Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells

Spinazzi, Marco; Casarin, Alberto; Pertegato, Vanessa; Salviati, Leonardo; Angelini, C.

doi:10.1038/nprot.2012.058

Cited by 828 publications

(732 citation statements)

References 34 publications

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“…Activities of NADH:coenzyme Q oxidoreductase (complex I), succinate dehydrogenase (complex II), ubiquinol:cytochrome c oxidoreductase (complex III), cytochrome c oxidase (complex IV), and citrate synthase (CS) were determined in mouse livers by spectrophotometric assays as described (Spinazzi, Casarin, Pertegato, Salviati & Angelini, 2012). …”

Section: Methodsmentioning

confidence: 99%

Overexpression of CYB5R3 and NQO1, two NAD⁺‐producing enzymes, mimics aspects of caloric restriction

et al. 2018

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SummaryCalorie restriction (CR) is one of the most robust means to improve health and survival in model organisms. CR imposes a metabolic program that leads to increased stress resistance and delayed onset of chronic diseases, including cancer. In rodents, CR induces the upregulation of two NADH‐dehydrogenases, namely NAD(P)H:quinone oxidoreductase 1 (Nqo1) and cytochrome b 5 reductase 3 (Cyb5r3), which provide electrons for energy metabolism. It has been proposed that this upregulation may be responsible for some of the beneficial effects of CR, and defects in their activity are linked to aging and several age‐associated diseases. However, it is unclear whether changes in metabolic homeostasis solely through upregulation of these NADH‐dehydrogenases have a positive impact on health and survival. We generated a mouse that overexpresses both metabolic enzymes leading to phenotypes that resemble aspects of CR including a modest increase in lifespan, greater physical performance, a decrease in chronic inflammation, and, importantly, protection against carcinogenesis, one of the main hallmarks of CR. Furthermore, these animals showed an enhancement of metabolic flexibility and a significant upregulation of the NAD +/sirtuin pathway. The results highlight the importance of these NAD + producers for the promotion of health and extended lifespan.

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Section: Methodsmentioning

confidence: 99%

Overexpression of CYB5R3 and NQO1, two NAD⁺‐producing enzymes, mimics aspects of caloric restriction

et al. 2018

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“…COX activity was measured in liver, heart, and WAT homogenates as described (Spinazzi, Casarin, Pertegato, Salviati & Angelini, 2012). The rate of oxidation of cytochrome c was calculated from the change in absorbance at 550 nm.…”

Section: Methodsmentioning

confidence: 99%

Lifelong reduction in complex IV induces tissue‐specific metabolic effects but does not reduce lifespan or healthspan in mice

et al. 2018

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SummaryLoss of SURF1, a Complex IV assembly protein, was reported to increase lifespan in mice despite dramatically lower cytochrome oxidase (COX) activity. Consistent with this, our previous studies found advantageous changes in metabolism (reduced adiposity, increased insulin sensitivity, and mitochondrial biogenesis) in Surf1 −/− mice. The lack of deleterious phenotypes in Surf1 −/− mice is contrary to the hypothesis that mitochondrial dysfunction contributes to aging. We found only a modest (nonsignificant) extension of lifespan (7% median, 16% maximum) and no change in healthspan indices in Surf1 −/− vs. Surf1 +/+ mice despite substantial decreases in COX activity (22%–87% across tissues). Dietary restriction (DR) increased median lifespan in both Surf1 +/+ and Surf1 −/− mice (36% and 19%, respectively). We measured gene expression, metabolites, and targeted expression of key metabolic proteins in adipose tissue, liver, and brain in Surf1 +/+ and Surf1 −/− mice. Gene expression was differentially regulated in a tissue‐specific manner. Many proteins and metabolites are downregulated in Surf1 −/− adipose tissue and reversed by DR, while in brain, most metabolites that changed were elevated in Surf1 −/− mice. Finally, mitochondrial unfolded protein response (UPRmt)‐associated proteins were not uniformly altered by age or genotype, suggesting the UPRmt is not a key player in aging or in response to reduced COX activity. While the changes in gene expression and metabolism may represent compensatory responses to mitochondrial stress, the important outcome of this study is that lifespan and healthspan are not compromised in Surf1 −/− mice, suggesting that not all mitochondrial deficiencies are a critical determinant of lifespan.

