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International audience; Computational phylogenetic analysis coupled to promoter sequence alignment was used to understand mechanisms of transcriptional regulation and to identify potentially coregulated genes. Our strategy was validated on the human ANT4 gene which encodes the fourth isoform of the mitochondrial adenine nucleotide translocator specifically expressed during spermatogenesis. The movement of sperm flagella is driven mainly by ATP generated by glycolytic pathways, and the specific induction of the mitochondrial ANT4 protein presented an interesting puzzle. We analysed the sequences of the promoters, introns and exons of 30 mammalian ANT4 genes and constructed regulatory models. The whole human genome and promoter database were screened for genes that were potentially regulated by the generated models. 80% of the identified co-regulated genes encoded proteins with specific roles in spermatogenesis and with functions linked to male reproduction. Our in silico study enabled us to precise the specific role of the ANT4 isoform in spermatozoid bioenergetics.

International audience; Background: BRCA1, the main breast and ovarian cancer susceptibility gene, has a key role in maintenance of genome stability, cell cycle and transcription regulation. Interestingly, some of the numerous proteins which interact with BRCA1 protein undergo conjugation with small ubiquitin-like modifiers (SUMO). This post-translational modification is related to transcription, DNA repair, nuclear transport, signal transduction, and to cell cycle stress response. Methods and results: Protein sequence analysis suggests that sumoylation target sites belong to the RING finger and BRCT domains (BRCA1 C-terminus), two crucial regions for BRCA1 function. Moreover putative SUMO interacting motifs are present in the sequence of many proteins of BRCA1 network. Using immunoprecipitations and western blotting, we show the conjugation of endogenous nuclear BRCA1 protein with SUMO-2/3. BRCA1 conjugation with SUMO-2/3 is linked to the cell cycle in a cell line dependent manner since no cell cycle dependence of sumoylation is observed in MCF7 breast cancer cells. In contrast, BRCA1 conjugation with SUMO-2/3 is linked to the oxidative stress independently to the cell line, in DU145, MCF7 and 293 T cells. Conclusion and general significance: Our data reveal a new BRCA1 regulation pathway implying sumoylation in response to cell cycle progression and oxidative stress, providing a possible mechanism for the involvement of BRCA1 gene in tumorigenesis. (C) 2010 Elsevier B.V. All rights reserved.

Methionine (Met) deprivation stress (MDS) is proposed in association with chemotherapy in the treatment of some cancers. A synergistic effect of this combination is generally acknowledged. However, little is known on the mechanism of the response to this therapeutic strategy. A model of B16 melanoma tumor in vivo was treated by MDS alone and in combination with chloroethylnitrosourea (CENU). It was applied recent developments in proton-NMR spectroscopy-based metabolomics for providing information on the metabolic response of tumors to MDS and combination with chemotherapy. MDS inhibited tumor growth during the deprivation period and growth resumption thereafter. The combination of MDS with CENU induced an effective time-dependent synergy on growth inhibition. Metabolite profiling during MDS showed a decreased Met content (P < 0.01) despite the preservation of the protein content, disorders in sulfur-containing amino acids, glutamine/proline, and phospholipid metabolism [increase of glycerophosphorylcholine (P < 0.01), decrease in phosphocholine (P < 0.05)]. The metabolic profile of MDS combined with CENU and ANOVA analysis revealed the implication of Met and phospholipid metabolism in the observed synergy, which may be interpreted as a Met-sparing metabolic reprogramming of tumors. It follows that combination therapy of MDS with CENU seems to intensify adaptive processes, which may set limitations to this therapeutic strategy.

Cancer cells mainly rely on glycolysis for energetic needs, and mitochondrial ATP production is almost inactive. However, cancer cells require the integrity of mitochondrial functions for their survival, such as the maintenance of the internal membrane potential gradient (DeltaPsim). It thus may be predicted that DeltaPsim regeneration should depend on cellular capability to produce sufficient ATP by upregulating glycolysis or recruiting oxidative phosphorylation (OXPHOS). To investigate this hypothesis, we compared the response to an anticancer agent chloroethylnitrosourea (CENU) of two transformed cell lines: HepG2 (hepatocarcinoma) with a partially differentiated phenotype and 143B (osteosarcoma) with an undifferentiated one. These cells types differ by their mitochondrial OXPHOS background; the most severely impaired being that of 143B cells. Treatment effects were tested on cell proliferation, O(2) consumption/ATP production coupling, DeltaPsim maintenance, and global metabolite profiling by NMR spectroscopy. Our results showed an OXPHOS uncoupling and a lowered DeltaPsim, leading to an increased energy request to regenerate DeltaPsim in both models. However, energy request could not be met by undifferentiated cells 143B, which ATP content decreased after 48 h leading to cell death, while partially differentiated cells (HepG2) could activate their oxidative metabolism and escape chemotherapy. We propose that mitochondrial OXPHOS background confers a survival advantage to more differentiated cells in response to chemotherapy. This suggests that the mitochondrial bioenergetic background of tumors should be considered for anticancer treatment personalization.

International audience; Mitochondrial respiratory chain deteriorates with age, mostly in tissues with high energy requirements. Damage to mitochondrial DNA (mtDNA) by reactive oxygen species is thought to contribute primarily to this impairment. However, the overall extent of random mtDNA mutations has still not been evaluated. We carried out molecular and biochemical analyses in muscle biopsies from healthy young and aged subjects. Deleted mtDNA accumulation was followed by both quantitative PCR analysis to quantify total mtDNA, and Southern-blotting, to determine deleted to full length mtDNA ratio. Enzymatic activities of the mitochondrial respiratory chain were measured in all subjects. Randomly deleted mtDNA appeared mainly in the oldest subjects (beyond 80 years old), affecting up to 70% of mtDNA molecules. The activities of complexes III and IV of the respiratory chain, complexes with mtDNA encoded subunits, are lower in the aged subjects. Physical activity could be one major parameter modulating the mitochondrial respiratory chain activity in aged muscle.

