Depletion of mitochondrial DNA alters glucose metabolism in SK-Hep1 cells

Maternally inherited mitochondrial DNA (mtDNA) has been suggested to be a genetic factor for diabetes. Reports have shown a decrease of mtDNA content in tissues of diabetic patients. We investigated the effects of mtDNA depletion on glucose metabolism by use of rho(0) SK-Hep1 human hepatoma cells, whose mtDNA was depleted by long-term exposure to ethidium bromide. The rho(0) cells failed to hyperpolarize mitochondrial membrane potential in response to glucose stimulation. Intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were decreased in rho(0) cells compared with parental rho(+) cells. Our results suggest that the quantitative reduction of mtDNA may suppress the expression of nuclear DNA-encoded glucose transporters and enzymes of glucose metabolism. Thus this may lead to diabetic status, such as decreased ATP production and glucose utilization.     

Sporulation of Saccharomyces cerevisiae in the Absence of a Functional Mitochondrial Genome

The role of the mitochondrial system during sporulation of Saccharomyces cerevisiae was studied. Addition of ethidium bromide (EthBr) to cells growing in acetate medium resulted in the quantitative (>98%) conversion of the culture to the petite genotype in one generation. The cells were respiratory active (derepressed) but contained no mitochondrial deoxyribonucleic acid (mtDNA) as demonstrated by analytical ultracentrifugation in CsCl. When transferred to acetate sporulation medium, the culture sporulated. Ascus production was only slightly below that of the control culture. Synthesis of mtDNA occurred during sporulation in the control but not in the EthBr-treated culture. Mitochondrial protein synthesis was virtually eliminated in the EthBr-treated culture. Therefore, completely derepressed cells can sporulate without a functional mitochondrial genetic system. When partially repressed cells were treated with EthBr, no ascus formation was observed after transfer to sporulation medium. Control cultures underwent respiratory adaptation in sporulation medium and then sporulated. Extensive derepression of the respiratory system is thus required for sporulation, and this adaptation is dependent on a functional mitochondrial system. Our results suggest that once the cells are fully derepressed no mitochondrial genetic information has to be expressed during meiosis and ascus formation.     

Human mitochondria and mitochondrial genome function as a single dynamic cellular unit

rho 0 HeLa cells entirely lacking mitochondrial DNA (mtDNA) and mitochondrial transfection techniques were used to examine intermitochondrial interactions between mitochondria with and without mtDNA, and also between those with wild-type (wt) and mutant-type mtDNA in living human cells. First, unambiguous evidence was obtained that the DNA-binding dyes ethidium bromide (EtBr) and 4',6-diamidino-2- phenylindole (DAPI) exclusively stained mitochondria containing mtDNA in living human cells. Then, using EtBr or DAPI fluorescence as a probe, mtDNA was shown to spread rapidly to all rho 0 HeLa mitochondria when EtBr- or DAPI-stained HeLa mitochondria were introduced into rho 0 HeLa cells. Moreover, coexisting wt-mtDNA and mutant mtDNA with a large deletion (delta-mtDNA) were shown to mix homogeneously throughout mitochondria, not to remain segregated by use of electron microscopic analysis of cytochrome c oxidase activities of individual mitochondria as a probe to identify mitochondria with predominantly wt- or delta- mtDNA in single cells. This rapid diffusion of mtDNA and the resultant homogeneous distribution of the heteroplasmic wt- and delta-mtDNA molecules throughout mitochondria in a cell suggest that the mitochondria in living human cells have lost their individuality. Thus, the actual number of mitochondria per cell is not of crucial importance, and mitochondria in a cell should be considered as a virtually single dynamic unit.     

Deletion of mtDNA disrupts mitochondrial function and structure, but not biogenesis

