Adenine Nucleotide Translocator Isoform Research Articles

Nicotinamide riboside alleviates exercise intolerance in ANT1-deficient mice

ObjectiveMitochondrial disorders are often characterized by muscle weakness and fatigue. Null mutations in the heart-muscle adenine nucleotide translocator isoform 1 (ANT1) of both humans and mice cause cardiomyopathy and myopathy associated with exercise intolerance and muscle weakness. Here we decipher the molecular underpinnings of ANT1-deficiency-mediated exercise intolerance. MethodsThis was achieved by correlating exercise physiology, mitochondrial function and metabolomics of mice deficient in ANT1 and comparing this to control mice. ResultsWe demonstrate a peripheral limitation of skeletal muscle mitochondrial respiration and a reduced complex I respiration in ANT1-deficient mice. Upon exercise, this results in a lack of NAD+ leading to a substrate limitation and stalling of the TCA cycle and mitochondrial respiration, further limiting skeletal muscle mitochondrial respiration. Treatment of ANT1-deficient mice with nicotinamide riboside increased NAD+ levels in skeletal muscle and liver, which increased the exercise capacity and the mitochondrial respiration. ConclusionIncreasing NAD+ levels with nicotinamide riboside can alleviate the exercise intolerance associated to ANT1-deficiency, indicating the therapeutic potential of NAD+-stimulating compounds in mitochondrial myopathies.

Multifactorial role of HIV-Vpr in cell apoptosis revealed by a naturally truncated 54aa variant.

Multifactorial role of HIV-Vpr in cell apoptosis revealed by a naturally truncated 54aa variant.

Abstract 486: Role of the Adenine Nucleotide Translocator Family in the Mitochondrial Permeability Transition Pore

Cardiomyocyte necrosis is dependent on the prolonged opening of the mitochondrial permeability transition pore (MPTP) which has escaped molecular identification for decades. Once thought to be comprised of the adenine nucleotide translocator (ANT) family, this hypothesis has fell by the way side when it was shown that liver mitochondria devoid of ANT 1 and 2 still display MPTP opening, even though these mitochondria were desensitized to MPTP opening and required a greater calcium bolus for it to engage. Thus, the ANT family has been labeled as a critical regulator of the MPTP and not the pore forming component. However, mice express three isoforms of ANT and only two of the three isoforms where deleted when ANTs were deemed to be non-essential. We hypothesize that the third ANT isoform may have compensated for the loss of the other two ANT family members during this analysis. Here, we have generated mice lacking all isoforms of ANT in a heart-, liver-, or cell-specific manner. Surprisingly, mice lacking all isoforms of ANT specifically in the liver are viable and are highly resistant to MPTP opening. Indeed, the amount of calcium required to engage the MPTP in mitochondria lacking all ANTs is 11 fold higher than wild type mitochondria and greater than 2 fold higher than wild type mitochondria treated with the MPTP inhibitor cyclosporine A (CsA). Furthermore, the treatment of CsA on ANT null liver mitochondria completely blocks MPTP opening suggesting that the ANT family is the primary pore in the liver. However, when subjected to extreme levels of calcium, a cyclophilin D (CypD) dependent pore engages. CypD is the MPTP trigger located in the matrix of the mitochondria which is inhibited by CsA and in wild type mitochondria high levels of calcium are enough to overcome the inhibition of CypD to open the pore. But, in ANT depleted liver mitochondria, extremely high levels of calcium do not override CypD inhibition which is suggestive of the presence of an additional pore. Further experimentation is underway to determine if MPTP-conductance is detectable by patch clamping mitoplasts lacking all isoforms of ANT.

