Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

Tenzin Tseyang; Jonathan Valeros; Paula Vo; Jessica B. Spinelli

doi:10.3791/65184

Sauerstoffunabhängige Assays zur Messung der mitochondrialen Funktion bei Säugetieren

JoVE Journal
Biochemistry

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JoVE Journal Biochemistry

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

Sauerstoffunabhängige Assays zur Messung der mitochondrialen Funktion bei Säugetieren

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05:59 min

May 19, 2023

DOI: 10.3791/65184-v

Tenzin Tseyang*¹, Jonathan Valeros*¹, Paula Vo*¹, Jessica B. Spinelli^1,2

¹Program in Molecular Medicine,University of Massachusetts Chan Medical School, ²Cancer Center,University of Massachusetts Chan Medical School

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Overview

This study presents a series of assays designed to measure mitochondrial function in mammalian cells, particularly under hypoxic conditions. The methods developed allow researchers to assess mitochondrial activity without relying solely on oxygen consumption.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Metabolism

Background

Mitochondria play a crucial role in cellular metabolism and energy production.
Hypoxia is a significant metabolic stressor affecting cell function.
Traditional assays may not accurately reflect mitochondrial function in low oxygen environments.
Understanding mitochondrial adaptation to stress is vital for insights into health and disease.

Purpose of Study

To develop improved assays for studying mitochondrial function in hypoxic conditions.
To explore the metabolic pathways utilized by mitochondria under stress.
To provide protocols that can be used by researchers in the field.

Methods Used

High-resolution mass spectrometry for metabolite analysis.
Stable isotope tracing to track metabolite fates.
Cell lysis and metabolite isolation techniques.
Liquid chromatography-mass spectrometry (LC-MS) for sample analysis.

Main Results

Mammalian mitochondria can maintain essential functions in low oxygen.
Fumarate serves as an alternative electron acceptor in the electron transport chain.
Assays developed can effectively measure mitochondrial function in hypoxia.
Results indicate differences in succinate dehydrogenase activity under varying conditions.

Conclusions

The study provides valuable protocols for assessing mitochondrial function.
Findings enhance understanding of mitochondrial adaptation to metabolic stress.
Future research can build on these methods to explore mitochondrial roles in health and disease.

Frequently Asked Questions

What is the significance of studying mitochondrial function?

Studying mitochondrial function is crucial for understanding energy metabolism and how cells respond to stress, which has implications for various diseases.

How does hypoxia affect mitochondrial activity?

Hypoxia can impair mitochondrial function, but this study shows that mitochondria can adapt by utilizing alternative metabolic pathways.

What techniques are used to analyze metabolites?

High-resolution mass spectrometry and liquid chromatography-mass spectrometry (LC-MS) are employed to analyze metabolites.

Why are traditional respirometry experiments insufficient?

Traditional respirometry may not accurately reflect mitochondrial function under hypoxic conditions, necessitating alternative assays.

What is fumarate's role in mitochondrial function?

Fumarate acts as an alternative electron acceptor in the electron transport chain, allowing mitochondria to function in low oxygen.

Can these assays be applied to other cell types?

Yes, the protocols can be adapted for various mammalian cell types to study mitochondrial function under different conditions.

Hier präsentieren wir eine Zusammenstellung von Assays zur direkten Messung der mitochondrialen Funktion in Säugetierzellen, unabhängig von ihrer Fähigkeit, molekularen Sauerstoff zu verbrauchen.

Unsere Forschung konzentriert sich darauf, wie Mitochondrien metabolische Stressoren wahrnehmen und sich an sie anpassen. Ein wichtiger metabolischer Stressfaktor ist Hypoxie, die auftritt, wenn Zellen unter Sauerstoffmangel leiden. Wir entwickeln bessere Assays, um die Funktion der Mitochondrien in sauerstoffarmen Umgebungen zu untersuchen.

Hochauflösende Massenspektrometrie wurde eingesetzt, um die Zell- und Gewebemetabolitenkonzentration zu untersuchen. Wir haben Stabilisotopen-Tracing-Studien verwendet, um das Schicksal bestimmter Metaboliten zu verfolgen und neue Stoffwechselwege aufzudecken, die für die Physiologie gesunder und kranker Säugetiere relevant sind. Die Gruppe hat herausgefunden, dass die Mitochondrien von Säugetieren in sauerstoffarmen Umgebungen wichtige Funktionen aufrechterhalten können.

Mechanistisch verwenden sie Fumarat als alternatives elektronisches Zepter für die Elektronentransportkette. Dieses Protokoll stellt mehrere Assays zusammen, mit denen Forscher die mitochondriale Funktion in Säugetierzellen besser beurteilen können. Wichtig ist, dass diese Protokolle verwendet werden können, um die mitochondriale Funktion bei Hypoxie zu untersuchen, da klassische Respirometrie-Experimente nicht ausreichen.