role of its product in the protection of the genome from the mutagenic effects of the oxidatively damaged purines.