La translocase des nucléotides adényliques (ANT) réalise l’échange ATP/ADP entre le cytoplasme et la mitochondrie. Les isoformes ANT1 (musculaire) et ANT3 (ubiquitaire) exportent l’ATP produit par les phosphorylations oxydatives mitochondriales. L’isoforme ANT2 est spécifiquement exprimée dans les cellules en prolifération, dotées d’un métabolisme majoritairement glycolytique. ANT2 est ainsi associée à la dédifférenciation cellulaire, caractéristique majeure de la cancérogenèse. Son rôle serait d’importer dans la mitochondrie l’ATP produit par la glycolyse, énergie indispensable à différentes fonctions intramitochondriales, notamment au maintien du gradient de potentiel membranaire qui conditionne la survie et la prolifération cellulaires. Le mécanisme de régénération de ce gradient pourrait impliquer trois protéines majeures : l’hexokinase II, l’ANT2 et la partie F1 de l’ATP synthétase mitochondriale. Ainsi, l’ANT2, grâce à son rôle déterminant dans la croissance de la cellule tumorale, pourrait être choisie comme cible dans une stratégie anticancéreuse., In the mitochondrial internal membrane, the adenine nucleotide translocator (ANT) carries out the ATP/ADP exchange between cytoplasm and mitochondrial matrix. Three isoforms with different kinetic properties are encoded from three different genes in Human : the muscle specific ANT1 and the ubiquitary ANT3 isoforms export ATP produced by mitochondrial oxidative phosphorylation (OXPHOS). The ANT2 isoform is specifically expressed in proliferative cells with a predominant glycolytic metabolism and is associated with cellular undifferentiation which is a major characteristic in carcinogenesis. Its role would be to import into mitochondria ATP produced by the glycolysis, energy essential to several intramitochondrial functions, particularly to maintenance of the membrne potential (ΔΨm), conditioning cellular survival and proliferation. The mechanism of regeneration of this ΔΨm gradient would involve at least three major proteins : the hexokinase II isoform, the ANT2 isoform and the F1 part of the mitochondrial ATP synthase complex. Taking into account this major role of ANT2 in cell proliferation and the very low expression of this isoform in differentiated tissues, this protein or its transcript could be chosen as a target for an anticancer strategy. Furthermore, previous studies showed that molecules of the cisplatin family, used as chemotherapeutic agents, led to the destruction of the mitochondrial membrane potential and thus to cell death. Does the anticancer effect of these molecules result, at least partially, from this mitochondrial aggression ? If it is the case, the ANT2 isoform, mainly involved in the generation of this potential by its ATP4–/ADP3– exchange, could be considered as a more specific targeting by an RNA interference approach.

Under hypoxic conditions, mitochondrial ATP production ceases, leaving cells entirely dependent on their glycolytic metabolism. The cytoplasmic and intramitochondrial ATP/ADP ratios, partly controlled by the adenine nucleotide translocator (ANT), are drastically modified. In dividing and growing cells that have a predominantly glycolytic metabolism, the ANT isoform 2, which has kinetic properties allowing ATP import into mitochondria, is over-expressed in comparison to control cells. We studied the cellular metabolic and proliferative response to hypoxia in two transformed human cell lines with different metabolic backgrounds: HepG2 and 143B, and in their rho(o) derivatives, i.e., cells with no mitochondrial DNA. Transformed 143B and rho(o) cells continued their proliferation whereas HepG2 cells, with a more differentiated phenotype, arrested their cell-cycle at the G(1)/S checkpoint. Hypoxia induced an increase in glycolytic activity, correlated to an induction of VEGF and hexokinase II (HK II) expression. Thus, according to their tumorigenicity, transformed cells may adopt one of two distinct behaviors to support hypoxic stress, i.e., proliferation or quiescence. Our study links the constitutive glycolytic activity and ANT2 expression levels of transformed cells with the loss of cell-cycle control after oxygen deprivation. ATP import by ANT2 allows cells to maintain their mitochondrial integrity while acquiring insensitivity to any alterations in the proteins involved in oxidative phosphorylation. This loss of cell dependence on oxidative metabolism is an important factor in the development of tumors.

It has been shown previously that human rho degrees cells, deprived of mitochondrial DNA and consequently of functional oxidative phosphorylation, maintain a mitochondrial membrane potential, which is necessary for their growth. The goal of our study was to determine the precise origin of this membrane potential in three rho degrees cell lines originating from the human HepG2, 143B, and HeLa S3 cell lines. Residual cyanide-sensitive oxygen consumption suggests the persistence of residual mitochondrial respiratory chain activity, about 8% of that of the corresponding parental cells. The fluorescence emitted by the three rho degrees cell lines in the presence of a mitochondrial specific fluorochrome was partially reduced by a protonophore, suggesting the existence of a proton gradient. The mitochondrial membrane potential is maintained both by a residual proton gradient (up to 45 to 50% of the potential) and by other ion movements such as the glycolytic ATP(4-) to mitochondrial ADP(3-) exchange. The ANT2 gene, encoding isoform 2 of the adenine nucleotide translocator, is overexpressed in rho degrees HepG2 and 143B cells strongly dependent on glycolytic ATP synthesis, as compared to the corresponding parental cells, which present a more oxidative metabolism. In rho degrees HeLa S3 cells, originating from the HeLa S3 cell line, which already displays a glycolytic energy status, ANT2 gene expression was not higher as in parental cells. Mitochondrial oxygen consumption and ANT2 gene overexpression vary in opposite ways and this suggests that these two parameters have complementary roles in the maintenance of the mitochondrial membrane potential in rho degrees cells.

We have examined the transcript levels of a variety of oxidative phosphorylation (OXPHOS) and associated bioenergetic genes in tissues of a patient carrying the myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) A3243G mitochondrial DNA (mtDNA) mutation and the skeletal muscles of 14 patients harboring other pathogenic mtDNA mutations. The patients' tissues, which harbored 88% or more mutant mtDNA, had increased levels of mtDNA transcripts, increased nuclear OXPHOS gene transcripts including the ATP synthase beta subunit and the heart-muscle isoform of the adenine nucleotide translocator, and increased ancillary gene transcripts including muscle mitochondrial creatine phosphokinase, muscle glycogen phosphorylase, hexokinase I, muscle phosphofructokinase, the E1alpha subunit of pyruvate dehydrogenase, and the ubiquinone oxidoreductase. A similar coordinate induction of bioenergetic genes was observed in the muscle biopsies of severe pathologic mtDNA mutations. The more significant coordinated expression was found in muscle from patients with the MELAS, myoclonic epilepsy with ragged red fibers, and chronic progressive external ophthalmoplegia deletion syndromes, with ragged red muscle fibers and mitochondrial paracrystalline inclusions. High levels of mutant mtDNAs were linked to a high induction of the mtDNA and nuclear OXPHOS genes and of several associated bioenergetic genes. These observations suggest that human tissues attempt to compensate for OXPHOS defects associated with mtDNA mutations by stimulating mitochondrial biogenesis, possibly mediated through redox-sensitive transcription factors.

Few cases of infantile liver disease associated with mitochondrial DNA (mtDNA) depletion have been reported. Most of the patients died before 1 year of age of severe liver failure. We describe a new case, a 28-month-old child, presenting with cholestasis at age 2 months, complicated by progressive portal and lobular liver fibrosis. Growth and psychomotor development are undisturbed. There is no clinical evidence of either myopathy or neurological involvement. Metabolic investigation in plasma revealed an abnormal oxido-reduction status after fasting and after carbohydrate-rich meals. Light microscopy performed on liver biopsies revealed steatosis, abnormal hepatocytes with an "oncocytic" appearance and extensive fibrosis. Electron microscopic investigation showed an increased number of mitochondria with rare or enlarged cristae. Biochemical studies of liver biopsies showed that the respiratory chain activities containing mtDNA-encoded subunits were severely decreased (complexes I, III and IV). In contrast, the complex II activity was normal and the citrate synthase activity was greatly increased. Southern blotting analysis revealed that the ratio of mtDNA to nuclear DNA in liver was only 15% and 20% of the mean control value at ages 12 and 21 months, respectively. For this mtDNA depletion syndrome which is clinically expressed in the liver, a liver transplantation is discussed.

The expression of several mitochondrial and nuclear genes involved in ATP production was examined in cells cultured from muscle biopsies of patients harboring mitochondrial pathologies. The transcript patterns in muscle cells from the patients affected by carnitine palmitoyl transferase II or 2-ketoglutarate dehydrogenase deficiencies were almost similar to control patterns. In the opposite, patterns were strikingly abnormal in all the other cell cultures from patients with defects in enzymatic complexes involved in oxidative phosphorylation: mitochondrial complex II and III deficiencies, two MELAS syndromes (myopathy, encephalopathy, lactic acidosis and stroke like episodes), a case of Kearns-Sayre syndrome and a case of chronic progressive external ophthalmoplegia. In cultured muscle cells from patients with mtDNA mutations, the percentage of mutated mtDNA was low as compared with those determined in the corresponding skeletal muscle biopsy. Moreover, the complex II defect resulting of a nuclear mutation was not expressed in the cell cultures. Thus, an undetermined transcriptional event, transmitted from muscle biopsies to cultured muscle cells, should be involved to account for such abnormal transcript patterns.

The expression of mitochondrial and nuclear genes involved in ATP production was examined in renal carcinomas, renal oncocytomas, and a salivary oncocytoma. Renal carcinomas were found to have a reduced mitochondrial DNA (mtDNA) content while oncocytomas had increased mtDNA contents. This parallels morphological changes in mitochondrial number in these tumours. In the carcinomas, mtDNA transcripts were decreased 5- to 10-fold relative to control kidneys, suggesting that mitochondrial transcript levels depend on the mtDNA content. In renal oncocytomas, mtDNA transcripts were slightly reduced in spite of a high mtDNA content. However, in the salivary gland oncocytoma, mtDNA transcripts were increased more than 10-fold in parallel with a 10-fold increase in mtDNA content. The expression of the nuclear DNA oxidative phosphorylation genes, ATPsynβ and ANT2, was reduced up to 4-fold in renal carcinoma. In contrast, the levels of these two nuclear gene transcripts were induced about 4-fold in renal oncocytoma and up to 30-fold in salivary gland oncocytoma. Moreover, the ANT2 precursors were observed to change in oncocytomas. These data suggest a coordinated regulation of nuclear and mitochondrial gene expression in renal carcinomas and the specific induction of nuclear OXPHOS gene expression in oncocytomas.

To investigate the regulation of genes encoding the proteins involved in energy metabolism in cancer cells, we studied the expression of several mitochondrial and nuclear genes involved in ATP production. Northern blot analysis was performed on renal tumors of different types: a clear cell carcinoma, an oncocytoma, and urothelial tumors at two different stages. The steady-state transcript patterns were compared with those observed in cell lines derived from renal tumors and in transformed cell lines. Striking differences were revealed among the three types of tumors, their respective controls, and the cultured renal cells. The levels of all mitochondrial transcripts were lower in tumor biopsies and tumoral cell lines than in the normal cell types. Moreover, a higher transcript level of nuclear genes involved in oxidative phosphorylation was observed in the oncocytomas and in the more malignant urothelial tumor. Different transcript patterns were observed in each of the tumoral and transformed cell lines, explaining the difference in metabolism between the different tumors and the tumoral or transformed cell lines. In particular, a high transcript level for the adenine nucleotide translocator isoform 2(ANT2) gene, which is usually not expressed in differentiated cells, was observed in oncocytoma and malignant urothelial renal tumor. This phenomenon was also observed in renal carcinoma cell lines and transformed cells. These data provide the first argument for the involvement of the ANT2 protein in glycolytic ATP uptake in cancer cell mitochondria and suggest a possible ANT2 antisense strategy for cancer therapy.

In response to different environmental stresses, eIF2 alpha phosphorylation represses global translation coincident with preferential translation of ATF4, a master regulator controlling the transcription of key genes essential for adaptative functions. Here, we establish that the eIF2 alpha/ATF4 pathway directs an autophagy gene transcriptional program in response to amino acid starvation or endoplasmic reticulum stress. The eIF2 alpha-kinases GCN2 and PERK and the transcription factors ATF4 and CHOP are also required to increase the transcription of a set of genes implicated in the formation, elongation and function of the autophagosome. We also identify three classes of autophagy genes according to their dependence on ATF4 and CHOP and the binding of these factors to specific promoter cis elements. Furthermore, different combinations of CHOP and ATF4 bindings to target promoters allow the trigger of a differential transcriptional response according to the stress intensity. Overall, this study reveals a novel regulatory role of the eIF2 alpha-ATF4 pathway in the fine-tuning of the autophagy gene transcription program in response to stresses.

Background The analysis of gene promoters is essential to understand the mechanisms of transcriptional regulation required under the effects of physiological processes, nutritional intake or pathologies. In higher eukaryotes, transcriptional regulation implies the recruitment of a set of regulatory proteins that bind on combinations of nucleotide motifs. We developed a computational analysis of promoter nucleotide sequences, to identify co-regulated genes by combining several programs that allowed us to build regulatory models and perform a crossed analysis on several databases. This strategy was tested on a set of four human genes encoding isoforms 1 to 4 of the mitochondrial ADP/ATP carrier ANT. Each isoform has a specific tissue expression profile linked to its role in cellular bioenergetics. Results From their promoter sequence and from the phylogenetic evolution of these ANT genes in mammals, we constructed combinations of specific regulatory elements. These models were screened using the full human genome and databases of promoter sequences from human and several other mammalian species. For each of transcriptionally regulated ANT1, 2 and 4 genes, a set of co-regulated genes was identified and their over-expression was verified in microarray databases. Conclusions Most of the identified genes encode proteins with a cellular function and specificity in agreement with those of the corresponding ANT isoform. Our in silico study shows that the tissue specific gene expression is mainly driven by promoter regulatory sequences located up to about a thousand base pairs upstream the transcription start site. Moreover, this computational strategy on the study of regulatory pathways should provide, along with transcriptomics and metabolomics, data to construct cellular metabolic networks.

International audience; Adenine nucleotide translocase (ANT), a mitochondria( protein that facilitates the exchange of ADP and ATP across the mitochondrial inner membrane, plays an essential role in cellular energy metabolism. Human ANT presents four isoforms (ANT1-4), each with a specific expression depending on the nature of the tissue, cell type, developmental stage and status of cell proliferation. Thus, ANTI is specific to muscle and brain tissues; ANT2 occurs mainly in proliferative, undifferentiated cells; ANT3 is ubiquitous; and ANT4 is found in germ cells. ANT1 and ANT3 export the ATP produced by oxidative phosphorylation (OxPhos) from the mitochondria into the cytosol while importing ADP. In contrast, the expression of ANT2, which is linked to the rate of glycolytic metabolism, is an important indicator of carcinogenesis. In fact, cancers are characterized by major metabolic changes that switch cells from the normally dual oxidative and glycolytic metabolisms to an almost exclusively glycolytic metabolism. When OxPhos activity is impaired, ANT2 imports glycolytically produced ATP into the mitochondria. In the mitochondrial matrix, the F1F0-ATPase complex hydrolyzes the ATP, pumping out a proton into the intermembrane space. The reverse operations of ANT2 and F1F0-ATPase under glycolytic conditions contribute to maintaining the mitochondrial membrane potential, ensuring cell survival and proliferation. Unlike the ANTI and ANT3 isoforms, ANT2 is not pro-apoptotic and may therefore contribute to carcinogenesis. Since the expression of ANT2 is closely linked to the mitochondrial bioenergetics of tumors, it should be taken into account for individualizing cancer treatments and for the development of anticancer strategies. This article is part of a Special Issue entitled: Bioenergetics of Cancer. (C) 2010 Elsevier B.V. All rights reserved.