The role of nuclear DNA (nDNA)-encoded proteins in the regulation of mitochondrial fission and fusion has been documented, yet the role of mitochondrial DNA (mtDNA) and encoded proteins in mitochondrial biogenesis remains unknown. Long-term treatment of a lymphoblastoid cell line Molt-4 with ethidium bromide generated mtDNA-deficient rho0 mutants. Depletion of mtDNA in rho0 cells produced functional and morphological changes in mitochondria without affecting the nuclear genome and encoded proteins. Indeed, the gene encoding subunit II of mitochondrial cytochrome c oxidase (COX II), a prototypical mitochondrial gene, was reduced in rho0 mutants blunting the activity of mitochondrial cytochrome coxidase. Yet, the amount of the nuclear beta-actin gene and the activity of citrate synthase, a mitochondrial matrix enzyme encoded by nDNA, remained unaffected in rho0 cells. Loss of mtDNA in rho0 cells was associated with significant distortion of mitochondrial structure, decreased electron density of the matrix and disorganized inner and outer membranes, resulting in the appearance of 'ghost-like' mitochondria. However, the number of mitochondria-like structures was not significantly different between mtDNA-deficient and parental cells. Thus, we conclude that cells lacking mtDNA still generate mitochondrial scaffolds, albeit with aberrant function.     

Metabolic capacity regulates iron homeostasis in endothelial cells

The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuates oxidative stress. Two respiration-deficient (rho(o)) endothelial cell lines with selective deletion of mitochondrial DNA (mtDNA) were created by exposing a parent endothelial cell line (EA) to ethidium bromide. Surviving cells were cloned and mtDNA-deficient cell lines were demonstrated to have diminished oxygen consumption. Total cellular and mitochondrial iron levels were measured, and iron uptake and compartmentalization were measured by inductively coupled plasma atomic emission spectroscopy. Iron transport and storage protein expression were analyzed by real-time polymerase chain reaction and Western blot or ELISA, and total and mitochondrial reactive oxygen species (ROS) generation was measured. Mitochondrial iron content was the same in all three cell lines, but both rho(o) lines had lower iron uptake and total cellular iron. Protein and mRNA expressions of major cytosolic iron transport constituents were down-regulated in rho(o) cells, including transferrin receptor, divalent metal transporter-1 (-IRE isoform), and ferritin. The mitochondrial iron-handling protein, frataxin, was also decreased in respiration-deficient cells. The rho(o) cell lines generated less mitochondrial ROS but released more extracellular H(2)O(2), and demonstrated significantly lower levels of lipid aldehyde formation than control cells. In summary, rho(o) cells with a minimal aerobic capacity had decreased iron uptake and storage. This work demonstrates that mitochondria regulate iron homeostasis in endothelial cells.     

Increase in mitochondrial DNA quantity and impairment of oxidative phosphorylation in bovine fibroblast cells treated with ethidium bromide for 15 passages in culture

Bovine fetal fibroblast cells were treated with ethidium bromide at a low concentration for 15 passages in culture to determine its effect on mitochondrial DNA copy number and on cell metabolism. Mitochondrial membrane potential and lactate production were estimated in order to characterize cell metabolism. In addition, mitochondrial DNA ND5 in proportion to a nuclear gene (luteinizing hormone receptor) was determined at the 1st, 2nd, 3rd, 10th, and 15th passages using semi-quantitative PCR amplification. Treated cells showed a lower mitochondrial membrane potential and higher levels of lactate production compared with control cells. However, the mitochondrial DNA/nuclear DNA ratio was higher in treated cells compared with control cells at the 10th and 15th passages. This ratio changed between the 3rd and 10th passages. Despite a clear impairment in mitochondrial function, ethidium bromide treatment did not lead to mitochondrial DNA depletion. It is possible that in response to a lower synthesis of ATP, due to an impairment in oxidative phosphorylation, treated cells develop a mechanism to resist the ethidium bromide effect on mtDNA replication, resulting in an increase in mitochondrial DNA copy number.     

Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses

Eukaryotic cells devoid of mitochondrial DNA (rho0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. Rho0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of rho0 cell lines, we developed an extremely mild, reliable and timesaving method to generate rho0 cell lines within 3-5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK- the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat rho0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK- rho0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human rho0 mitochondria.     

Incompatibility of linear DNA killer plasmids pGKL1 and pGKL2 from Kluyveromyces lactis with mitochondrial DNA from Saccharomyces cerevisiae.

Two linear killer plasmids (pGKL1 and pGKL2) from Kluyveromyces lactis stably replicated and expressed the killer phenotype in a neutral petite mutant [( rho0]) of Saccharomyces cerevisiae. However, when cytoplasmic components were introduced by cytoduction from a wild-type [( rho+]) strain of S. cerevisiae, the linear plasmids became unstable and were frequently lost from the cytoductant cells during mitosis, giving rise to nonkiller clones. The phenomenon was ascribed to the incompatibility with the introduced S. cerevisiae mitochondrial DNA (mtDNA), because the plasmid stability was restored by [rho0] mutations in the cytoductant cells. Incompatibility with mtDNA was also apparent for the transmission of plasmids into diploid progeny in crosses between killer cells carrying the pGKL plasmids and [rho+] nonkiller cells lacking the plasmids. High-frequency transmission of the plasmids was observed in crosses lacking mtDNA [( rho0] by [rho0] crosses) and in crosses involving mutated mtDNA with large deletions of various regions of mitochondrial genome. In contrast, mutated mtDNA from various mit- mutations also exerted the incompatibility effect on the transmission of plasmids. Double-stranded RNA killer plasmids were stably maintained and transmitted in the presence of wild-type mtDNA and stably coexisted with pGKL killer plasmids in [rho0] cells of S. cerevisiae.     

Inducible expression of a dominant negative DNA polymerase-gamma depletes mitochondrial DNA and produces a rho0 phenotype

We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-gamma to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-gamma harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t1/2 = 2-3 days) when expression of the transgene was induced. Concurrent reduction of mitochondrial encoded mRNA and protein, decreased cellular growth rate, and compromised respiration and mitochondrial membrane potential were observed. mtDNA depletion was reversible, as demonstrated by restoration of mtDNA copy number to normal within 10 days when the expression of POLGdn was suppressed following a 3-day induction period. Long term (20 days) expression of POLGdn completely eliminated mtDNA from the cells, resulting in rho0 cells that were respiration-deficient, lacked electron transport complex activities, and were auxotrophic for pyruvate and uridine. Fusion of the rho0 cells with human platelets yielded clonal cybrid cell lines that were populated exclusively with donor-derived mtDNA. Respiratory function, mitochondrial membrane potential, and electron transport activities were restored to normal in the cybrid cells. Inducible expression of a dominant negative DNA polymerase-gamma can yield mtDNA-deficient cell lines, which can be used to study the impact of specific mtDNA mutations on cellular physiology, and to investigate mitochondrial genome function and regulation.     

Reduced stimulation of recombinant DNA polymerase γ and mitochondrial DNA (mtDNA) helicase by variants of mitochondrial single-stranded DNA-binding protein (mtSSB) correlates with defects in mtDNA replication in animal cells

The mitochondrial single-stranded DNA-binding protein (mtSSB) is believed to coordinate the functions of DNA polymerase γ (pol γ) and the mitochondrial DNA (mtDNA) helicase at the mtDNA replication fork. We generated five variants of the human mtSSB bearing mutations in amino acid residues specific to metazoans that map on the protein surface, removed from the single-stranded DNA (ssDNA) binding groove. Although the mtSSB variants bound ssDNA with only slightly different affinities, they exhibited distinct capacities to stimulate the DNA polymerase activity of human pol γ and the DNA unwinding activity of human mtDNA helicase in vitro. Interestingly, we observed that the variants with defects in stimulating pol γ had unaltered capacities to stimulate the mtDNA helicase; at the same time, variants showing reduced stimulation of the mtDNA helicase activity promoted DNA synthesis by pol γ similarly to the wild-type mtSSB. The overexpression of the equivalent variants of Drosophila melanogaster mtSSB in S2 cells in culture caused mtDNA depletion under conditions of mitochondrial homeostasis. Furthermore, we observed more severe reduction of mtDNA copy number upon expression of these proteins during recovery from treatment with ethidium bromide, when mtDNA replication is stimulated in vivo. Our findings suggest that mtSSB uses distinct structural elements to interact functionally with its mtDNA replisome partners and to promote proper mtDNA replication in animal cells.     

POLG mutations cause decreased mitochondrial DNA repopulation rates following induced depletion in human fibroblasts

Disorders of mitochondrial DNA (mtDNA) maintenance have emerged as an important cause of human genetic disease, but demonstrating the functional consequences of de novo mutations remains a major challenge. We studied the rate of depletion and repopulation of mtDNA in human fibroblasts exposed to ethidium bromide in patients with heterozygous POLG mutations, POLG2 and TK2 mutations. Ethidium bromide induced mtDNA depletion occurred at the same rate in human fibroblasts from patients and healthy controls. By contrast, the restoration of mtDNA levels was markedly delayed in fibroblasts from patients with compound heterozygous POLG mutations. Specific POLG2 and TK2 mutations did not delay mtDNA repopulation rates. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation within intact cells, and provide a potential method of demonstrating the functional consequences of putative pathogenic alleles causing a defect of mtDNA synthesis.     

Mutations in Mgi Genes Convert Kluyveromyces Lactis into a Petite-Positive Yeast

Following targeted disruption of the unique CYC1 gene, the petite-negative yeast, Kluyveromyces lactis, was found to grow fermentatively in the absence of cytochrome c-mediated respiration. This observation encouraged us to seek mitochondrial mutants by treatment of K. lactis with ethidium bromide at the highest concentration permitting survival. By this technique, we isolated four mtDNA mutants, three lacking mtDNA and one with a deleted mitochondrial genome. In the three isolates lacking mtDNA, a nuclear mutation is present that permits petite formation. The three mutations occur at two different loci, designated MGI1 and MGI2 (for Mitochondrial Genome Integrity). The mgi mutations convert K. lactis into a petite-positive yeast. Like bakers' yeast, the mgi mutants spontaneously produce petites with deletions in mtDNA and lose this genome at high frequency on treatment with ethidium bromide. We suggest that the MGI gene products are required for maintaining the integrity of the mitochondrial genome and that, petite-positive yeasts may be naturally altered in one or other of these genes.     

The F1-ATPase inhibitor Inh1 (IF1) affects suppression of mtDNA loss-lethality in Kluyveromyces lactis

Loss of mtDNA by the petite-negative yeast Kluyveromyces lactis is lethal (rho(o)-lethality). However, mutations in the alpha, beta and gamma subunits of F(1)-ATPase can suppress lethality by increasing intramitochondrial hydrolysis of ATP. Increased hydrolysis of ATP can also occur on inactivation of Inh1, the natural inhibitor of F(1)-ATPase. However, not all strains of K. lactis show suppression of rho(o)-lethality on inactivation of INH1. Genetic analysis indicates that one or more alleles of modifying factors are required for suppression. Papillae showing enhanced resistance to ethidium bromide (EB) in INH1 disruptants have mutations in the alpha, beta and gamma subunits of F(1)-ATPase. Increased growth of double mutants on EB has been investigated by disruption of INH1 in previously characterized atp suppressor mutants. Inactivation of Inh1, with one exception, results in better growth on EB and increased F(1)-ATPase activity, indicating that suppression of rho(o)-lethality is not due to atp mutations preventing Inh1 from interacting with the F(1)-complex. By contrast, in suppressor mutants altered in Arg435 of the beta subunit, disruption of INH1 did not change the kinetic properties of F(1)-ATPase or alter growth on EB. Consequently, Arg435 appears to be required for interaction of Inh1 with the beta subunit. In a previous study, a mex1-1 allele was found to enhance mgi(atp) expression. In accord with results from double mutants, it has been found that mex1-1 is a frameshift mutation in INH1 causing inactivation of Inh1p.     

A "petite obligate" mutant of Saccharomyces cerevisiae: functional mtDNA is lethal in cells lacking the delta subunit of mitochondrial F1-ATPase

Within the mitochondrial F(1)F(0)-ATP synthase, the nucleus-encoded delta-F(1) subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the delta subunit gene (Deltadelta) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% rho(-)/rho degrees petites (i.e. cells missing a large portion (>50%) of the mtDNA (rho(-)) or totally devoid of mtDNA (rho degrees )). Previous work has suggested that the absence of complete mtDNA (rho(+)) in Deltadelta yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8(m)) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Deltadelta yeast. We further show that combined with a nuclear mutation (Deltaatp4) preventing the ATP synthase proton channel assembly, a lack of delta subunit fails to destabilize the mtDNA, and rho(+) Deltadelta cells become viable. We conclude that Deltadelta yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the rho(-)/rho degrees mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae.     

Distinct nuclear gene expression profiles in cells with mtDNA depletion and homoplasmic A3243G mutation

The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA (rho0 cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in rho0 cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and rho0 cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and rho0 cells. Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease.     