Functional Insights on the Human Adenine Nucleotide Translocator Interactome

The adenine nucleotide translocator (ANT), by mediating the exchange of ADP for ATP across the inner mitochondrial membrane, lies at the core of oxidative phosphorylation (OXPHOS). Discrepancies in ANT expression and/or activity resulting to reduced energy metabolism are linked to a wide spectrum of pathologies, including lactic acidosis, progressive external opthalmoplegia, muscular dystrophy and cardiomyopathy. In yeast, the major isoform was modeled for a long time to exist in isolation, but was demonstrated to physically interact with respiratory supercomplexes (RSCs) exclusively in the context of the phospholipid cardiolipin (CL). OXPHOS in CL‐null yeast is remarkably decreased in optimal situations and completely eliminated under stressful conditions, pointing to the physiological significance of these protein‐protein associations. In this study, we determined that human ANTs also have the capacity to assemble into high molecular weight complexes. Additionally, we defined the interactome of two human ANT isoforms using an unbiased proteomics approach. Similar to the yeast orthologue, we saw RSCs and other solute carriers as binding partners, which was surprising given that the organization of yeast and mammalian ANTs is different, with Complex I missing in yeast. To probe the physiological relevance of the ANT‐RSC interaction, we deleted the major ANT isoform in HEK293, which unexpectedly only had a moderate effect on OXPHOS, indicating that the minimal levels of the other isoforms can still support respiratory capacity. As a reciprocal inquiry, we ablated OXPHOS function and found that ADP/ATP transport by ANTs is impaired. These results provide insights regarding the physiological relevance of evolutionarily conserved interactions of proteins important for mitochondrial function.Support or Funding InformationThis work was supported by the National Institutes of Health (R01HL108882) to S.M.C and pre‐doctoral fellowships from the American Heart Association (16PRE31140006 to M.G.A. and 12PRE11910004 to Y‐W.L.).

Mitochondrial energy deficiency leads to hyperproliferation of skeletal muscle mitochondria and enhanced insulin sensitivity

Diabetes is associated with impaired glucose metabolism in the presence of excess insulin. Glucose and fatty acids provide reducing equivalents to mitochondria to generate energy, and studies have reported mitochondrial dysfunction in type II diabetes patients. If mitochondrial dysfunction can cause diabetes, then we hypothesized that increased mitochondrial metabolism should render animals resistant to diabetes. This was confirmed in mice in which the heart-muscle-brain adenine nucleotide translocator isoform 1 (ANT1) was inactivated. ANT1-deficient animals are insulin-hypersensitive, glucose-tolerant, and resistant to high fat diet (HFD)-induced toxicity. In ANT1-deficient skeletal muscle, mitochondrial gene expression is induced in association with the hyperproliferation of mitochondria. The ANT1-deficient muscle mitochondria produce excess reactive oxygen species (ROS) and are partially uncoupled. Hence, the muscle respiration under nonphosphorylating conditions is increased. Muscle transcriptome analysis revealed the induction of mitochondrial biogenesis, down-regulation of diabetes-related genes, and increased expression of the genes encoding the myokines FGF21 and GDF15. However, FGF21 was not elevated in serum, and FGF21 and UCP1 mRNAs were not induced in liver or brown adipose tissue (BAT). Hence, increased oxidation of dietary-reducing equivalents by elevated muscle mitochondrial respiration appears to be the mechanism by which ANT1-deficient mice prevent diabetes, demonstrating that the rate of mitochondrial oxidation of calories is important in the etiology of metabolic disease.

ATP/ADP Turnover and Import of Glycolytic ATP into Mitochondria in Cancer Cells Is Independent of the Adenine Nucleotide Translocator

Non-proliferating cells oxidize respiratory substrates in mitochondria to generate a protonmotive force (Δp) that drives ATP synthesis. The mitochondrial membrane potential (ΔΨ), a component of Δp, drives release of mitochondrial ATP(4-) in exchange for cytosolic ADP(3-) via the electrogenic adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane, which leads to a high cytosolic ATP/ADP ratio up to >100-fold greater than matrix ATP/ADP. In rat hepatocytes, ANT inhibitors, bongkrekic acid (BA), and carboxyatractyloside (CAT), and the F1FO-ATP synthase inhibitor, oligomycin (OLIG), inhibited ureagenesis-induced respiration. However, in several cancer cell lines, OLIG but not BA and CAT inhibited respiration. In hepatocytes, respiratory inhibition did not collapse ΔΨ until OLIG, BA, or CAT was added. Similarly, in cancer cells OLIG and 2-deoxyglucose, a glycolytic inhibitor, depolarized mitochondria after respiratory inhibition, which showed that mitochondrial hydrolysis of glycolytic ATP maintained ΔΨ in the absence of respiration in all cell types studied. However in cancer cells, BA, CAT, and knockdown of the major ANT isoforms, ANT2 and ANT3, did not collapse ΔΨ after respiratory inhibition. These findings indicated that ANT was not mediating mitochondrial ATP/ADP exchange in cancer cells [corrected]. We propose that suppression of ANT contributes to low cytosolic ATP/ADP, activation of glycolysis, and a Warburg metabolic phenotype in proliferating cells.

