Chicco 2022 Abstract Bioblast

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Chicco Adam
Chicco Adam, Zilhaver PT, Whitcomb LA, Fresa KJ, Izon CS, Gonzalez-Franquesa A, Izon CS, Dometita C, Irving BA, Garcia-Roves PM (2022) Elucidating the complexity of substrate-uncoupler-inhibitor titration protocols.
Bioblast 2022: BEC Inaugural Conference. In: https://doi.org/10.26124/bec:2022-0001
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Link: Bioblast 2022: BEC Inaugural Conference

Chicco Adam J, Zilhaver Philip T, Whitcomb Luke A, Fresa Kyle J, Izon Cheyanne S, Gonzalez-Franquesa Alba, Dometita Crystal, Irving Brian A, Garcia-Roves Pablo Miguel (2022)

Event: Bioblast 2022

In mitochondria (mt) of most tissues and cells, NADH-linked substrates (N: pyruvate, glutamate, malate) in combination with succinate (S) support higher respiratory capacities in the OXPHOS or electron transfer (ET) state compared to the separate N- or S-pathway capacities supported by either N-substrates or succinate&rotenone. NS-substrate combinations are required for partial reconstitution of TCA cycle function in mt-preparations, to compensate for metabolite depletion into the incubation medium. NS in combination exerts an additive effect on OXPHOS or ET capacity (P or E, respectively) of convergent electron-input into the Q-junction. Substrate-uncoupler-inhibitor titration (SUIT) protocols are used to resolve the relative contributions of the N- and S-pathway to the NS-supported oxygen consumption rate (JO2,NS = NSP or NSE). In SUIT protocols starting with N-pathway capacity, addition of succinate exerts a stimulatory effect (NS > N). Then the selective CI inhibitor rotenone is utilized to eliminate the contribution of the N-pathway to NS-pathway capacity, typically revealing an inhibitory effect on JO2 and thus allowing quantification of S-pathway capacity (NS > S). However, in some types of mitochondria, rotenone added in the NS-state elicits a paradoxical increase in JO2, revealing a complex interaction of N- and S-pathway substrate oxidation on JO2.

We demonstrate an inhibitory effect of >1 mM malate or 4 µM malonate (a CII inhibitor) on JO2,N supported by pyruvate (PM) and/or glutamate (PGM or GM). Collectively, our studies suggest that under these conditions, succinate formation is not completely prevented by the loss of TCA cycle intermediates. Thus, the S-pathway contributes to some extent to PM-, PGM-, or GM-supported respiration by oxidation of succinate formed endogenously in the TCA cycle and interacts with malate- and further fumarate- and oxaloacetate-concentrations to potently regulate JO2 supported by exogenous N-substrates in a tissue-specific manner. Potential mechanisms are discussed to stimulate further experimentation aimed at elucidating the biological bases for variations in NS-pathway flux in SUIT protocols used to study the additive effect of convergent electron flow at the Q-junction.

Keywords: Mitochondrial respiration, electron transfer system, succinate, glutamate, oxidative phosphorylation, high-resolution respirometry Bioblast editor: Gnaiger E O2k-Network Lab: US CO Fort Collins Chicco AJ, US LA Baton Rouge Irving BA, ES Barcelona Garcia-Roves PM


Affiliations

Chicco AJ1, Zilhaver PT1, Whitcomb LA1, Fresa KJ1, Izon CS1, Gonzalez-Franquesa A2, Dometita C3, Irving BA3,4, Garcia-Roves PM5
  1. Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA - [email protected]
  2. NovoNordisk, Copenhagen, Denmark
  3. Geisinger Obesity Institute, Danville, Pennsylvania, USA
  4. Department of Kinesiology, Louisiana State University, Baton Rouge, Louisiana, USA
  5. Department of Physiological Sciences, University of Barcelona, Barcelona, Spain

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