Difference between revisions of "Sumbalova 2014 Abstract MiP2014"

From Bioblast
Jump to navigation Jump to search
(15 intermediate revisions by 5 users not shown)
Line 1: Line 1:
{{Abstract
{{Abstract
|title=Optimization of malate concentration for high-resolution respirometry: mitochondria from rat liver and brain.
|title=Optimization of malate concentration for high-resolution respirometry: mitochondria from rat liver and brain.
|info=[[File:Person.JPG|240px|right|Name]] [http://www.mitophysiology.org/index.php?mip2014 MiP2014], [[Laner 2014 Mitochondr Physiol Network MiP2014|Book of Abstracts Open Access]]
|info=[[File:Sumbalova_Z.jpg|180px|right|Sumbalova Z]] [[Laner 2014 Mitochondr Physiol Network MiP2014|Mitochondr Physiol Network 19.13]] - [http://www.mitophysiology.org/index.php?mip2014 MiP2014]
|authors=Sumbalova Z, Vancova O, Krumschnabel G, Gnaiger E
|authors=Sumbalova Z, Vancova O, Krumschnabel G, Gnaiger E
|year=2014
|year=2014
|event=MiP2014
|event=MiP2014
|abstract=Complex substrate-uncoupler-inhibitor titration (SUIT) [1] protocols, used in high-resolution respirometry, are designed to determine respiration with C<sub>I</sub>-linked and C<sub>II</sub>-linked substrates in a single experiment. Observations on multiple types of mitochondria revealed that malate, a substrate used to fuel C<sub>I</sub>-linked respiration, inhibits C<sub>II</sub>-linked (succinate+rotenone; coupled and noncoupled) respiration at 2 mM concentration. Mutual interference between succinate and malate was already described by Harris and Manger [2], and the effect was attributed to accumulation of oxaloacetate, an inhibitor of succinate dehydrogenase (SDH) and fumarate [1,3].
|abstract=Complex substrate-uncoupler-inhibitor titration (SUIT) [1] protocols, used in high-resolution respirometry, are designed to determine respiration with CI-linked and CII-linked substrates in a single experiment. Observations on multiple types of mitochondria revealed that malate, a substrate used to fuel CI-linked respiration, inhibits CII-linked (succinate+rotenone; coupled and noncoupled) respiration at 2 mM concentration. Mutual interference between succinate and malate was already described by Harris and Manger [2], and the effect was attributed to accumulation of oxaloacetate, an inhibitor of succinate dehydrogenase (SDH) and fumarate [1,3].


In this study, we examined the effect of various concentrations of malate on C<sub>I</sub>-linked and C<sub>II</sub>-linked respiration, as well as on the involvement of SDH as a constitutive part of Krebs cycle, in the respiration with C<sub>I</sub>-linked substrate combinations, in mitochondria from rat liver and brain. In both liver and brain mitochondria, 0.5 mM malate, added in combination with 5 mM pyruvate and 10 mM glutamate, supported >90% of maximal C<sub>I</sub>-linked respiration observed with saturating 2 mM malate. Conversely, when malate was added to noncoupled mitochondria fueled by C<sub>II</sub>-linked substrate succinate+rotenone, it inhibited ETS capacity  by 6% and 8% at 0.5 mM, 22% and 25% at 2 mM, and 33% and 37% at 5 mM malate concentration, in mitochondria from liver and brain, respectively. A similar degree of inhibition of noncoupled C<sub>II</sub>-linked respiration by malate was observed when mitochondria were previously exposed to C<sub>I+II</sub>–linked substrates followed by inhibition of C<sub>I</sub> with rotenone. Assuming that this effect is caused by an indirect impact of malate on SDH, lower inhibition of respiration at lower malate concentration would be suggestive of a higher involvement of SDH in respiration with C<sub>I</sub>-linked substrate combinations. Indeed, this involvement, as determined by inhibition of SDH with 5 mM malonate after ADP-stimulated respiration with pyruvate+glutamate+malate, amounted to more than 50% without malate, ~35% with 0.5 mM, ~20% with 2 mM, and ~15% with 5 mM malate concentration in both types of mitochondria.
In this study, we examined the effect of various concentrations of malate on CI-linked and CII-linked respiration, as well as on the involvement of SDH as a constitutive part of Krebs cycle, in the respiration with CI-linked substrate combinations, in mitochondria from rat liver and brain. In both liver and brain mitochondria, 0.5 mM malate, added in combination with 5 mM pyruvate and 10 mM glutamate, supported >90% of maximal CI-linked respiration observed with saturating 2 mM malate. Conversely, when malate was added to noncoupled mitochondria fueled by CII-linked substrate succinate+rotenone, it inhibited ET capacity  by 6% and 8% at 0.5 mM, 22% and 25% at 2 mM, and 33% and 37% at 5 mM malate concentration, in mitochondria from liver and brain, respectively. A similar degree of inhibition of noncoupled CII-linked respiration by malate was observed when mitochondria were previously exposed to CI&II–linked substrates followed by inhibition of CI with rotenone. Assuming that this effect is caused by an indirect impact of malate on SDH, lower inhibition of respiration at lower malate concentration would be suggestive of a higher involvement of SDH in respiration with CI-linked substrate combinations. Indeed, this involvement, as determined by inhibition of SDH with 5 mM malonate after ADP-stimulated respiration with pyruvate&glutamate&malate, amounted to more than 50% without malate, ~35% with 0.5 mM, ~20% with 2 mM, and ~15% with 5 mM malate concentration in both types of mitochondria.


