SUIT-017 O2 mt D046

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SUIT-017 O2 mt D046

Description

1OctM;2D;2c;3G;4S;5U;6Rot;7Ama.png

Abbreviation: FNS(Oct,GM)

Reference: A: SUIT-017

SUIT number: D046_1OctM;2D;3G;3c;4S;5U;6Rot;7Ama

O2k-Application: O2

MitoPedia: SUIT
SUIT-category: FNS(Oct,GM)
SUIT protocol pattern: diametral 1OctM;2D;3G;4S;5U;6Rot-

SUIT-017 O2 mt D046 gives information on F-pathway in LEAK state and OXPHOS state avoiding fatty acid oxidation (FAO) overestimation in the presence of anaplerotic pathways. In addition, the pathway control of FN and FNS in OXPHOS state and of FNS and S in ET state is evaluated.

Communicated by Krumschnabel G, Cardoso LHD, Antunes D, Iglesias-Gonzalez J and Gnaiger E (last update 2019-07-31)
MitoPedia: SUIT

Steps and respiratory states

1OctM;2D;2c;3G;4S;5U;6Rot;7Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1OctM OctML(n) F(N) FAO 1OctM

Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(n), in the absence of ADP, ATP, AMP (no adenylates).

2D OctMP F(N) FAO 1OctM;2D

Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.

2c OctGMcP FN FAO 1OctM;2D;2c

NADH-linked substrates (type N-pathway to Q). Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state. Addition of cytochrome c yields a test for integrity of the mtOM. Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (P, OXPHOS-capacity with saturating [ADP]).

3G OctGMP FN F&CI 1OctM;2D;2c;3G;

NADH-linked substrates (type N-pathway to Q). Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.

4S OctGMSP FNS F&CI&II 1OctM;2D;2c;3G;4S

Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.

5U OctGMSE FNS F&CI&II 1OctM;2D;2c;3G;4S;5U

Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. Noncoupled electron transfer state, ET-state, with ET-capacity, E.

6Rot SE S CII 1OctM;2D;2c;3G;4S;5U;6Rot

Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway if fatty acids are present. Noncoupled electron transfer state, ET-state, with ET-capacity, E.

7Ama ROX 1OctM;2D;2c;3G;4S;5U;6Rot;7Ama

Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.

Step Respiratory state Pathway control ET-Complex entry into Q-junction Comment
## AsTm AsTmE CIV CIV
## Azd ROX


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Strengths and limitations

  • Comparison of GM- with PM-capacity yields important information on N-pathway respiratory control upstream of CI (Lemeux et al 2017; Votion et al 2012).
+ Glutamate is easier to prepare compared to pyruvate.
+ Reasonable duration of the experiment.
+ This protocol can be extended with the Complex IV module, which can prolong the experimental time with ~30 min.

Compare SUIT protocols

  • SUIT-002: A more comprehensive protocol incuding additional substrate states.
  • SUIT-005: A comparable protocol evaluating P instead of G.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry