Fluxes of Nicotinamide Adenine Dinucleotides through Mitochondrial Membranes in Human Cultured Cells

Pierre Rustin,Béatrice Parfait,Dominique Chretien,Thomas Bourgeron,Fatima Djouadi,Jean Bastin,Agnès Rötig,Arnold Munnich

doi:10.1074/jbc.271.25.14785

Pierre Rustin, Béatrice Parfait + Show 6 more

Open Access

https://doi.org/10.1074/jbc.271.25.14785

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Abstract

We report on the loss of mitochondrial nicotinamide adenine dinucleotides in human cultured cells along with cell culture and acidification of the culture medium. This was established both by the direct measurement of the decrease in the mitochondrial NAD content and by the alteration of the oxidative properties of the mitochondria. In situ, this loss could be reversed in less than 2 h by changing the culture medium or by readjusting the pH of the medium at physiological pH values. By studying the oxidative properties of intact, but NAD-depleted, mitochondria in digitonin-permeabilized cells, we found that a rapid influx of NAD could replenish the mitochondrial NAD pool. This allowed the restoration of an active NAD+-dependent substrate oxidation. Depletion of mitochondrial NAD in cells grown under quiescent conditions was further confirmed by fluorimetric measurement of mitochondrial NAD, as was the influx of NAD+ into the mitochondrial matrix. These data constitute the first evidence of rapid fluxes of NAD through mitochondrial membranes in animal cells. They also point to the possible confusion between a loss of mitochondrial NAD and a defect of respiratory chain complex I in the context of screening procedures for respiratory chain disorder in human.

Highlights

By studying the oxidative properties of intact, but NAD-depleted, mitochondria in digitonin-permeabilized cells, we found that a rapid influx of NAD could replenish the mitochondrial NAD pool
We report on the following: (i) the decrease of intact cell respiration and of NADϩ-linked substrate oxidation in digitonin-permeabilized cells during human B-lymphoblastoid cell line culture, concomitant with a decrease of the NADϩ content of the mitochondria; (ii) the in vitro restoration of an active oxidation of NADϩ-linked substrates by exogenous NADϩ, associated with an influx of NADϩ into the mitochondrial matrix; and (iii) the role of the culture medium pH in controlling NADϩ influx into the mitochondria
The above results address both fundamental and applied aspects of mitochondria-devoted research. These results first question the idea of the mitochondrial inner membrane impermeability to nicotinamide adenine dinucleotides

Summary

Introduction

By studying the oxidative properties of intact, but NAD-depleted, mitochondria in digitonin-permeabilized cells, we found that a rapid influx of NAD could replenish the mitochondrial NAD pool. This allowed the restoration of an active NAD؉-dependent substrate oxidation. Depletion of mitochondrial NAD in cells grown under quiescent conditions was further confirmed by fluorimetric measurement of mitochondrial NAD, as was the influx of NAD؉ into the mitochondrial matrix These data constitute the first evidence of rapid fluxes of NAD through mitochondrial membranes in animal cells. In order to rule out any artifact related to the isolation of mitochondria, we studied mitochondrial properties in digitonin-permeabilized cells that were shown, at the digitonin concentration used, to keep their mitochondria fully intact [7]

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