ATP synthesis and citrate synthase assays

We measured in vitro ATP production from isolated mitochondria following methods from [42, 55]. For each sample, ten males and ten females were homogenized in 800 μL isolation buffer (400 mM sucrose, 100 mM KCl, 6 mM EGTA, 3 mM EDTA, 70 mM HEPES, 1% w/v BSA, pH 7.6), and their mitochondria were isolated by sequential centrifugation. The isolated mitochondrial pellet was resuspended in ~55μL assay buffer (560 mM sucrose, 100mM KCL, 10mM KH₂PO₄, and 70mM HEPES). Suspended mitochondria (25μL) from each sample were then added to 5μL of either Complex I (CI) substrate (1 mM ADP, 2 mM malate, 10 mM glutamate, and assay buffer) or Complex II (CII) substrate [1 mM ADP, 10 mM succinate, 0.5 μM rotenone (Complex I inhibitor)]. Samples were incubated for 10 minutes at 20°C to allow for ATP synthesis. After the incubation period, 25μL of each sample was added to a 96-well assay plate with 25μL of CellTitre-Glo (Promega)—which halts ATP synthesis. Luminescence of the samples and ATP standards was measured on the same plate using a Fluoroskan Ascent FL plate reader (Thermo Labysystems).

A citrate synthase (CS) activity assay was performed on the remaining mitochondrial suspension to estimate mitochondrial volume and standardize ATP production per sample following the methods of [56]. Mitochondrial volume (or mitochondrial content) represents the fraction area of cell volume taken up by the mitochondria [57, 58]. CS activity correlates strongly with the fraction of mitochondrial surface area to total cell surface area in the cell [57], thus we use it as a proxy for mitochondrial content or mitochondrial volume [58] when standardizing ATP production [56]. CS activity was determined as follows: a 5μL aliquot of the mitochondrial suspension was added to 50μL of 200mM Tris buffer with 0.2% Triton-X (Sigma), 24μL DI water, 10μL 1mM DTNB (5, 5’-dithiobis (2-nitrobenzoic acid)), and 6μL acetyl coenzyme A (0.3mM). After estimating background activity, 5μL of 10mM oxaloacetic acid was added to each sample and absorbance read at 412nm. The change in absorbance measured over 5 minutes was used to calculate CS activity.