Background: Chronic renal disease (CKD) is characterized by complex changes in cell metabolism leading to an increased production of oxygen radicals, that, in turn has been suggested to play a key role in numerous clinical complications of this pathological condition. Several reports have focused on the identification of biological elements involved in the development of systemic biochemical alterations in CKD, but this abundant literature results fragmented and not exhaustive.

The question of whether and to what extent the in vivo cytochrome c oxidase (COX) capacity in mammalian cells exceeds that required to support respiration is still unresolved. In the present work, to address this question, a newly developed approach for measuring the rate of COX activity, either as an isolated step or as a respiratory chain-integrated step, has been applied to a variety of human cell types, including several tumor-derived semidifferentiated cell lines, as well as specialized cells removed from the organism. KCN titration assays, carried out on intact uncoupled cells, have clearly shown that the COX capacity is in low excess (16 -40%) with respect to that required to support the endogenous respiration rate. Furthermore, measurements of O 2 consumption rate supported by 0.4 mM tetramethyl-p-phenylenediamine in antimycin-inhibited uncoupled intact cells have given results that are fully consistent with those obtained in the KCN titration experiments. Similarly, KCN titration assays on digitonin-permeabilized cells have revealed a COX capacity that is nearly limiting (7-22% excess) for ADP ؉ glutamate/malate-dependent respiration. The present observations, therefore, substantiate the conclusion that the in vivo control of respiration by COX is much tighter than has been generally assumed on the basis of experiments carried out on isolated mitochondria. This conclusion has important implications for understanding the role of physiological or pathological factors in affecting the COX threshold.In recent years, the metabolic control of oxidative phosphorylation has received growing attention, and the approach based on the "control of flux" theory (1, 2) has been increasingly applied to the study of mitochondrial metabolism since its first use for such purpose (3). The discovery that mitochondrial DNA mutations can cause diseases in humans (4, 5), affecting either components of the translation apparatus or subunits of various respiratory complexes, and the increasing evidence that a reduction in the activities of some respiratory chain complexes is associated with aging or neurodegenerative diseases have raised fundamental questions as to the degree of control that the individual steps of oxidative phosphorylation exert on the rate of mitochondrial respiration. Most of the experimental work aimed at answering these questions has been carried out by inhibitor titration experiments on isolated mitochondria and has led to the conclusion that the activity of the various components of the respiratory chain is in excess, sometimes a large excess (2-4-fold), with evidence for a tissue-specific pattern, over the rate required to support the endogenous respiration rate (6 -11). However, the issue has been recently raised as to how accurately a metabolic control analysis applied to isolated mitochondria can reflect the in vivo situation, considering the possible loss of essential metabolites during organelle isolation and the disruption of the normal interactions of mitochondria with the cytoskeleton, which may b...

Alterations of hepatic metabolism are critical to the development of liver disease. The PPAR gamma 1 coactivators (PGC-1) are able to orchestrate, on a transcriptional level, different aspects of liver metabolism, such as mitochondrial oxidative phosphorylation, gluconeogenesis and fatty acid synthesis. As modifications affecting both mitochondrial and lipid metabolism contribute to the initiation and/or progression of liver steatosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC), a link between disrupted PGC-1 pathways and onset of these pathological conditions has been postulated. However, despite the large quantity of studies, the scenario is still not completely understood and some issues remain controversial. Here, we discuss the roles of PGC-1s in healthy liver, shedding a light on their contribution to the pathogenesis and future therapy of NASH and eventually HCC.

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is a transcriptional coactivator able to up-regulate mitochondrial biogenesis, respiratory capacity, oxidative phosphorylation, and fatty acid β-oxidation with the final aim of providing a more efficient pathway for aerobic energy production. In the continuously renewed intestinal epithelium, proliferative cells in the crypts migrate along the villus axis and differentiate into mature enterocytes, increasing their respiratory capacity and finally undergoing apoptosis. Here we show that in the intestinal epithelial surface, PGC1α drives mitochondrial biogenesis and respiration in the presence of reduced antioxidant enzyme activities, thus determining the accumulation of reactive oxygen species and fostering the fate of enterocytes toward apoptosis. Combining gain-and loss-offunction genetic approaches in human cells and mouse models of intestinal cancer, we present an intriguing scenario whereby PGC1α regulates enterocyte cell fate and protects against tumorigenesis.colon cancer | medical physiology | metabolism | mitochondria | nuclear receptors