In summary, our observations showed that despite tremendous physiological differences between liver and brain mitochondria, malate affected their respiratory patterns in a similar manner, suggesting that this may be a more general phenomenon. Therefore, we recommend that when malate is used in complex SUIT protocols, a concentration of 0.5 mM should be used rather than previously applied higher concentrations (2 mM), balancing its stimulatory and inhibitory effects on mitochondrial respiration.
In summary, our observations showed that despite tremendous physiological differences between liver and brain mitochondria, malate affected their respiratory patterns in a similar manner, suggesting that this may be a more general phenomenon. Therefore, we recommend that when malate is used in complex SUIT protocols, a concentration of 0.5 mM should be used rather than previously applied higher concentrations (2 mM), balancing its stimulatory and inhibitory effects on mitochondrial respiration.
 
|mipnetlab=SK Bratislava Sumbalova Z, AT Innsbruck Gnaiger E, AT Innsbruck Oroboros
|mipnetlab=SK Bratislava Sumbalova Z, AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS
}}
}}
{{Labeling
{{Labeling
Line 17: Line 16:
|organism=Rat
|organism=Rat
|tissues=Nervous system, Liver
|tissues=Nervous system, Liver
|preparations=Isolated Mitochondria
|preparations=Isolated mitochondria
|topics=Inhibitor, Substrate, Uncoupler
|topics=Inhibitor, Substrate, Uncoupler
|couplingstates=OXPHOS, ETS
|couplingstates=OXPHOS, ET
|substratestates=CI, CII, CI+II
|pathways=F, N, S, NS
|instruments=Oxygraph-2k, Protocol
|instruments=Oxygraph-2k, O2k-Protocol
|event=B1, Oral
|additional=MiP2014
|additional=MiP2014
}}
}}
== Affiliation ==
== Affiliation ==
1-Pharmacobiochem Lab, Fac Medicine, Comenius Univ, Bratislava, Slovakia; 2-OROBOROS INSTRUMENTS, Innsbruck, Austria; 3-Dep Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria. – [email protected]
1-Pharmacobiochem Lab, Fac Medicine, Comenius Univ, Bratislava, Slovakia; 2-Oroboros Instruments, Innsbruck, Austria; 3-Dep Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria. – [email protected]


== References ==
== References ==
# Gnaiger E (2012) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 3rd ed. Mitochondr Physiol Network 17.18. OROBOROS MiPNet Publications, Innsbruck: 64 pp.
# Gnaiger E (2012) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 3rd ed. Mitochondr Physiol Network 17.18. Oroboros MiPNet Publications, Innsbruck: 64 pp.
# Harris EJ, Manger JR (1968) Intramitochondrial substrate concentration as a factor controlling metabolism. The role of interanion competition. Biochem J 109: 239-46.
# Harris EJ, Manger JR (1968) Intramitochondrial substrate concentration as a factor controlling metabolism. The role of interanion competition. Biochem J 109: 239-46.
# Gunter TE, Gerstner B, Lester T, Wojtovich AP, Malecki J, Swarts SG, Brookes PS, Gavin CE, Gunter KK (2010) An analysis of the effects of Mn<sup>2+</sup> on oxidative phosphorylation in liver, brain, and heart mitochondria using State 3 oxidation rate assays. Toxicol Appl Pharmacol 249: 65-75.
# Gunter TE, Gerstner B, Lester T, Wojtovich AP, Malecki J, Swarts SG, Brookes PS, Gavin CE, Gunter KK (2010) An analysis of the effects of Mn<sup>2+</sup> on oxidative phosphorylation in liver, brain, and heart mitochondria using State 3 oxidation rate assays. Toxicol Appl Pharmacol 249: 65-75.

Revision as of 12:05, 23 January 2019

Optimization of malate concentration for high-resolution respirometry: mitochondria from rat liver and brain.

Link:

Sumbalova Z

Mitochondr Physiol Network 19.13 - MiP2014

Sumbalova Z, Vancova O, Krumschnabel G, Gnaiger E (2014)

Event: MiP2014

Complex substrate-uncoupler-inhibitor titration (SUIT) [1] protocols, used in high-resolution respirometry, are designed to determine respiration with CI-linked and CII-linked substrates in a single experiment. Observations on multiple types of mitochondria revealed that malate, a substrate used to fuel CI-linked respiration, inhibits CII-linked (succinate+rotenone; coupled and noncoupled) respiration at 2 mM concentration. Mutual interference between succinate and malate was already described by Harris and Manger [2], and the effect was attributed to accumulation of oxaloacetate, an inhibitor of succinate dehydrogenase (SDH) and fumarate [1,3].

In this study, we examined the effect of various concentrations of malate on CI-linked and CII-linked respiration, as well as on the involvement of SDH as a constitutive part of Krebs cycle, in the respiration with CI-linked substrate combinations, in mitochondria from rat liver and brain. In both liver and brain mitochondria, 0.5 mM malate, added in combination with 5 mM pyruvate and 10 mM glutamate, supported >90% of maximal CI-linked respiration observed with saturating 2 mM malate. Conversely, when malate was added to noncoupled mitochondria fueled by CII-linked substrate succinate+rotenone, it inhibited ET capacity by 6% and 8% at 0.5 mM, 22% and 25% at 2 mM, and 33% and 37% at 5 mM malate concentration, in mitochondria from liver and brain, respectively. A similar degree of inhibition of noncoupled CII-linked respiration by malate was observed when mitochondria were previously exposed to CI&II–linked substrates followed by inhibition of CI with rotenone. Assuming that this effect is caused by an indirect impact of malate on SDH, lower inhibition of respiration at lower malate concentration would be suggestive of a higher involvement of SDH in respiration with CI-linked substrate combinations. Indeed, this involvement, as determined by inhibition of SDH with 5 mM malonate after ADP-stimulated respiration with pyruvate&glutamate&malate, amounted to more than 50% without malate, ~35% with 0.5 mM, ~20% with 2 mM, and ~15% with 5 mM malate concentration in both types of mitochondria.

In summary, our observations showed that despite tremendous physiological differences between liver and brain mitochondria, malate affected their respiratory patterns in a similar manner, suggesting that this may be a more general phenomenon. Therefore, we recommend that when malate is used in complex SUIT protocols, a concentration of 0.5 mM should be used rather than previously applied higher concentrations (2 mM), balancing its stimulatory and inhibitory effects on mitochondrial respiration.


O2k-Network Lab: SK Bratislava Sumbalova Z, AT Innsbruck Gnaiger E, AT Innsbruck Oroboros


Labels: MiParea: Respiration, Instruments;methods 


Organism: Rat  Tissue;cell: Nervous system, Liver  Preparation: Isolated mitochondria 

Regulation: Inhibitor, Substrate, Uncoupler  Coupling state: OXPHOS, ET  Pathway: F, N, S, NS  HRR: Oxygraph-2k, O2k-Protocol  Event: B1, Oral  MiP2014 

Affiliation

1-Pharmacobiochem Lab, Fac Medicine, Comenius Univ, Bratislava, Slovakia; 2-Oroboros Instruments, Innsbruck, Austria; 3-Dep Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria. – [email protected]

References

  1. Gnaiger E (2012) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 3rd ed. Mitochondr Physiol Network 17.18. Oroboros MiPNet Publications, Innsbruck: 64 pp.
  2. Harris EJ, Manger JR (1968) Intramitochondrial substrate concentration as a factor controlling metabolism. The role of interanion competition. Biochem J 109: 239-46.
  3. Gunter TE, Gerstner B, Lester T, Wojtovich AP, Malecki J, Swarts SG, Brookes PS, Gavin CE, Gunter KK (2010) An analysis of the effects of Mn2+ on oxidative phosphorylation in liver, brain, and heart mitochondria using State 3 oxidation rate assays. Toxicol Appl Pharmacol 249: 65-75.