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“…9 Activities of NADH:coenzyme Q 1 oxidoreductase (complex I), cytochrome C oxidoreductase (complex III), NADH:cytochrome C reductase (complex I+III), succinate:cytochrome C reductase (complex II+ III) and citrate synthase (CS) were determined in cell extracts using previously described spectrophotometric methods. 10 Results are expressed as specific activity (μmol/ min/mg protein; mean ± SD). Mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) generation were carried out as described.…”

Section: Mitochondrial Biochemistrymentioning

confidence: 99%

“…4,5 In contrast to the already described cases of mitochondrial diseases showing CoQ 10 deficiency, we describe here two unrelated children that present a fatal neurological disorder, associated to genetic variants in PDH genes. The two cases presented high levels of both CoQ 10 and mitochondria in fibroblasts and skeletal muscle, with low efficient mitochondria that show defective homeostasis, and high levels of antioxidant enzymes.…”

Section: Introductionmentioning

confidence: 95%

“…6 Among tissues, CoQ content in brain is very stable and highly independent of environment. 7 Furthermore, mutations of genes that are not part of respiratory chain also cause mitochondrial dysfunctions and may induce an adaptive decrease of CoQ 10 , named secondary deficiency, although the mechanisms are totally unknown. 4,5 In contrast to the already described cases of mitochondrial diseases showing CoQ 10 deficiency, we describe here two unrelated children that present a fatal neurological disorder, associated to genetic variants in PDH genes.…”

Section: Introductionmentioning

confidence: 99%

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Severe encephalopathy associated to pyruvate dehydrogenase mutations and unbalanced coenzyme Q10 content

et al. 2015

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Coenzyme Q 10 (CoQ 10 ) deficiency is associated to a variety of clinical phenotypes including neuromuscular and nephrotic disorders. We report two unrelated boys presenting encephalopathy, ataxia, and lactic acidosis, who died with necrotic lesions in different areas of brain. Levels of CoQ 10 and complex II+III activity were increased in both skeletal muscle and fibroblasts, but it was a consequence of higher mitochondria mass measured as citrate synthase. In fibroblasts, oxygen consumption was also increased, whereas steady state ATP levels were decreased. Antioxidant enzymes such as NQO1 and MnSOD and mitochondrial marker VDAC were overexpressed. Mitochondria recycling markers Fis1 and mitofusin, and mtDNA regulatory Tfam were reduced. Exome sequencing showed mutations in PDHA1 in the first patient and in PDHB in the second. These genes encode subunits of pyruvate dehydrogenase complex (PDH) that could explain the compensatory increase of CoQ 10 and a defect of mitochondrial homeostasis. These two cases describe, for the first time, a mitochondrial disease caused by PDH defects associated with unbalanced of both CoQ 10 content and mitochondria homeostasis, which severely affects the brain. Both CoQ 10 and mitochondria homeostasis appears as new markers for PDH associated mitochondrial disorders.

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Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells

Cited by 828 publications

References 34 publications

Overexpression of CYB5R3 and NQO1, two NAD⁺‐producing enzymes, mimics aspects of caloric restriction

Overexpression of CYB5R3 and NQO1, two NAD⁺‐producing enzymes, mimics aspects of caloric restriction

Lifelong reduction in complex IV induces tissue‐specific metabolic effects but does not reduce lifespan or healthspan in mice

Severe encephalopathy associated to pyruvate dehydrogenase mutations and unbalanced coenzyme Q10 content

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Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells

Cited by 828 publications

References 34 publications

Overexpression of CYB5R3 and NQO1, two NAD+‐producing enzymes, mimics aspects of caloric restriction

Overexpression of CYB5R3 and NQO1, two NAD+‐producing enzymes, mimics aspects of caloric restriction

Lifelong reduction in complex IV induces tissue‐specific metabolic effects but does not reduce lifespan or healthspan in mice

Severe encephalopathy associated to pyruvate dehydrogenase mutations and unbalanced coenzyme Q10 content

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Overexpression of CYB5R3 and NQO1, two NAD⁺‐producing enzymes, mimics aspects of caloric restriction

Overexpression of CYB5R3 and NQO1, two NAD⁺‐producing enzymes, mimics aspects of caloric restriction