Deficiency in the mouse mitochondrial adenine nucleotide translocator isoform 2 gene is associated with cardiac noncompaction

The mouse fetal and adult hearts express two adenine nucleotide translocator (ANT) isoform genes. The predominant isoform is the heart–muscle–brain ANT-isoform gene 1 (Ant1) while the other is the systemic Ant2 gene. Genetic inactivation of the Ant1 gene does not impair fetal development but results in hypertrophic cardiomyopathy in postnatal mice. Using a knockin X-linked Ant2 allele in which exons 3 and 4 are flanked by loxP sites combined in males with a protamine 1 promoter driven Cre recombinase we created females heterozygous for a null Ant2 allele. Crossing the heterozygous females with the Ant2fl, PrmCre(+) males resulted in male and female ANT2-null embryos. These fetuses proved to be embryonic lethal by day E14.5 in association with cardiac developmental failure, immature cardiomyocytes having swollen mitochondria, cardiomyocyte hyperproliferation, and cardiac failure due to hypertrabeculation/noncompaction. ANTs have two main functions, mitochondrial–cytosol ATP/ADP exchange and modulation of the mitochondrial permeability transition pore (mtPTP). Previous studies imply that ANT2 biases the mtPTP toward closed while ANT1 biases the mtPTP toward open. It has been reported that immature cardiomyocytes have a constitutively opened mtPTP, the closure of which signals the maturation of cardiomyocytes. Therefore, we hypothesize that the developmental toxicity of the Ant2 null mutation may be the result of biasing the cardiomyocyte mtPTP to remain open thus impairing cardiomyocyte maturation and resulting in cardiomyocyte hyperproliferation and failure of trabecular maturation. This article is part of a Special Issue entitled ‘EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2–6, 2016’, edited by Prof. Paolo Bernardi.

Abstract 59: A Protective Role Of Adenine Nucleotide Translocator 1 In The Inflammatory Response Following Myocardial Infarction

Background: Inflammation following myocardial infarction (MI) contributes significantly to the pathogenesis of heart failure. Although the initial stage of inflammatory response is necessary for the repair and healing of myocardium, excessive inflammation post MI causes adverse remodeling leading to heart failure. Recent studies suggest that mitochondrial dysfunction plays an important role in inflammation. However, the molecular target in cardiac mitochondria and the mechanisms of action remain unclear. We hypothesized that mitochondrial ADP/ATP carrier adenine nucleotide translocator isoform 1 (ANT1) is protective in post MI inflammation. Methods and Results: MI was induced by permanent occlusion of left anterior descending artery in C57BL/6 mice. In the inflamed heart following MI, ANT1 protein levels were significantly decreased in the left ventricle (0.48±8.94%), indicating ANT1 down-regulation occurs in myocardial inflammation following MI. To delineate the mechanisms involved, we performed siRNA knockdown of ANT1 in myocardium-derived H9c2 cells and cardiomyocytes, and examined tumor necrosis factor alpha (TNFα)-induced pro-inflammatory response. Knocking down ANT1 significantly increased the expression of TNFα (~10-fold) and interleukin-6 (IL-6) (~3.6-fold) concomitant with increased reporter gene activity of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) (~61±1.22%) in response to TNFα. These effects were not due to mitochondria loss because no changes were detected for voltage-dependent anion channel, or cyclophilin D when ANT1 is knocked down. Confocal microscopy analysis indicated that ANT1 knockdown increased TNFα-induced donut/blob-shaped mitochondria (~4.9-fold). Interestingly, exogenous TNFα and lipopolysaccharide (LPS) in turn significantly decreased ANT1 protein levels (~37±13%; ~37±6%, respectively) suggesting a feed forward regulation of pro-inflammatory cytokine expression activated by ANT1 down-regulation. Conclusion: These data suggest that cardiac inflammation is dependent on, in part, the function of ANT1. Preventing ANT1 down-regulation can be a novel molecular target to attenuate post MI inflammation and heart failure.

Translocation of Glycolytic ATP into Mitochondria of Cancer Cells does not Utilize the Adenine Nucleotide Transporter

BACKGROUND: Cancer cells utilize aerobic glycolysis rather than oxidative phosphorylation to generate most cellular ATP (Warburg phenomenon). In non-transformed differentiated cells, the adenine nucleotide translocator (ANT) catalyzes exchange of ATP for ADP across the mitochondrial inner membrane. Bongkrekic acid (BA) and carboxyatractyloside (CATR) specifically inhibit ANT. Here, our AIM was to assess whether mitochondrial ATP translocation in cancer cells depends on ANT.METHODS: Mitochondrial membrane potential (ΔΨ) was assessed by confocal microscopy of tetramethylrhodamine methylester (TMRM) fluorescence. Respiration by HepG2 and A549 cells was determined with a Seahorse XF24 Analyzer. RESULTS: In rat hepatocytes, respiratory inhibition by myxothiazol (MYX) slightly decreased ΔΨ, but subsequent oligomycin (OL), BA or CATR collapsed ΔΨ, indicating that mitochondrial hydrolysis of glycolytic (cytosolic) ATP sustains ΔΨ. In HepG2 and A549 cells, MYX also slightly decreased ΔΨ, and subsequent OL collapsed ΔΨ. By contrast to hepatocytes, BA and CATR added after MYX did not collapse ΔΨ, whereas 2-deoxyglucose (2-DG), a glycolytic inhibitor, added after MYX, MYX+BA and MYX+CATR did collapse ΔΨ. OL but not BA or CATR alone decreased respiration in both cell lines, whereas BA and CATR inhibited hepatocyte respiration. ANT2 is the predominant ANT isoform expressed in cancer cells, and 2-DG after MYX in ANT2 knockdown cells depolarized mitochondria.CONCLUSION: In cancer cells ANT is not the principal ATP transporter responsible for mitochondrial uptake of glycolytic ATP from the cytosol. Moreover, ANT2 deficiency does not alter uptake of glycolytic ATP into mitochondria. Warburg metabolism, therefore, appears to utilize an alternative pathway for entry of ATP into mitochondria.

The Interaction between Cell Adhesion Molecule L1, Matrix Metalloproteinase 14, and Adenine Nucleotide Translocator at the Plasma Membrane Regulates L1-Mediated Neurite Outgrowth of Murine Cerebellar Neurons

We have identified the adenine nucleotide translocator (ANT) isoforms ANT1 and ANT2 that are present in the plasma membrane of mouse cerebellar neurons as novel binding partners of the cell adhesion molecule L1. The direct interaction between ANT and L1 is mediated by sites within the fibronectin type III domains of L1 and the first and third extracellular loops of the ANT proteins. We also show that L1 interacts with the ANT binding partner matrix metalloprotease 14 (MMP14) and that the ANT proteins bind directly to the L1 interaction partner glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, we provide evidence that the functional interplay between L1, ANT proteins, MMP14, and GAPDH at the plasma membrane mediates L1-induced neurite outgrowth of cerebellar neurons. Disruption of this interplay by ANT inhibitors, ANT-derived synthetic peptides, and/or function-blocking MMP14 and ANT antibodies leads to alterations in L1-dependent neurite outgrowth. Stimulation of L1-mediated signaling in cerebellar neurons triggers transient ATP secretion via ANT proteins and leads to transient src family-dependent tyrosine phosphorylation of L1, ANT1, ANT2, and MMP14. Thus, our results indicate that plasma membrane-localized ANT1 and ANT2 regulate L1-mediated neurite outgrowth in conjunction with MMP14.

