Assessing bioenergetic functions from isolated mitochondria in Drosophila melanogaster

Aw, Wen C.; Bajracharya, Rijan; Towarnicki, Samuel G.; Ballard, J. William O.

doi:10.14440/jbm.2016.112

Cited by 14 publications

(13 citation statements)

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“…We predicted that functionally significant mutations would reduce the activity of the complex. In vitro mitochondrial oxygen consumption is increasingly recognised as a fundamental measure of mitochondrial function [ 89 , 90 ] and we assayed the rate from extracted mitochondria using complex I and II substrates [ 91 ]. Complex I substrates assay the combined mitochondrial functions of complexes I, III and IV, while complex II substrates assay the collective functions of complexes II, III and IV.…”

Section: Resultsmentioning

confidence: 99%

“…Here, we test the hypothesis that rates of β-oxidation differed between the mitotypes and add Etomoxir to the diet. β-oxidation of fatty acids generates NADH and FADH 2 and thereby partially bypasses complex I of the electron transport system [ 91 ]. Etomoxir inhibits entry of long-chain fatty acids into the mitochondrion via the carnitine shuttle and we predicted its addition would result in loss of the selective advantage to Dahomey.…”

Section: Resultsmentioning

confidence: 99%

“…The sequence of the A + T rich region showed no differences from the published short-read sequences [ 84 ] and no differences in repeat number between mitotypes (NCBI Project accession #PRJNA397821). Mitochondria were extracted from adult flies [ 91 ]. The mtDNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen 69582).…”

Section: Methodsmentioning

confidence: 99%

“…Mitochondrial oxygen consumption of mitochondria isolated from third instar wandering female larvae was assayed using the XF24 Extracellular Flux Analyser (Seahorse Bioscience, MA, USA) at 23° C using complex I and complex II substrates [ 91 ]. Respiration rates were expressed as oxygen consumption rate with the unit of pmol of oxygen consumption per min per unit of citrate synthase activity (pmol.…”

Section: Methodsmentioning

confidence: 99%

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Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness

Towarnicki

Melvin

et al. 2018

PLoS Genet

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Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. All these interactions have the potential to alter the frequencies of mtDNA haplotypes (mitotypes) in nature and may impact human health. In a model laboratory system, we fed four diets varying in Protein: Carbohydrate (P:C) ratio (1:2, 1:4, 1:8 and 1:16 P:C) to four homoplasmic Drosophila melanogaster mitotypes (nuclear genome standardised) and assayed their frequency in population cages. When fed a high protein 1:2 P:C diet, the frequency of flies harbouring Alstonville mtDNA increased. In contrast, when fed the high carbohydrate 1:16 P:C food the incidence of flies harbouring Dahomey mtDNA increased. This result, driven by differences in larval development, was generalisable to the replacement of the laboratory diet with fruits having high and low P:C ratios, perturbation of the nuclear genome and changes to the microbiome. Structural modelling and cellular assays suggested a V161L mutation in the ND4 subunit of complex I of Dahomey mtDNA was mildly deleterious, reduced mitochondrial functions, increased oxidative stress and resulted in an increase in larval development time on the 1:2 P:C diet. The 1:16 P:C diet triggered a cascade of changes in both mitotypes. In Dahomey larvae, increased feeding fuelled increased β-oxidation and the partial bypass of the complex I mutation. Conversely, Alstonville larvae upregulated genes involved with oxidative phosphorylation, increased glycogen metabolism and they were more physically active. We hypothesise that the increased physical activity diverted energy from growth and cell division and thereby slowed development. These data further question the use of mtDNA as an assumed neutral marker in evolutionary and population genetic studies. Moreover, if humans respond similarly, we posit that individuals with specific mtDNA variations may differentially metabolise carbohydrates, which has implications for a variety of diseases including cardiovascular disease, obesity, and perhaps Parkinson’s Disease.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness

Towarnicki

Melvin

et al. 2018

PLoS Genet

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“…Isolation of Mitochondria 156Mitochondria were isolated from larvae following a protocol modified fromAw et al, 2016.157 100-200 larvae were collected and rinsed with larval wash buffer (0.7% NaCl and 0.1% Triton 158 X-100). Larvae were very gently homogenized in 300-500 µL of chilled isolation buffer (154 159 mM KCl, 1 mM EDTA, pH 7.4) in a glass-teflon Thomas® homogenizer on ice.…”

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confidence: 99%

Genetic variation for ontogenetic shifts in metabolism underlies physiological homeostasis inDrosophila

Matoo

Julick

Montooth

2018

Preprint

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12Organismal physiology emerges from metabolic pathways and structures that can vary across 13 development and among individuals. Here we tested whether genetic variation at one level of 14 physiology can be buffered at higher levels during development by the inherent capacity for 15 homeostasis in physiological systems. We found that the fundamental scaling relationship 16 between mass and metabolic rate, as well as the oxidative capacity per mitochondria, differed 17 significantly across development in the fruit fly Drosophila. However, mitochondrial respiration 18 rate was maintained across development at similar levels. Furthermore, genotypes clustered into 19 two types-those that switched to aerobic, mitochondrial ATP production before the second 20 instar and those that relied on anaerobic production of ATP via glycolysis through the second 21 instar. Despite genetic variation for the timing of this metabolic shift, second-instar metabolic 22 rate was more robust to genetic variation than was the metabolic rate of other instars. We also 23 found that a mitochondrial-nuclear genotype with disrupted mitochondrial function both 24 increased aerobic capacity more through development and relied more heavily on anaerobic ATP 25 production relative to wildtype genotypes. By taking advantage of both ways of making ATP, 26 this genotype maintained mitochondrial respiratory capacity, but also generated more free 27 radicals and had decreased mitochondrial membrane potential, potentially as a physiological-28 defense mechanism. Taken together, the data revealed that genetic defects in core physiology can 29 be buffered at the organismal level via physiological compensation and that natural populations 30 likely harbor genetic variation for distinct metabolic strategies in development that generate 31 similar organismal outcomes. 32 33 Keywords: mtDNA, metabolism, oxidative phosphorylation, reactive oxygen species 34 A challenge to connecting genetic variation in biochemical processes to metabolic 57 performance, is that metabolism is an emergent property of interacting biochemical, structural, 58 regulatory and physiological systems, often arranged in hierarchical functional modules 59 (Barabási and Oltvai, 2004;Jeong et al., 2000;Ravasz et al., 2002;Strogatz, 2001). In addition, 60 metabolic enzymes and metabolites have potential "moonlighting" functions in the signaling that 61 underlies metabolic homeostasis (Marden, 2013;Boukouris et al., 2016). The capacity for 62 homeostasis in physiological systems, also suggests that genetic variation in biochemical 63 processes at one level of the energetic hierarchy may not necessarily result in organismal fitness 64 variation. In other words, the physiological regulatory processes that maintain energy 65 homeostasis may provide stability in metabolic trajectories, in an analogous way to the canalized 66 developmental trajectories envisioned by Waddington (Meiklejohn and Hartl, 2002; 67 Waddington, 1942 67 Waddington, , 1957. Furthermore, a diversity of biochemical pa...

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Differential manifestation of RONS and antioxidant enzymes in response to singular versus combinatorial stress in Chironomus ramosus

Bomble

Nath

2022

Stress Biology

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In nature, organisms face multiple abiotic stress concurrently. Our previous study has indicated how threshold level of lethality depends on the type and combination of stressors. Many mechanisms exist by which organisms respond to stressors and maintain homeostasis. We examined the homeostatic pliability in an extremophilic oriental midge Chironomus ramosus larvae under various combinatorial stress conditions of desiccation (DS), heat (HS) and starvation (SS). Exposure to these stressors led to activation of a common response pathway of oxidative stress. Abundance of antioxidant enzymes like superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase along with selective as well as stressor specific increase in total antioxidant capacity were reflected from the corresponding level of reactive oxygen and nitrogen species (RONS) in larvae exposed to various combinatorial stress. Additionally, we found stressor specific increment in lipid peroxidation level, protein carbonyl content and advanced oxidative protein products during the stress regime. Further investigation revealed a sharp decline in the activity of mitochondrial aconitase enzyme activity in response to abiotic stress induced oxidative stress. The combinatorial stressor specific comparative study based on biochemical and fluorescence based redox-endpoint assays confirmed that the generation of oxidative stress is the consequential convergent pathway of DS, HS and SS, but the quantum of RONS decides the redox potential of homeostatic response and survival rate.

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Assessing bioenergetic functions from isolated mitochondria in Drosophila melanogaster

Cited by 14 publications

References 14 publications

Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness

Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness

Genetic variation for ontogenetic shifts in metabolism underlies physiological homeostasis inDrosophila

Differential manifestation of RONS and antioxidant enzymes in response to singular versus combinatorial stress in Chironomus ramosus

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