We have examined the capacity of calf thymus DNA polymerases a, f, 6, and e to perform in vitro translesion synthesis on a substrate containing a single d(GpG)-cisplatin adduct placed on codon 13 of the human HRAS gene. We found that DNA synthesis catalyzed by DNA polymerases ca, 6, and E was blocked at the base preceding the lesion Among the possible mechanisms of mutagenesis is error-prone replication by cellular DNA polymerases past a DNA lesion followed by fixation of the mutation during subsequent rounds of replication. In eukaryotic cells, it is still unclear whether the replicative DNA polymerases a, 8, and E can carry out translesion synthesis either alone or with the help of accessory proteins. Alternatively, a separate DNA polymerase may be required for this process. For example, genetic evidence in the yeast Saccharomyces cerevisiae indicates that a putative DNA polymerase, the product of the REV3 gene, may be involved in error-prone translesion synthesis but not in normal DNA replication (1). DNA polymerase (3 is one of the five mammalian polymerases identified to date and is believed to function primarily in the repair of damaged DNA (2). However, DNA polymerase a may also have a role in replicative synthesis, since the enzyme can substitute for DNA polymerase I during DNA replication in Escherichia coli (3), and it is required for the conversion of single-stranded M13 DNA to double-stranded DNA in Xenopus oocytes and in oocyte nuclear extracts (4). cis-Diamminedichloroplatinum(II) (cisplatin) is an anticancer agent widely used in the treatment of ovarian, testicular, head, and neck carcinomas (5). It is believed that this compound exerts its cytotoxic properties by forming stable lesions on DNA, primarily intrastrand cross-links at the N-7 positions of adjacent guanine bases [d(GpG)-cisplatin or Pt-d(GpG)] (6). Replicative bypass of cisplatin adducts has been described in bacteria (7,8) and in eukaryotic cells (9). Recent work in our laboratory has demonstrated that a single-stranded DNA The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.vector bearing a unique intrastrand bifunctional adduct Ptd(GpG) at codon 13 of the human protooncogene HRAS is replicated in simian COS-7 cells and that such translesion synthesis may be mutagenic (10).To our knowledge, the capacities of the major mammalian replication enzymes to bypass the Pt-d(GpG) lesion have not been compared. To address this question, we have investigated the ability of purified calf thymus DNA polymerases a, (3, 8, and s to catalyze in vitro the bypass synthesis of a single Pt-d(GpG) adduct placed on codon 13 of the human HRAS protooncogene, the same sequence used for our previous in vivo studies (10). Results show that only DNA polymerase 3 is capable of in vitro translesional synthesis and indicate that its ability to initiate DNA replication opposite the cisplatin adduct may ...

Mutations in the parkin gene are expected to play an essential role in autosomal recessive Parkinson's disease. Recent studies have established an impact of parkin mutations on mitochondrial function and autophagy. In primary skin fibroblasts from two patients affected by an early onset Parkinson's disease, we identified a hitherto unreported compound heterozygous mutation del exon2-3/del exon3 in the parkin gene, leading to the complete loss of the full-length protein. In both patients, but not in their heterozygous parental control, we observed severe ultrastructural abnormalities, mainly in mitochondria. This was associated with impaired energy metabolism, deregulated reactive oxygen species (ROS) production, resulting in lipid oxidation, and peroxisomal alteration. In view of the involvement of parkin in the mitochondrial quality control system, we have investigated upstream events in the organelles' biogenesis. The expression of the peroxisome proliferator-activated receptor gamma-coactivator 1-alpha (PGC-1α), a strong stimulator of mitochondrial biogenesis, was remarkably upregulated in both patients. However, the function of PGC-1α was blocked, as revealed by the lack of its downstream target gene induction. In conclusion, our data confirm the role of parkin in mitochondrial homeostasis and suggest a potential involvement of the PGC-1α pathway in the pathogenesis of Parkinson's disease. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

The metabolic control of respiration is still poorly understood, due mainly to the lack of suitable approaches for studying it in vivo. Experiments on isolated mammalian mitochondria have indicated that a relatively small fraction of each of several components of the electron transport chain is sufficient to sustain a normal O 2 consumption rate. These experiments, however, may not ref lect accurately the in vivo situation, due to the lack in the mitochondrial fraction of essential cytosolic components and to the use of excess of substrates in the in vitro assays. An approach is described here whereby the control of respiration by cytochrome c oxidase (COX; EC 1.9.3.1) was analyzed in intact cultured human osteosarcoma 143B.TK ؊ cells and other wild-type cells and in mitochondrial DNA mutation-carrying human cell lines. Surprisingly, in wild-type cells, only a slightly higher COX capacity was detected than required to support the endogenous respiration rate, pointing to a tighter in vivo control of respiration by COX than generally assumed. Cell lines carrying the MERRF mitochondrial tRNA Lys gene mutation, which causes a pronounced decrease in mitochondrial protein synthesis and respiration rates, revealed, in comparison, a significantly greater COX capacity relative to the residual endogenous respiration rate, and, correspondingly, a higher COX inhibition threshold above which the overall respiratory f lux was affected. The observed relationship between COX respiratory threshold and relative COX capacity and the potential extension of the present analysis to other respiratory complexes have significant general implications for understanding the pathogenetic role of mutations in mtDNA-linked diseases and the tissue specificity of the mutation-associated phenotype.The rapid accumulation of knowledge concerning mitochondrial diseases, especially those caused by mitochondrial DNA (mtDNA) mutations, has stimulated in recent years a strong interest in the metabolic control of oxidative phosphorylation (OXPHOS). In particular, the discovery of threshold effects in the capacity of a mtDNA mutation to produce an OXPHOS defect in the presence of varying amounts of wild-type mtDNA has called attention to the degree of control that a particular step exerts in the OXPHOS pathway. It has been emphasized that application of the metabolic control theory (1, 2) to the study of mitochondrial metabolism can be a valuable approach for determining the level of control exerted by different OXPHOS steps on the rate of mitochondrial respiration (3), and for identifying and quantifying enzymatic defects caused in the OXPHOS machinery by mitochondrial or nuclear DNA mutations (4-7). Recently, this approach has been applied to isolated rat tissue mitochondria, by using increasing concentrations of inhibitors of complex I (rotenone), complex III (myxothiazol or antimycin A), or complex IV (potassium cyanide) to mimic the effects of mutations affecting these complexes (4-7). These experiments have produced results suggesting that ...

Eukaryotic cells devoid of mitochondrial DNA (ρ0 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. ρ0 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 ρ0 cell lines, we developed an extremely mild, reliable and timesaving method to generate ρ0 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 ρ0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK− ρ0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human ρ0 mitochondria.