Mitochondrial DNA replication in petite mutants of yeast: Resistance to inhibition by ethidium bromide, berenil and euflavine

Summary Mitochondrial DNA (mtDNA) replication in petite mutants ofSaccharomyces cerevisiae is generally less sensitive to inhibition by ethidium bromide than in grande (respiratory competent) cells. In every petite that we have examined, which retain a range of different grande mtDNA sequences, this general phenomenon has been demonstrated by measurements of the loss of mtDNA from cultures grown in the presence of the drug. The resistance is also demonstrable by direct analysis of drug inhibition of mtDNA replication in isolated mitochondria. Furthermore, the resistance to ethidium bromide is accompanied, in every case tested, by cross-resistance to berenil and euflavine, although variations in the levels of resistance are observed.In one petite the level of in vivo resistance to the three drugs was very similar (4-fold over the grande parent) whilst another petite was mildly resistant to ethidium bromide and berenil (each 1.6-fold over the parent) and strongly resistant (nearly 8-fold) to inhibition of mtDNA replication by euflavine. The level of resistance to ethidium bromide in several other petite clones tested was found to vary markedly. Using genetic techniques it is possible to identify those petites which display an enhanced resistance to ethidium bromide inhibition of mtDNA replication.It is considered that the general resistance of petites arises because a product of mitochondrial protein synthesis is normally involved in facilitating the inhibitory action of these drugs on mtDNA synthesis in grande cells. The various levels of resistance in petites may be modulated by the particular mtDNA sequences retained in each petite.     

Regulation of energy metabolism in human cells in aging and diabetes: FoF(1), mtDNA, UCP, and ROS

Recent advances in bioenergetics consist of discoveries related to rotational coupling in ATP synthase (FoF(1)), uncoupling proteins (UCP), reactive oxygen species (ROS) and mitochondrial DNA (mtDNA). As shown in cloned sheep, mammalian genomes are composed of both nuclear DNA (nDNA) and maternal mtDNA. Oxidative phosphorylation (oxphos) varies greatly depending on cellular activities, and is regulated by both gene expression and the electrochemical potential difference of H(+) (Delta muH(+)). The expression of both mtDNA (by mtTFA) and nDNA for oxphos and UCP (by NRFs, etc.) is coordinated by a factor called PGC-1. The Delta muH(+) rotates an axis in FoF(1) that is regulated by inhibitors and ATP-sensitive K(+)-channels. We cultured human rho(o) cells (cells without mtDNA) in synthetic media and elucidated relationships among mtDNA, nDNA, Delta muH(+), UCPs, ROS, and apoptosis. These cells lack oxphos-dependent ROS formation and survive under conditions of high O(2). Cells cultured in the absence of ROS scavengers have proliferated for 40 years. UCPs lower Delta muH(+) and prevent ROS formation and resulting apoptosis. These results were applied to diabetology and gerontology. The pancreatic rho(o) cells did not secrete insulin, and mtDNA mutations caused diabetes, owing to the deficient Delta muH(+). Insulin resistance was closely related to UCPs and other energy regulators. The resulting high-glucose environment caused glycation of proteins and ROS-mediated apoptosis in vascular cells involved in diabetic complications. Telomeres, oxphos, and ROS are determinants in cellular aging. Cell division and ROS shortened telomeres and accelerated aging. In aged cells, Delta muH(+) was reduced by the slow respiration, and this change induced apoptosis. Cybrids made from aged cytoplasts and rho(o) cells showed that both decreased expression of nDNA, and somatic mutations of mtDNA are involved in the slowing of respiration in aged cells.     

POLG mutations cause decreased mitochondrial DNA repopulation rates following induced depletion in human fibroblasts

Disorders of mitochondrial DNA (mtDNA) maintenance have emerged as an important cause of human genetic disease, but demonstrating the functional consequences of de novo mutations remains a major challenge. We studied the rate of depletion and repopulation of mtDNA in human fibroblasts exposed to ethidium bromide in patients with heterozygous POLG mutations, POLG2 and TK2 mutations. Ethidium bromide induced mtDNA depletion occurred at the same rate in human fibroblasts from patients and healthy controls. By contrast, the restoration of mtDNA levels was markedly delayed in fibroblasts from patients with compound heterozygous POLG mutations. Specific POLG2 and TK2 mutations did not delay mtDNA repopulation rates. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation within intact cells, and provide a potential method of demonstrating the functional consequences of putative pathogenic alleles causing a defect of mtDNA synthesis.