The expression of nuclear and mitochondrial oxidative phosphorylation (OXPHOS) genes was examined in the skeletal muscle of patients with Kearns-Sayre syndrome (KSS), myoclonic epilepsy associated with ragged red fibers (MERRF), and myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and compared with controls. In KSS muscle, mtDNA transcripts outside the deletion were elevated, while those within the deletion were reduced according to the percentage of deleted mtDNA molecules. In MERRF and MELAS muscle, mitochondrial transcripts levels were increased, but the increase was greater in MERRF muscle. The processing of mtDNA transcripts was reduced in all pathogenic muscles. This was true for full-length heavy and light strand transcripts as well as for the 16 S rRNA + tRNA(Leu)+ND1 transcript. However, the tRNA(Lys) level was reduced in all three muscles. In MELAS muscle, our results are not consistent with an impairment of transcription termination at the end of the 16 S mitochondrial rRNA. Finally, the transcription of the nuclear ATPsyn.beta and ANT1 genes was induced in parallel with the high level of mtDNA transcripts in MERRF and MELAS muscle, but was repressed in KSS muscle. The results demonstrate that the expression of nuclear and cytoplasmic OXPHOS genes is coordinated and that OXPHOS gene expression increases to compensate for respiratory deficiency. The repression of nuclear genes in KSS muscle could be a consequence of the segmental distribution of deleted mtDNA molecules in muscle cells.

The mitochondrial heart-skeletal muscle adenine nucleotide translocator (ANT1) was regionally mapped to 4q35-qter using somatic cell hybrids containing deleted chromosome 4. The regional location was further refined through family studies using ANT1 intron and promoter nucleotide polymorphisms recognized by the restriction endonucleases MboII, NdeI, and HaeIII. Two alleles were found, each at a frequency of 0.5. The ANT1 locus was found to be closely linked to D4S139, D4S171, and the dominant skeletal muscle disease locus facioscapulohumeral muscular dystrophy (FSHD). A crossover that separated D4S171 and ANT1 from D4S139 was found. Since previous studies have established the chromosome 4 map order as centromere-D4S171-D4S139-FSHD, it was concluded that ANT1 is located on the side of D4S139, that is opposite from FSHD. This conclusion was confirmed by sequencing the exons and analyzing the transcripts of ANT1 from several FSHD patients and finding no evidence of aberration.

NMR spectroscopy-based metabolomics still needs development in quantification procedures. A method was designed for quantitative two-dimensional high resolution magic angle spinning (HRMAS) proton-NMR spectroscopy-based metabolite profiling of intact cells. It uses referencing of metabolite-related NMR signals to protein-related NMR signals and yields straightforward and automatable metabolite profiling. The method enables exploitation of only two-dimensionally visible metabolites and combination of one- and two-dimensional spectra, thus providing an appreciable number of screened metabolites. With this procedure, 32 intracellular metabolites were attributed and quantified in human normal fibroblasts and tumor cells. The phenotype of several tumor cell lines (MCF7, PC3, 143B, and HepG2) was characterized by high levels of glutathione in cell lines with the higher proliferation rate, high levels of creatine, low levels of free amino acids, increased levels of phospholipid derivatives (mostly phosphocholine), and lower lactate content in cell lines with the higher proliferation rate. Other metabolites such as fatty acids differed widely among tumor cell lines. The response of tumor cell lines to chemotherapy also was evaluated by differential metabolite profiling, bringing insights into drug cytotoxicity and tumor cell adaptive mechanisms. The method may prove widely applicable to tumor cell phenotyping. Magn Reson Med 63:1172–1183, 2010. © 2010 Wiley-Liss, Inc.

The OXBOX promoter regions of the genes for the muscle-specific adenine nucleotide translocator (ANT1) and the beta subunit of the ATPsynthase (ATPsyn beta) have been implicated in the increased transcription of these nuclear-encoded oxidative phosphorylation (OXPHOS) genes in heart and skeletal muscle. DNA binding, electrophoretic mobility shift (gel-shift) assays now reveal that the OXBOX region has two unique but overlapping elements, the 13-base pair (bp) OXBOX and an 8-bp REBOX. The OXBOX binding factors are found only in myogenic cell lines, whereas the REBOX factors are ubiquitous. Methylation interference experiments have defined the boundaries of the OXBOX and REBOX elements, confirmed that the OXBOX factors are muscle-specific, and shown that the OXBOX and REBOX factors do not bind concurrently. The binding of the REBOX factors was found to be sensitive to NADH and thyroxine, suggesting that it may modulate OXPHOS gene expression in response to environmental and hormonal changes. Hence, the OXBOX/REBOX complex provides one mechanism by which mammalian energy metabolism can be adapted to developmental and environmental demands.

The adenine nucleotide translocator (ANT) catalyzes the exchange of ADP and ATP across the mitochondrial internal membrane. Its three isoforms, ANT1, ANT2, and ANT3 are coded by differentially regulated nuclear genes. The patterns of expression of these genes in human, bovine, and mouse tissue are similar. ANT1 is highly expressed in heart and skeletal muscle and is induced during myoblast differentiation. It is coordinately regulated with the nuclear gene for the mitochondrial ATP synthase beta subunit, with which it shares the positive muscle cis element, the OXBOX. ANT2 is either absent or weakly expressed in all tissues. ANT3 is ubiquitously expressed in all tissues, and its transcript level is proportional to the level of oxidative metabolism. The tissue-specific expression of the ANT gene family thus provides insight into the molecular basis of the differential reliance of mammalian tissues on oxidative phosphorylation.

Cancer cells mainly rely on glycolysis for energetic needs, and mitochondrial ATP production is almost inactive. However, cancer cells require the integrity of mitochondrial functions for their survival, such as the maintenance of the internal membrane potential gradient (DeltaPsim). It thus may be predicted that DeltaPsim regeneration should depend on cellular capability to produce sufficient ATP by upregulating glycolysis or recruiting oxidative phosphorylation (OXPHOS). To investigate this hypothesis, we compared the response to an anticancer agent chloroethylnitrosourea (CENU) of two transformed cell lines: HepG2 (hepatocarcinoma) with a partially differentiated phenotype and 143B (osteosarcoma) with an undifferentiated one. These cells types differ by their mitochondrial OXPHOS background; the most severely impaired being that of 143B cells. Treatment effects were tested on cell proliferation, O(2) consumption/ATP production coupling, DeltaPsim maintenance, and global metabolite profiling by NMR spectroscopy. Our results showed an OXPHOS uncoupling and a lowered DeltaPsim, leading to an increased energy request to regenerate DeltaPsim in both models. However, energy request could not be met by undifferentiated cells 143B, which ATP content decreased after 48 h leading to cell death, while partially differentiated cells (HepG2) could activate their oxidative metabolism and escape chemotherapy. We propose that mitochondrial OXPHOS background confers a survival advantage to more differentiated cells in response to chemotherapy. This suggests that the mitochondrial bioenergetic background of tumors should be considered for anticancer treatment personalization.

International audience; The three adenine nucleotide translocator (ANT1 to ANT3) isoforms, differentially expressed in human cells, play a crucial role in cell bioenergetics by catalyzing ADP and ATP exchange across the mitochondrial inner membrane. In contrast to differentiated tissue cells, transformed cells, and their rho(0) derivatives, i.e. cells deprived of mitochondrial DNA, sustain a high rate of glycolysis. We compared the expression pattern of ANT isoforms in several transformed human cell lines at different stages of the cell cycle. The level of ANT2 expression and glycolytic ATP production in these cell lines were in keeping with their metabolic background and their state of differentiation. The sensitivity of the mitochondrial inner membrane potential (Delta psi) to several inhibitors of glycolysis and oxidative phosphorylation confirmed this relationship. We propose a new model for ATP uptake in cancer cells implicating the ANT2 isoform, in conjunction with hexokinase II and the beta subunit of mitochondrial ATP synthase, in the Delta psi maintenance and in the aggressiveness of cancer cells.

In the mitochondrial internal membrane, the adenine nucleotide translocator (ANT) carries out the ATP/ADP exchange between cytoplasm and mitochondrial matrix. Three isoforms with different kinetic properties are encoded from three different genes in Human: the muscle specific ANT1 and the ubiquitary ANT3 isoforms export ATP produced by mitochondrial oxidative phosphorylation (OXPHOS). The ANT2 isoform is specifically expressed in proliferative cells with a predominant glycolytic metabolism and is associated with cellular undifferentiation which is a major characteristic in carcinogenesis. Its role would be to import into mitochondria ATP produced by the glycolysis, energy essential to several intramitochondrial functions, particularly to maintenance of the membrane potential (Delta Psi m), conditioning cellular survival and proliferation. The mechanism of regeneration of this Delta Psi m gradient would involve at least three major proteins: the hexokinase II isoform, the ANT2 isoform and the F1 part of the mitochondrial ATP synthase complex. Taking into account this major role of ANT2 in cell proliferation and the very low expression of this isoform in differentiated tissues, this protein or its transcript could be chosen as a target for an anticancer strategy. Furthermore, previous studies showed that molecules of the cisplatin family, used as chemotherapeutic agents, led to the destruction of the mitochondrial membrane potential and thus to cell death. Does the anticancer effect of these molecules result, at least partially, from this mitochondrial aggression? If it is the case, the ANT2 isoform, mainly involved in the generation of this potential by its ATP4-/ADP3- exchange, could be considered as a more specific targeting by an RNA interference approach.

Under hypoxic conditions, mitochondrial ATP production ceases, leaving cells entirely dependent on their glycolytic metabolism. The cytoplasmic and intramitochondrial ATP/ADP ratios, partly controlled by the adenine nucleotide translocator (ANT), are drastically modified. In dividing and growing cells that have a predominantly glycolytic metabolism, the ANT isoform 2, which has kinetic properties allowing ATP import into mitochondria, is over-expressed in comparison to control cells. We studied the cellular metabolic and proliferative response to hypoxia in two transformed human cell lines with different metabolic backgrounds: HepG2 and 143B, and in their rho(o) derivatives, i.e., cells with no mitochondrial DNA. Transformed 143B and rho(o) cells continued their proliferation whereas HepG2 cells, with a more differentiated phenotype, arrested their cell-cycle at the G(1)/S checkpoint. Hypoxia induced an increase in glycolytic activity, correlated to an induction of VEGF and hexokinase II (HK II) expression. Thus, according to their tumorigenicity, transformed cells may adopt one of two distinct behaviors to support hypoxic stress, i.e., proliferation or quiescence. Our study links the constitutive glycolytic activity and ANT2 expression levels of transformed cells with the loss of cell-cycle control after oxygen deprivation. ATP import by ANT2 allows cells to maintain their mitochondrial integrity while acquiring insensitivity to any alterations in the proteins involved in oxidative phosphorylation. This loss of cell dependence on oxidative metabolism is an important factor in the development of tumors.

National audience

Background: Many mitochondrial pathologies are quantitative disorders related to tissue-specific deletion, depletion, or overreplication of mitochondrial DNA (mtDNA). We developed an assay for the determination of mtDNA copy number by real-time quantitative PCR for the molecular diagnosis of such alterations.Methods: To determine altered mtDNA copy number in muscle from nine patients with single or multiple mtDNA deletions, we generated calibration curves from serial dilutions of cloned mtDNA probes specific to four different mitochondrial genes encoding either ribosomal (16S) or messenger (ND2, ND5, and ATPase6) RNAs, localized in different regions of the mtDNA sequence. This method was compared with quantification of radioactive signals from Southern-blot analysis. We also determined the mitochondrial-to-nuclear DNA ratio in muscle, liver, and cultured fibroblasts from a patient with mtDNA depletion and in liver from two patients with mtDNA overreplication.Results: Both methods quantified 5–76% of deleted mtDNA in muscle, 59–97% of mtDNA depletion in the tissues, and 1.7- to 4.1-fold mtDNA overreplication in liver. The data obtained were concordant, with a linear correlation coefficient (r2) between the two methods of 0.94, and indicated that quantitative PCR has a higher sensitivity than Southern-blot analysis.Conclusions: Real-time quantitative PCR can determine the copy number of either deleted or full-length mtDNA in patients with mitochondrial diseases and has advantages over classic Southern-blot analysis.

Thyroid oncocytoma is characterized by the presence of oncocytes containing abnormally large numbers of mitochondria. However, the relationship between the abundance of mitochondria and the pathogenesis of the tumors is unknown. Recently, a new cell line, named XTC.UC1, has been derived from a metastasis of thyroid oncocytoma. We have studied the metabolism and the gene expression profile of the mitochondria in XTC.UC1 cells, using B-CPAP cells as controls. There were no signs of mitochondrial respiratory chain defects or uncoupling between the respiratory chain and adenosine triphosphate (ATP) production. In XTC.UC1 cells, mtDNA transcripts were increased more than fivefold than in controls, in parallel with a 3.6-fold increase in mtDNA content. Finally, in spite of the glycolytic metabolism induced by the culture medium, the mitochondria of XTC.UC1 cells possess the phenotype of oncocytic cells with hypertrophic mitochondria, higher respiratory enzyme activity and higher mtDNA content than in controls. XTC.UC1 cells may therefore offer a useful model for investigating the coordination of the nuclear and mitochondrial genomes, in the context of thyroid tumors.