The hepatoprotective effects of adenine nucleotide translocator-2 against aging and oxidative stress

Mitochondrial adenine nucleotide translocator (ANT) plays important roles in the regulation of mitochondrial permeability transition and cell bioenergetics. The mouse has three ANT isoforms (1, 2 and 4) showing tissue-specific expression patterns. Although ANT1 is known to have a pro-apoptotic property, the specific functions of ANT2 have not been well determined. In the present study, ANT2 expression was significantly lower in the aged rat liver and in a liver fibrosis model. To explore the protective role of ANT2 in the liver, we established a hepa1c1c7 cell line overexpressing ANT2. Overexpression of ANT2 caused hepa1c1c7 cells to be more resistant to oxidative stress, and mitochondrial membrane potential (MMP, ∆Ψm) was relatively intact in ANT2-overexpressing cells under oxidative stress. In addition, ANT2 was found to increase ATP production by influencing mitochondrial bioenergetics. These results imply that the hepatoprotective effect of ANT2 is due to the stabilization of MMP and enhanced ATP production, and thus, maintaining ANT2 levels in the liver might be important to enhance resistance to aging and oxidative stress.

Computational analysis of the transcriptional regulation of the adenine nucleotide translocator isoform 4 gene and its role in spermatozoid glycolytic metabolism

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.

Abstract 4078: ANT-ligands small molecules induce apoptosis of cancer cells by inhibiting ATP/ADP translocation

Abstract Since fifteen years ago, the mitochondrion has been progressively recognized as an integrator-coordinator of apoptosis. In this context recent anti-cancer drugs in development are targeting mitochondrial apoptotic machinery to induce tumor cell death. By this strategy, drugs can interact with apoptosis-regulating proteins of Bcl-2 family to trigger Bax/Bak oligomerization and mitochondrial membrane permeabilization. This event conducts to the release of pro-apoptotic factors such as cytochrome c into the cytosol that are required for caspases activation and apoptosis process. In this study, we selected another strategy and investigated the effect of ANT-targeting small molecules to induce cancer cells apoptosis. The adenine nucleotide translocator (ANT) is a mitochondrial protein of the inner membrane involved in the exchange of ADP and ATP between the mitochondrial matrix and the cytosol. Medicinal chemistry approach coupled with in silico studies yield to several small organic compounds, which proved to be specific for ANT and fulfill druggability criteria (good cell penetration and biodisponibility). We identified a family of compounds that inhibited ATP translocation and induced apoptosis by a pathway involving Bax-dependent cytochrome c release and caspases activation. One of the hit compound was optimized to obtain molecules able to induce apoptosis of HT-29 and BxPC3 cells at 100 nM. We are currently investigating which ANT isoform(s) is (are) targeted in cells. In vivo tumor regression experiments will be performed to achieve the proof of concept of ANT-ligands small molecules as an interesting anti-cancer strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4078. doi:10.1158/1538-7445.AM2011-4078

The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1-4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.

Carboxyatractyloside effects on brown-fat mitochondria imply that the adenine nucleotide translocator isoforms ANT1 and ANT2 may be responsible for basal and fatty-acid-induced uncoupling respectively

In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1-/- mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1-/- brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration ('proton leak'). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1-/- mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.

ANT2 Isoform Required for Cancer Cell Glycolysis

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 (Deltapsi) 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 Deltapsi maintenance and in the aggressiveness of cancer cells.

ANT2 expression under hypoxic conditions produces opposite cell-cycle behavior in 143B and HepG2 cancer cells.

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.

Mobilization of Adenine Nucleotide Translocators as Molecular Bases of the Biochemical Threshold Effect Observed in Mitochondrial Diseases

The existence of a biochemical threshold effect in the metabolic expression of oxidative phosphorylation deficiencies has considerable implications for the understanding of mitochondrial bioenergetics and the study of mitochondrial diseases. However, the molecular bases of this phenomenon remain unclear. We report here a new mechanism to explain this threshold effect, based on a reserve of enzymes not initially participating in the respiratory rate that can be activated either to respond to a flux increase or to compensate for a defect induced by a mutation. We show that this mobilization occurs through 1) the assembly of inactive adenine nucleotide translocator isoform 1 subunits into oligomeric active carriers or 2) conformational changes in the adenine nucleotide translocator isoform 1 in a permeability transition pore-like structure. We discuss how these transitions are sensitive to the steady state of oxidative phosphorylation functioning or tissue and analyze their consequences on the threshold effect.

The adenine nucleotide translocator in apoptosis

Alteration of mitochondrial membrane permeability is a central mechanism leading invariably to cell death, which results, at least in part, from the opening of the permeability transition pore complex (PTPC). Indeed, extended PTPC opening is sufficient to trigger an increase in mitochondrial membrane permeability and apoptosis. Among the various PTPC components, the adenine nucleotide translocator (ANT) appears to act as a bi-functional protein which, on the one hand, contributes to a crucial step of aerobic energy metabolism, the ADP/ATP translocation, and on the other hand, can be converted into a pro-apoptotic pore under the control of onco- and anti-oncoproteins from the Bax/Bcl-2 family. In this review, we will discuss recent advances in the cooperation between ANT and Bax/Bcl-2 family members, the multiplicity of agents affecting ANT pore function and the putative role of ANT isoforms in apoptosis control.

Animal models for mitochondrial disease.

Mutations in mitochondrial genes encoded by both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA have been implicated in a wide range of degenerative diseases. MtDNA base substitution and rearrangement mutations can cause myopathy, cardiomyopathy, ophthalmological defects, growth retardation, movement disorders, dementias, and diabetes. nDNA mutations can affect mtDNA replication and transcription, increase mtDNA mutations through defects in the adenine nucleotide translocator isoform 1 (ANT1), or cause Leigh's syndrome, as a result of defects in oxidative phosphorylation (OXPHOS) structural genes. Mouse models of mtDNA base substitution mutations have been created by introducing the mtDNA 16S rRNA chloramphenicol (CAP)-resistance mutation into the mouse female germline. This resulted in ophthalmological defects in chimeras and perinatal lethality resulting from myopathy and cardiomyopathy in mutant animals. Mouse models of mtDNA rearrangements have resulted in animals with myopathy, cardiomyopathy, and nephropathy. Conditional inactivation of the mouse nDNA mitochondrial transcription factor (Tfam) gene in the heart caused neonatal lethal cardiomyopathy, whereas its inactivation in the pancreatic beta-cells caused diabetes. Mutational inactivation of the mouse Ant1 gene resulted in myopathy, cardiomyopathy, and multiple mtDNA deletions in association with elevated reactive oxygen species (ROS) production. This suggests that multiple mtDNA deletion syndrome can be caused by increased ROS damage. The inactivation of the uncoupler protein genes (Ucp) 1-3 resulted in alterations in delta mu H+ and increased ROS production. Inactivation of the Ucp2 gene, which is expressed in the pancreatic beta-cells, resulted in increased islet ATP, increased serum insulin levels, and suppression of the diabetes of the ob/ob mouse genotype. Transgenic mice with altered beta-cell ATP-sensitive K+ channels (KATP) also developed diabetes. Mutational inactivation of the mitochondrial antioxidant genes for glutathione peroxidase (GPx1) and Mn superoxide dismutase (Sod2) caused reduced energy production and neonatal lethal dilated cardiomyopathy, respectively, the later being ameliorated by treatment with MnSOD mimics. Partial Sod2 deficiency (+/-) resulted in mice with increased mitochondrial damage during aging, and treatment of C. elegans with catalytic antioxidant drugs can extend their life-span. Mice deficient in cytochrome-c died early in embryogenesis, but cells derived from these embryos had a complete deficiency in mitochondrial apoptosis. Mice lacking the proapoptotic Bax and Bak genes were not able to release cytochrome-c from the mitochondrion and were blocked in apoptosis. Mice lacking Apaf1, Cas9, and Cas3 did release mitochondrial cytochrome-c and were blocked in the downstream steps of apoptosis. These animal studies confirm that alterations in mitochondrial energy generation, ROS production, and apoptosis can all contribute to the pathophysiology of mitochondrial disease.