SUIT-016

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SUIT-016

Description

Abbreviation: FNS(Oct,GM)

Reference: A »Versions

SUIT-category: FNS(Oct,GM)

SUIT protocol pattern: diametral/complex 1OctM;2D;3G;4S;5Rot;6Omy;7U-

SUIT-016 gives information on F-pathway in LEAK state and OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. In addition, the pathway control of FN and FNS in OXPHOS state and of S in LEAK state and ET state is evaluated.

Communicated by Doerrier C, Huete-Ortega M and Gnaiger E (last update 2019-01-30)

Specific SUIT protocols

References

	Year	Reference	Organism	Tissue;cell
Gnaiger 2015 Scand J Med Sci Sports	2015	Gnaiger E, Boushel R, Søndergaard H, Munch-Andersen T, Damsgaard R, Hagen C, Díez-Sánchez C, Ara I, Wright-Paradis C, Schrauwen P, Hesselink M, Calbet JAL, Christiansen M, Helge JW, Saltin B (2015) Mitochondrial coupling and capacity of oxidative phosphorylation in skeletal muscle of Inuit and caucasians in the arctic winter. https://doi.org/10.1111/sms.12612	Human	Skeletal muscle

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1OctM	OctM_L(n)	F(N)	CETF	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	OctM_P	F(N)	CETF	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	OctGM_P	FN	CETF&I	1OctM;2D;3G 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. & NADH-linked substrates (type N-pathway to Q). Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4S	OctGMS_P	FNS	CETF&CI&II	1OctM;2D;3G;4S 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. & NADH-linked substrates (type N-pathway to Q). & Succinate, S ( type S-pathway to Q). Respiratory stimulation by simultaneous action of the F-pathway, N-pathway, and S-pathway, with convergent electron flow in the FNS-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5Rot	S_P	S	CII	1OctM;2D;3G;4S;5Rot Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway. Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
6Omy	S_L(Omy)	S	CII	1OctM;2D;3G;4S;5Rot;6Omy Succinate, S ( type S-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
7U	S_E	S	CII	1OctM;2D;3G;4S;5Rot;6Omy;7U Succinate, S ( type S-pathway to Q). Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency. Noncoupled electron transfer state, ET state, with ET capacity E.
8Ama	ROX			1OctM;2D;3G;4S;5Rot;6Omy;7U;8Ama Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).

Step	Respiratory state	Pathway control	ET-Complex	Comment
## AsTm	AsTm_E	CIV	CIV
## Azd	CHB

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Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

Strengths and limitations

+ This protocol provides information on FAO capacity in the absence of other potentially interfering pathways both in LEAK and OXPHOS coupling control states.

+ FNS OXPHOS capacity comprises the most important pathways in many cell types and thus provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.

+ FNS ET capacity is a good estimate of overall ET capacity in many cell types.

+ This is a good protocol to analyse coupling control at S-pathway, which is an advantage compared to SUIT-015 and SUIT-017.

+ Glutamate is easier to prepare compared to pyruvate.

- In some tissues GM is not sufficient to fully support N-pathway capacity

- SRot(E) may be underestimated if S is not saturating.

- SRot(E) may be underestimated in the presence of OMY in some tissues and cell types

- CIV activity is not measured, to save experimental time.

Compare SUIT protocols

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry