SUIT-017 O2 pfi D049

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SUIT-017 O2 pfi D049

Description

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

Abbreviation: FNS(Oct,GM)

Reference: A: SUIT-017

SUIT number: D049_1OctM;2D;2c;3G;4S;5U;6Rot;7Ama

O2k-Application: O2

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

SUIT-017 O2 pfi D049 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. Permeabilized muscle fibers are sensitive to oxygen supply due to limited diffusion of oxygen to the fiber bundle core. To counteract this limitation, hyperoxic conditions (400-250 µM O2) must be employed. To set the optimal oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.

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

Steps and respiratory states

1OctM;2D;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.
3G OctGMP FN F&CI 1OctM;2D;3G
4S OctGMSP FNS F&CI&II 1OctM;2D;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;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;3G;4S;5U;6Rot
7Ama ROX 1OctM;2D;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 (Lemieux 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.

Chemicals and syringes

Step Chemical(s) and link(s) Comments
1OctM Octanoylcarnitine (Oct) and Malate (M)
2D ADP (D)
2c Cytochrome c (c)
3G Glutamate (G)
4S Succinate (S)
5U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
6Rot Rotenone (Rot)
7Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.

References

 YearReferenceOrganismTissue;cell
Pesta 2012 Methods Mol Biol2012Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58.HumanSkeletal muscle
Other cell lines
HEK
Fibroblast
HUVEC


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry