Effects of essential fatty acid deficiency on mitochondria and peroxisomes in rat hepatocytes with special reference to a partially hydrogenated fish oil diet

Feeding male rats a high cal% partially hydrogenated fish oil diet induced morphological and biochemical changes in hepatocytes at the mitochondrial and peroxisomal level. At the mitochondrial level, formation of megamitochondria was related to the development of an essential fatty acid deficiency, as measured by a high 20:3/20:4 fatty acid ratio. These mitochondrial changes were fully prevented by adding linoleic acid to the partially hydrogenated fish oil diet. The megamitochondria revealed a normal specific content of respiratory chain pigments, normal specific respiratory rates and a normal energy coupling. At the peroxisomal level, feeding of the partially hydrogenated fish oil diet caused a considerable proliferation, which was unrelated to essential fatty acid deficiency. The total number of peroxisomes increased 1.9-fold, and 2.6-fold in the presence of added linoleic acid. Essential fatty acid deficiency seemed to result in an inhibition of peroxisomal biogenesis. It was concluded that the induction of megamitochondria by partially hydrogenated fish oil was fully attributable to essential fatty acid deficiency, whereas peroxisomal proliferation must be attributed to other factors in the diet.     

Rtg2 protein links metabolism and genome stability in yeast longevity

Mitochondrial dysfunction induces a signaling pathway, which culminates in changes in the expression of many nuclear genes. This retrograde response, as it is called, extends yeast replicative life span. It also results in a marked increase in the cellular content of extrachromosomal ribosomal DNA circles (ERCs), which can cause the demise of the cell. We have resolved the conundrum of how these two molecular mechanisms of yeast longevity operate in tandem. About 50% of the life-span extension elicited by the retrograde response involves processes other than those that counteract the deleterious effects of ERCs. Deletion of RTG2, a gene that plays a central role in relaying the retrograde response signal to the nucleus, enhances the generation of ERCs in cells with (grande) or in cells without (petite) fully functional mitochondria, and it curtails the life span of each. In contrast, overexpression of RTG2 diminishes ERC formation in both grandes and petites. The excess Rtg2p did not augment the retrograde response, indicating that it was not engaged in retrograde signaling. FOB1, which is known to be required for ERC formation, and RTG2 were found to be in converging pathways for ERC production. RTG2 did not affect silencing of ribosomal DNA in either grandes or petites, which were similar to each other in the extent of silencing at this locus. Silencing of ribosomal DNA increased with replicative age in either the presence or the absence of Rtg2p, distinguishing silencing and ERC accumulation. Our results indicate that the suppression of ERC production by Rtg2p requires that it not be in the process of transducing the retrograde signal from the mitochondrion. Thus, RTG2 lies at the nexus of cellular metabolism and genome stability, coordinating two pathways that have opposite effects on yeast longevity.     

Frataxin deficiency and mitochondrial dysfunction

Friedreich ataxia (FA) is an inherited recessive disorder characterized by progressive neurological disability and heart abnormalities. The Friedreich ataxia gene (FRDA) encodes a small mitochondrial protein, frataxin, which is produced in insufficient amounts in the disease as a consequence of a GAA triplet repeat expansion in the first intron of the gene. Frataxin deficiency leads to excessive free radical production, dysfunction of Fe-S center containing enzymes (in particular respiratory complexes I, II and III, and aconitase), and progressive iron accumulation in mitochondria. Frataxin may be a mitochondrial iron-binding protein that prevents this metal from participating in Fenton chemistry to generate toxic hydroxyl radicals. We investigated whether frataxin deficiency may in addition interfere with signaling pathways. First, we showed that exposure of FA fibroblasts to iron fails to produce the normally observed increase in expression of the stress defense protein manganese superoxide dismutase. This impaired induction involves a nuclear factor-kappaB-independent pathway that does not require free radical signaling intermediates. We also examined the role of frataxin in neuronal differentiation by using stably transfected clones of P19 embryonic carcinoma cells with antisense or sense frataxin constructs. We found that during retinoic acid-induced neurogenesis frataxin deficiency enhances apoptosis and reduces the number of terminally differentiated neuronal-like cells. The addition of the antioxidant N-acetyl-cysteine only rescues cells non-committed to the neuronal lineage, indicating that frataxin deficiency impairs differentiation mechanisms and survival responses through different mechanisms. Both studies suggest that some abnormalities in frataxin-deficient cells are related to free radical independent signaling pathways.     

Peroxisomal L-bifunctional enzyme (Ehhadh) is essential for the production of medium-chain dicarboxylic acids

L-bifunctional enzyme (Ehhadh) is part of the classical peroxisomal fatty acid β-oxidation pathway. This pathway is highly inducible via peroxisome proliferator-activated receptor α (PPARα) activation. However, no specific substrates or functions for Ehhadh are known, and Ehhadh knockout (KO) mice display no appreciable changes in lipid metabolism. To investigate Ehhadh functions, we used a bioinformatics approach and found that Ehhadh expression covaries with genes involved in the tricarboxylic acid cycle and in mitochondrial and peroxisomal fatty acid oxidation. Based on these findings and the regulation of Ehhadh's expression by PPARα, we hypothesized that the phenotype of Ehhadh KO mice would become apparent after fasting. Ehhadh mice tolerated fasting well but displayed a marked deficiency in the fasting-induced production of the medium-chain dicarboxylic acids adipic and suberic acid and of the carnitine esters thereof. The decreased levels of adipic and suberic acid were not due to a deficient induction of ω-oxidation upon fasting, as Cyp4a10 protein levels increased in wild-type and Ehhadh KO mice.We conclude that Ehhadh is indispensable for the production of medium-chain dicarboxylic acids, providing an explanation for the coordinated induction of mitochondrial and peroxisomal oxidative pathways during fasting.     

p21WAF1/CIP1 deficiency induces mitochondrial dysfunction in HCT116 colon cancer cells

p21^WAF1/CIP1 is a critical regulator of cell cycle progression. However, the role of p21 in mitochondrial function remains poorly understood. In this study, we examined the effect of p21 deficiency on mitochondrial function in HCT116 human colon cancer cells. We found that there was a significant increase in the mitochondrial mass of p21^?/? HCT116 cells, as measured by 10-N-nonyl-acridine orange staining, as well as an increase in the mitochondrial DNA content. In contrast, p53^?/? cells had a mitochondrial mass comparable to that of wild-type HCT116 cells. In addition, the expression levels of the mitochondrial biogenesis regulators PGC-1α and TFAM and AMPK activity were also elevated in p21^?/? cells, indicating that p21 deficiency induces the rate of mitochondrial biogenesis through the AMPK-PGC-1α axis. However, the increase in mitochondrial biogenesis in p21^?/? cells did not accompany an increase in the cellular steady-state level of ATP. Furthermore, p21^?/? cells exhibited significant proliferation impairment in galactose medium, suggesting that p21 deficiency induces a defect in the mitochondrial respiratory chain in HCT116 cells. Taken together, our results suggest that the loss of p21 results in an aberrant increase in the mitochondrial mass and in mitochondrial dysfunction in HCT116 cells, indicating that p21 is required to maintain proper mitochondrial mass and respiratory function.     

Overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance

In yeast the UPF1, UPF2 and UPF3 genes encode three interacting factors involved in translation termination and nonsense-mediated mRNA decay (NMD). UPF1 plays a central role in both processes. In addition, UPF1 was originally isolated as a multicopy suppressor of mitochondrial splicing deficiency, and its deletion leads to an impairment in respiratory growth. Here, we provide evidence that inactivation of UPF2 or UPF3, like that of UPF1, leads to an impairment in respiratory competence, suggesting that their products, Upf1p, Upf2p and Upf3p, are equivalently involved in mitochondrial biogenesis. In addition, however, we show that only Upf1p acts as a multicopy suppressor of mitochondrial splicing deficiency, and its activity does not require either Upf2p or Upf3p. Mutations in the conserved cysteine- and histidine-rich regions and ATPase and helicase motifs of Upf1p separate the ability of Upf1p to complement the respiratory impairment of a Deltaupf1 strain from its ability to act as a multicopy suppressor of mitochondrial splicing deficiency, indicating that distinct pathways express these phenotypes. In addition, we show that, when overexpressed, Upf1p is not detected within mitochondria, suggesting that its role as multicopy suppressor of mitochondrial splicing deficiency is indirect. Furthermore, we provide evidence that cells overexpressing certain upf1 alleles accumulate a phosphorylated isoform of Upf1p. Altogether, these results indicate that overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance, which relies on Upf1p-Upf2p-Upf3p functional interplay.     

Peroxisomal ABC transporters and beta-oxidation during the life cycle of the filamentous fungus Podospora anserina

ATP-binding cassette transporters are ubiquitous proteins that facilitate transport of diverse substances across a membrane. However, their exact role remains poorly understood. In order to test their function in a fungus life cycle, we deleted the two Podospora anserina peroxisomal ABC transporter pABC1 and pABC2 genes as well as the three genes involved in peroxisomal (fox2) and mitochondrial (scdA and echA) beta-oxidation. Analysis of the single and double mutants shows that fatty acid beta-oxidation occurs in both organelles. Furthermore, the peroxisomal and mitochondrial fatty acid beta-oxidation pathways are both dispensable for vegetative and sexual development. They are, however, differently required for ascospore pigmentation and germination, this latter defect being restored in a DeltapABC1 and DeltapABC2 background. We report also that lack of peroxisomal ABC transporters does not prevent peroxisomal long-chain fatty acid oxidation, suggesting the existence of another pathway for their import into peroxisomes. Finally, we show that some aspects of fatty acid degradation are clearly fungus species specific.