Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

Our research focuses on how mitochondria sense and adapt to metabolic stressors. One key metabolic stressor is hypoxia, which occurs when cells are starved of oxygen. We are developing better assays to study mitochondrial function in low oxygen environments.

High resolution mass spectrometry was employed to study cell and tissue metabolite levels. We use stable isotope tracing studies to track the fate of specific metabolites and uncover novel metabolic pathways relevant to healthy and diseased mammalian physiology. Group has found that mammalian mitochondria can sustain critical functions in low oxygen environments.

Mechanistically, they employ fumarate as an alternative electronic scepter for the electron transport chain. This protocol compiles several assays that researchers can use to better assess mitochondrial function in mammalian cells. Importantly, these protocols can be used to study mitochondrial function in hypoxia, as classical respirometry experiments will not be sufficient.

To begin, see the 143B human osteosarcoma cells in a six well plate. Once cells become 75%confluent, replace the medium in each well with the medium containing 10 millimolar, 13C4 aspartate and incubate for eight hours to achieve a steady state for labeling the cells. For isolating metabolites, cool the prepared buffer overnight at 80 degrees Celsius or place it on dry ice.

Meanwhile, take a plate from the incubator and aspirate the medium from each well using a pipette. Wash it with PBS twice and then remove the residual PBS before proceeding further. Now place the plate on dry ice and add 800 microliters of prepared buffer to each well.

To facilitate cell lysis, incubate the plate for 15 minutes at 80 degrees Celsius. After incubation, scrape each well on dry ice using a cell lifter and transfer the lysate to a 15 milliliter micro centrifuge tube placed on dry ice. Vortex the lysate for 10 minutes at 4 degrees Celsius and centrifuge it at 4 degrees Celsius and 17, 000 G for 10 minutes.

Then transfer the supernatant to a 1.5 milliliter micro centrifuge tube and dry it in a 4 degree Celsius vacuum concentrator with a cold trap under a high vacuum for approximately six hours. Store the dried metabolite pellet at 80 degrees Celsius until further use. For analyzing the sample using liquid chromatography mass spectrometry or LCMS, add 100 microliters of HPLC grade water to the dried pellet and vortex as demonstrated.

Centrifuge the samples at 4 degrees Celsius for 10 minutes at maximum speed. Then transfer 25 microliters of supernatant to the LCMS vial and inject two microliters into the LCMS system. To perform liquid chromatography, use a constant flow rate of 0.15 milliliter per minute under the gradient mode of a mobile phase B from 80%to 20%for 20 minutes, 20%to 80%for 0.5 minutes, and the hold at 80%for 7.5 minutes against mobile phase A.Next, perform mass spectroscopy by choosing a full scan between MZ 70 to 1000 Dalton and resolution at 70, 000.

Set AGC target of 1 x 10 to the 6 and a maximum injection time of 20 milliseconds. Next, set the runtime to 16.5 minutes. Polarity set to positive.

AGC target set to 1 to the power 5. Maximum IT set to 20 milliseconds and scan range set to 70 to 1000 mass to charge ratio. Then set the spray voltage at 3.0 kilovolts and the heated capillary at 275 degrees Celsius.

Select the sheath gas flow at 40 units, the auxiliary gas flow at 15 units, and the sweep gas flow at 1 unit. For 13C5 glutamine isotope tracing, once the cells reach 75%confluency, change the medium to 2 millimolar 13C5 glutamine containing medium and incubate to achieve a steady state of labeling the cells of interest. Isolate and analyze the metabolites as previously demonstrated.

After isolating and analyzing the metabolites, analyze the succinate dehydrogenase, or SDH activity by calculating the percent labeling of 13C3 fumarate, 13C4 fumarate, 13C3 succinate, and 13C4 succinate. Then evaluate succinate oxidation and fumarate reduction using the formula. In vehicle treated conditions, the SDH complex favored the forward activity and the incorporation of 13C4 succinate into 13C4 fumarate was higher than 13C3 fumarate into 13C3 succinate.

In antimycin treated osteosarcoma cells, the SDH complex favored the reverse activity and the incorporation of 13C3 fumarate into 13C3 succinate was more significant than 13C4 succinate into 13C4 fumarate.