We have previously described two cases of children with a liver mitochondrial DNA (mtDNA) depletion syndrome, characterised by a low ratio of mtDNA to nuclear DNA. Light microscopy performed on liver biopsy showed abnormal hepatocytes with a characteristic 'oncocytic' appearance, indicative of perturbed oxidative phosphorylation. The adenine nucleotide translocator (ANT), the last step in oxidative phosphorylation catalyses the exchange of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) between the cytosol and mitochondria. The ANT2 gene, which is not normally expressed in human tissues, encodes an isoform preferentially expressed under conditions of glycolytic metabolism. ANT2 gene expression is regulated by a transcription factor involved in a molecular mechanism selecting for the import of glycolytic ATP into the mitochondrial matrix. This ATP import is required in highly proliferative cells, such as tumour cells, which are highly dependent on glycolysis for ATP synthesis. We postulated that, as a result of the defective oxidative phosphorylation observed in these patient biopsies, the ANT2 expression would be induced. We simultaneously quantified the mtDNA depletion and the ANT2 gene expression in liver biopsies from the two patients and six controls. ANT2 mRNA levels were significantly increased in the two patient liver biopsies. Moreover, in one patient, the liver mtDNA depletion was found to be partially reversed after less than 4 years and this reversion was coupled to a concomitant decrease of the ANT2 expression. These results suggest that dysfunction of oxidative phosphorylation could lead to a switch from mitochondrial to glycolytic ATP production, to restore tissue-specific energy requirements.

The protective activity of small stress proteins (sHsp) against H2O2-mediated cell death in the highly sensitive murine L929 fibroblast has been analyzed. We report here that the human Hsp27- and murine Hsp25-mediated rise in glutathione (GSH) levels as well as the maintenance of this redox modulator in its reduced form was directly responsible for the protection observed at the level of cell morphology and mitochondrial membrane potential. sHsp expression also buffered the increase in protein oxidation following H2O2 treatment and protected several key enzymes against inactivation. In this case, however, the protection necessitated both an increase in GSH and the presence of sHsp per se since the pattern of protection against protein oxidation mediated by a simple GSH increase was different from that induced by sHsp expression. Among the enzymes analyzed, we noticed that sHsp significantly increased glucose-6-phosphate dehydrogenase (G6PD) activity and to a lesser extent glutathione reductase and glutathione transferase activities. Moreover, an increased GSH level was observed in G6PD-overexpressing L929 cell clones. Taken together our results suggest that sHsp protect against oxidative stress through a G6PD-dependent ability to increase and uphold GSH in its reduced form and by using this redox modulator as an essential parameter of their in vivo chaperone activity against oxidized proteins.

The transcript levels of nuclear and mitochondrial genes involved in oxidative phosphorylation were quantified in human myoblasts and myotubes cultured from biopsies of patients harboring either heteroplasmic point mutation or deletion of mitochondrial DNA. The transcript patterns were determined by two different methodologies, competitive reverse-transcription polymerase chain reaction and classical Northern blot analysis, both referred to the mitochondrial to nuclear DNA ratio. In myoblasts from the patients with MELAS (myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) and KSS (Kearns-Sayre) syndromes, both methodologies revealed an increase of mtDNA transcript levels. A higher level of the nuclear ATP synthase beta transcript was observed in the MELAS patient cells and could be the consequence of a feedback effect of the mitochondrial DNA mutation. Moreover, the nuclear and mitochondrial transcript accumulation is more pronounced after myoblast differentiation. Thus, the OXPHOS expression is specifically altered in patients with mitochondrial diseases. The competitive RT-PCR, a rapid and sensitive technique, could be applied to investigation of mitochondrial myopathies.The transcript levels of nuclear and mitochondrial genes involved in oxidative phosphorylation were quantified in human myoblasts and myotubes cultured from biopsies of patients harboring either heteroplasmic point mutation or deletion of mitochondrial DNA. The transcript patterns were determined by two different methodologies, competitive reverse-transcription polymerase chain reaction and classical Northern blot analysis, both referred to the mitochondrial to nuclear DNA ratio. In myoblasts from the patients with MELAS (myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) and KSS (Kearns-Sayre) syndromes, both methodologies revealed an increase of mtDNA transcript levels. A higher level of the nuclear ATP synthase beta transcript was observed in the MELAS patient cells and could be the consequence of a feedback effect of the mitochondrial DNA mutation. Moreover, the nuclear and mitochondrial transcript accumulation is more pronounced after myoblast differentiation. Thus, the OXPHOS expression is specifically altered in patients with mitochondrial diseases. The competitive RT-PCR, a rapid and sensitive technique, could be applied to investigation of mitochondrial myopathies.

International audience; The adenine nucleotide translocator (ANT) is the most abundant mitochondrial inner membrane protein which catalyses the exchange of ADP and ATP between cytosol and mitochondria. The human ANT protein has three isoforms encoded by three differentially regulated nuclear genes. The ANT gene expression was examined in several human cells. The gene encoding the ANT2 isoform was found specifically induced in Simian virus 40 (SV40)-transformed, tumoral and mtDNA lacking rho degrees cell lines. Moreover, the ANT2 gene was preferentially expressed under a glycolytic metabolism. Functional complementation of a Saccharomyces cerevisiae mutant revealed that the human ANT2 protein specifically restores yeast cell growth under anaerobic conditions. Sequence analysis of the ANT2 proximal promoter in comparison to that of the third yeast adenine nucleotide translocator (AAC3) led us to identify a new motif termed GRBOX. Promoter-deletion transfection and mobility gel-shift assays revealed that this motif is recognized by a negative transcriptional regulator. This transcription factor might be involved in a molecular mechanism which selects the import of the glycolytic ATP in the mitochondrial matrix. This ATP import is required in highly proliferative cells, such as tumour cells, which depend strongly on glycolysis for ATP synthesis.The adenine nucleotide translocator (ANT) is the most abundant mitochondrial inner membrane protein which catalyses the exchange of ADP and ATP between cytosol and mitochondria. The human ANT protein has three isoforms encoded by three differentially regulated nuclear genes. The ANT gene expression was examined in several human cells. The gene encoding the ANT2 isoform was found specifically induced in Simian virus 40 (SV40)-transformed, tumoral and mtDNA lacking rho degrees cell lines. Moreover, the ANT2 gene was preferentially expressed under a glycolytic metabolism. Functional complementation of a Saccharomyces cerevisiae mutant revealed that the human ANT2 protein specifically restores yeast cell growth under anaerobic conditions. Sequence analysis of the ANT2 proximal promoter in comparison to that of the third yeast adenine nucleotide translocator (AAC3) led us to identify a new motif termed GRBOX. Promoter-deletion transfection and mobility gel-shift assays revealed that this motif is recognized by a negative transcriptional regulator. This transcription factor might be involved in a molecular mechanism which selects the import of the glycolytic ATP in the mitochondrial matrix. This ATP import is required in highly proliferative cells, such as tumour cells, which depend strongly on glycolysis for ATP synthesis.

We describe an accurate procedure for a rapid diagnosis of heteroplasmic mtDNA deletions based on the polymerase chain reaction (PCR). For a selective amplification of deleted mtDNA across the breakpoints of the deletion, we used seven combinations of primers surrounding the most common deleted region between the two origins of mtDNA replication. This procedure was performed on muscle biopsies of twenty patients harboring a single mtDNA deletion and one patient with multiple mtDNA deletions. The results were compared with Southern-blotting analysis. We conclude that this PCR procedure is a sensitive and convenient screening method for the detection of mtDNA deletions. (Mol Cell Biochem 174: 221–225, 1997)

We have identified a new mitochondrial (mt) cytochrome b mutation in a 29-year-old man with progressive exercise muscle intolerance associated with a marked deficiency of complex III activity and a decreased amount of mitochondrial-encoded cytochrome b. This G to A transition at mtDNA position 15615 leads to the substitution (G290D) of a very highly conserved amino acid of cytochrome b during evolution. The mutant mtDNA was heteroplasmic (80% mutant) in patient muscle but was undetectable in blood from the patient and his healthy mother and sisters. A maternally inherited cytochrome b polymorphism was also identified in this patient. Molecular screening of 150 individuals showed that the G290D mutation associated with the described phenotype. We suggest that this molecular defect is the primary cause of the muscle disease in this patient.

The steady state levels of both mitochondrial and nuclear transcripts were examined in a Kearns-Sayre syndrome patient harboring a heteroplasmic 7.7 kb mitochondrial DNA deletion. Transcripts originating from the genes located outside of the deletion were present in similar amounts to those of control samples, with the transcript levels of each tissue linked to its oxidative phosphorylation capacities. Transcripts originating from genes within the deletion were reduced according to the percentage of mtDNA deleted molecules in the tissue. The fusion transcript resulting from the rearranged genome is expressed in all the tissues tested and its level is related to the amount of the deleted mtDNA. The RNA levels from three nuclear genes encoding two of the Adenine Nucleotide Translocator isoforms (ANT1 and 2) and the β subunit of the ATPsynthase (ATPsyn β) were significantly induced in the different tissues independently of the percentage of deleted mtDNA molecules. In contrast, the ANT1 and ATPsyn β levels were decreased in skeletal muscle. This result could be related to the different distribution of the deleted molecules in tissues.

We have previously shown using proton NMR spectroscopy-based metabolomics that mice bearing B16 melanoma tumors treated by chloroethylnitrosourea (CENU) had a strong intratumoral decrease in glycolysis involving the accumulation of intracellular glucose and glucose-6-phosphate, together the accumulation of glutamate derivatives, despite no significant lactate content change (1). To get insights into the metabolic pathway followed by glucose carbons in CENU-treated tumor, we used double labelled glucose ([1,2-13C]glucose) and investigated the obtained isotopomers of lactate, the final product of glycolysis. Mice bearing B16 melanoma tumors were treated by CENU or saline solution (control) at days 11, 14 and 17 at dose 15µg/g weight. At day 23, mice were injected intraperitoneally with [1,2-13C]glucose at 25 mg in 150µl saline solution. Animals were sacrificed 30 minutes after glucose injection. Tumors were removed and kept at −80°C until use. The analysis of lactate isotopomers, a fluxomics approach, was done at 500 MHz using high resolution magic angle spinning (HRMAS) 1H-NMR spectroscopy by exploiting 1H-13C coupling signals. The two lactate signals, the methyl (C3) signal centered at 1.33 ppm and the methine (C2) signal centered at 4.12 ppm were differentially analyzed, and quantified from spectra witout and with broadband 13C-decoupling. In addition, the activity of pyruvate-kinase was measured using enzyme assay. In comparison with control tumors, CENU-treated tumors exhibited an overall decrease of labelled lactate both at C3 and C2 position, together with a significant increase in the relative proportion of C2 to C3 labelling (0.65±0.10 vs 0.91±0.13, P Altogether these fluxomics data provide evidence of glycolysis adaptation that may be correlated to reduced proliferation, decreased aggressiveness, and redifferentiation, as previously shown in this model (2). In addition, this study suggests that metabolic targeting of glycolysis-related ATP production may improve CENU efficacy. 1- Morvan D, Demidem A. Cancer Res, 67; 2150-59, 2007 2- Demidem A et al. Cancer Res, 61; 2294-300, 2001 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 62.

Objective. —Oxidative phosphorylation (OXPHOS) deficiency due to hypoxemia or other causes was hypothesized to increase oxygen radical generation, damage mitochondrial DNA (mtDNA), and reduce adenosine triphosphate synthesis, resulting in compensatory OXPHOS gene induction. Therefore, we investigated the levels of mtDNA damage and OXPHOS transcripts in normal and ischemic hearts, and then in other forms of heart disease. Design. —DNA was extracted from the heart and the levels of the common 4977 base pair mtDNA deletion were quantitated as an index for mtDNA damage. Total RNA was extracted from hearts and analyzed for OXPHOS transcript levels. Results. —In control hearts, the 4977 base pair mtDNA deletion appeared at age 40 years and reached a maximum deletion of 0.0035%. Much higher levels were found in ischemic hearts (0.02% to 0.85%), as well as in three of 10 cases with other types of heart disease (0.017% to 0.16%). The OXPHOS transcripts were increased in all diseased hearts. Conclusion. —Ischemic hearts have increased mtDNA damage and OXPHOS gene expression, suggesting that mtDNA damage is associated with OXPHOS deficiency. Oxidative phosphorylation defects may also play a role in some other forms of cardiac disease. (JAMA. 1991;266:1812-1816)

Treatment of Drosophila melanogaster Kc 0% cells with juvenile hormone (JH), which is of crucial importance to insect physiology, leads to a specific, early apparent increase in mitochondrial protein synthesis and to a later increase in cytochrome oxidase activity. This increase is at a maximum after a 12-h treatment with JH concentrations ranging from 10 −12 to 10 −9 M. Electrophoretic analysis of the mitochondrial translation products shows that all the mitochondria but a stimulating effect by a simultaneously. We saw no hormonal effect on isolated mitochondria but a stimulating effect by a post-mitochondrial supernatant from induced cells, strongly suggests that the action of JH is indirect and may result from a nuclear effect. 2-D electrophoretic analysis of the total mitochondrial proteins shows that at least two polypeptides coded by nuclear genome are affected. Such results suggest that despite the absence of morphological cellular modification, JH does have an active influence on energy metabolism.

In order to study the interrelation between nuclear genome and mitochondria during biogenesis of mitochondrial proteins, mitochondria of D. melanogaster were isolated from whole insects. The fraction obtained showed respiratory control by ADP in the presence of pyruvate-malate substrates of up to 6–7. Conditions of in vitro uptake of ( 35 S) methionine in polypeptides were studied. The energy necessary for protein synthesis is supplied by endogenous ATP. Bacterial and fungal contamination was severely repressed. Incorporation rates of 50–100 picomoles of( 35 S) methionine mg protein −1 hour −1 could be measured under these conditions. Electrophoresis in the presence of SDS of proteins of the internal membrane showed, after 50 min of incubation, a significantly labelled band of proteins at 22 Kd. Other bands at 80, 52 and 40 Kd were labelled to a lesser degree.

Adresse de correspondance: Patrick.Herpin@rennes.inra.fr; National audience; Implication of mitochondria in muscular biology: a key role during early development, growth and muscular atrophy. Mitochondria are implicated in the regulation of biological process controlling development, growth and muscle properties in farm animals, as well as sarcopenia during aging in humans. Recent results obtained at INRA on these aspects are reviewed in this paper. First, the relationship between oxidative metabolism, mitochondria density and muscle contractile type is confirmed, and the specificity of mitochondria properties according to myosin isotypes is demonstrated. Contribution of mitochondria to the acquisition of muscle contractile types and to myoblast differentiation, through the mitochondria-nuclear cross-talk, is described, and the key roles of c-Myc, Calcineurine and myogenin in this process is clearly established. The involvement of the direct T3 mitochondrial pathway in the regulation of mitochondrial biogenesis and activity is confirmed both in vitro and in vivo. No clear relation is found between mitochondrial activity and the amount of intramuscular fat, a key component of meat quality. In fact, muscle lipid content appears to be more related to intra-muscular adipocyte differentiation and lipid turnover rate than to any metabolic pathway. We also show in pigs and chickens that uncoupling proteins, UCP2, UCP3 and avUCP are differentially regulated in the muscle, but it is still difficult to conclude on their physiological roles. UCP3 and avUCP are clearly implicated in the thermogenic effect of T3 and probably play a role in the control of basal metabolism, whereas avUCP can be seen as a key metabolic sensor in the chicken because its expression is modified by cold or warm exposure and correlated to the intensity of lipid metabolism. Finally, mitochondrial H(2)O(2) production increases during healthy aging but has no short-term effect on muscle aging (sarcopenia). In fact, accumulation of mitochondrial DNA deletions and alteration of the expression of mitochondrial or nuclear genes are only observed in very old patients (older than 80). However, sensitivity of mitochondrial respiration to calcium clearly increases with aging, a process that could contribute to the reduction of aged muscle resistance to stress and to the development of apoptosis.; Cette synthèse concerne les recherches récemment effectuées à l’INRA sur le rôle des mitochondries dans la régulation du développement, de la croissance et des propriétés du muscle des animaux d’élevage et dans le déterminisme de la fonte musculaire au cours du vieillissement chez l’homme. La relation entre activité mitochondriale et développement musculaire a été confirmée. Au-delà de la relation entre métabolisme oxydatif, densité mitochondriale et type contractile des fibres, nous montrons que les caractéristiques intrinsèques des organites diffèrent en fonction des isoformes de myosine présentes. Les mitochondries participent à la définition du type contractile et à la différenciation des myoblastes, via le dialogue mitochondries-noyau auquel participent trois gènes cibles majeurs de l’organite : c-Myc (répresseur), Calcineurine et myogénine (stimulateurs). L’importance de la voie d’action mitochondriale de la T3 pour la régulation de la itochondriogenèse et de l’activité de l’organite est confirmée à la fois in vitro et in vivo. Aucune relation claire n’a été mise en évidence entre la quantité de lipides intramusculaires (LIM), un déterminant important de la qualité des viandes, et l’activité mitochondriale. La teneur en LIM semble davantage corrélée avec la différenciation des adipocytes intramusculaires et l’importance des flux de lipides dans le muscle qu’avec une voie métabolique particulière. Les protéines découplantes, UCP2, UCP3 (chez le porc) et avUCP (chez le poulet) sont régulées différemment mais il reste difficile de conclure sur leur rôle physiologique. UCP3 et avUCPs sont des effecteurs importants de l’effet thermogénique de la T3 et pourraient jouer un rôle dans le contrôle du métabolisme basal, tandis qu’avUCP semble être un régulateur métabolique majeur chez le poulet. Les travaux concernant l’activité mitochondriale en relation avec le vieillissement ont produit un certain nombre de résultats divergents par rapport à la littérature : absence d’accumulation de délétions de l’ADN mitochondrial et d’altération de l’expression de gènes mitochondriaux ou nucléaires avant un âge très avancé. Pourtant, la production mitochondriale d’H2O2 augmente avec l’âge et le vieillissement accroît la sensibilité de la respiration mitochondriale au calcium, deux phénomènes susceptibles de participer à l’induction « facilitée » d’une mort cellulaire. En conclusion, que ce soit chez l’animal ou l’homme, les mitochondries musculaires participent à de nombreuses fonctions physiologiques en liaison avec leurs différentes activités biologiques.

Current systems biology relies on high-throughput biological analysis techniques such as transcriptomics or metabolomics. However, these techniques may generate errors. By crossing results from different analysis techniques, we hope to avoid at least part of these limits. For that purpose, we started to develop a modeling platform, MPSA (Metabolic Pathways Software Analyzer). MPSA allows integrating biological data on metabolic pathways. MPSA also ensures the display of metabolic pathways graphs, the simulation of models based on ordinary differential equations systems solving and the study of network structures using elementary flux modes. We have developed several web applications allowing on the one hand to interpret biological results by using databases, and on the other hand to export these data to MPSA. The main database of this work is myKegg. It includes all human KEGG metabolic pathways and a list of synonyms for human KEGG entries. This base allows to identify metabolic pathways from a list of biological compounds and to import them in MPSA. Another database, BioNMR, has been developed to organize the data extracted from NMR spectra. Another web application named GeneProm has been developed to analyze gene promoters. A promotology protocol was developed and tested on a set of four genes coding for the four ANT (adenine nucleotide translocator) protein isoforms. Each ANT isoform has a specific expression profile and role in cell bioenergetics. The promotology study of these four genes led us to construct specific regulatory models from identified regulatory elements in their promoter sequence. Potentially co-regulated genes were deduced from these models. Then they can be exported to our MPSA platform. This whole development will be included in the project of Integrative Biology platform in the INRA Human Nutrition Unit., La biologie des systèmes actuelle s’appuie sur des techniques d’analyse biologique à haut débit comme la transcriptomique ou la métabolomique. Cependant, ces techniques haut débit ont leurs limites et peuvent générer des erreurs. En croisant les résultats de différentes techniques d’analyse biologique, nous espérons pallier une partie de leurs limites. À cet effet, nous avons commencé à développer une plateforme de modélisation, MPSA (Metabolic Pathways Software Analyzer), permettant d’intégrer les données générées à des réseaux métaboliques. MPSA permet de représenter les graphes de voies métaboliques, d’effectuer des simulations basées sur la résolution de systèmes d’équations différentielles et d’étudier la structure des réseaux métaboliques par le calcul et la représentation des modes élémentaires. Nous avons développé différentes applications web permettant, d’une part, l’interprétation des résultats biologiques en utilisant des bases de données et, d’autre part, leur export vers MPSA. La base de données centrale de ce développement est myKegg, incluant l’ensemble des voies métaboliques humaines de la base de données publique KEGG ainsi qu’une base de synonymes construite elle aussi à partir de KEGG. Cette base permet d’identifier des voies métaboliques et de les importer dans MPSA. Une base de données de métabolomique, BioNMR, a aussi été construite spécifiquement pour organiser les résultats générés à partir de spectres de RMN. Une autre application web, GeneProm, a été développée pour l’analyse de promoteurs de gènes ou promotologie. Un protocole d’étude a été mis au point et testé sur un groupe de 4 gènes codant pour les isoformes 1 à 4 de la protéine ANT, transporteur mitochondrial d’ATP, chacune ayant un rôle et un profil d’expression spécifique dans la bioénergétique cellulaire. L’étude par promotologie de ces 4 gènes a permis d’identifier des éléments de régulation spécifiques dans leurs séquences promotrices et d’identifier des gènes potentiellement co-régulés. Ces gènes peuvent ensuite être exportés vers notre plateforme MPSA. L’ensemble de ce développement sera inclus au projet de plateforme intégrative de l’Unité de Nutrition Humaine de l’INRA.