Timon-Gomez 2023 MiP2023
Substrate-uncoupler-inhibitor titration protocol for analyzing carbohydrate and fatty acid metabolism |
Link: MiP2023 Obergurgl AT
Timon-Gomez Alba (2023)
Event: MiP2023 Obergurgl AT
Authors: Timon-Gomez Alba, Cardoso Luiza HD, Doerrier Carolina, Garcia-Souza Luiz F, Gnaiger Erich
Introduction: Mitochondrial dysfunction in muscle tissue is associated with obesity (mitObesity) and its comorbidities. Many drugs and nutraceuticals used to treat these conditions target mitochondria. Early diagnosis of mitObesity is crucial for understanding the link between obesity, mitochondrial dysfunction, and its associated chronic comorbidities. Respirometry of mitochondrial preparations can assess electron transfer pathways and coupling in oxidative metabolism with high diagnostic resolution [1].
Methods: We developed a standardized protocol for functional diagnosis of mitochondrial defects using high-resolution respirometry [2]. This substrate-uncoupler-inhibitor titration (SUIT) protocol analyzes fatty acid oxidation (FAO) by adding 0.1 mM malate and octanoylcarnitine, with consideration of malate-linked anaplerosis to avoid overestimation of FAO [3-4]. The protocol is extended to stimulate the NADH-linked pathway by adding pyruvate and glutamate. Then succinate and glycerophosphate are titrated to investigate convergent CoQ-reducing pathways. A stepwise titration of uncoupler CCCP allows quantification of the electron transfer capacity. Residual oxygen consumption is assessed after inhibition by rotenone and antimycin A.
Results and discussion: To quantify FAO, malate was needed to avoid inhibition by accumulating acetyl-CoA. However, in the presence of mitochondrial malic enzyme, 2 mM malate stimulated respiration through the NADH-linked pathway in liver and brain mitochondria. Anaplerotic activity above endogenous respiration was minimized at a low (0.1 mM) malate concentration and subtracted from respiration obtained after addition of octanoylcarnitine. This SUIT reference protocol can be used as a general diagnostic tool for bioenergetic profiling in various sample preparations from different cell types, tissues, and organisms.
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β’ Keywords: fatty acid oxidation, SUIT, reference protocol, mitObesity
β’ O2k-Network Lab: AT Innsbruck Oroboros
Affiliatoin
- Timon-Gomez Alba, Cardoso LHD, Doerrier C, Garcia-Souza LF, Gnaiger E
- Oroboros Instruments, Innsbruck, AT
- Corresponding author: alba.timon@oroboros.at
Labels:
Pathology: Obesity
Regulation: Fatty acid
HRR: O2k-Protocol
Event: Oral