Metabolic profiling of alternative NAD biosynthetic routes in mouse tissues.

Valerio Mori,Francesca Mazzola,Giulio Magni,Nadia Raffaelli,Laura Conforti,Michele Di Stefano,Silverio Ruggieri,Adolfo Amici,Giuseppe Orsomando,Valerie De Crécy-Lagard

doi:10.1371/journal.pone.0113939

Abstract

NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the “amidated” and “deamidated” routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT), which in mammals comprises three distinct isozymes, and NAD synthetase (NADS). First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes'rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme's substrate NaAD (nicotinic acid adenine dinucleotide). In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.

Highlights

nicotinamide adenine dinucleotide (NAD) is pivotal for cell life, first as a reusable redox coenzyme for energy production, second as a consumable substrate in enzymatic reactions regulating crucial biological processes, including gene expression, DNA repair, cell death and lifespan, calcium signaling, glucose homeostasis, and circadian rhythms [1,2,3,4,5]
nicotinamide mononucleotide adenylyltransferase (NMNAT) activity was ubiquitously distributed in contrast to NAD synthetase (NADS), whose activity levels were barely detectable in brain, and below the detection limit both in lung and skeletal muscle, suggesting a limited NAD synthesis capability through the deamidated route in these tissues
Two exceptions were represented by small intestine, where NADS was rather similar to NMNAT, in keeping with the selective, high-level mRNA expression in this tissue [44], and by blood, where NMNAT activity showed the lowest absolute value, even lower than local NADS (Figure 3A, B insets)

Summary

Introduction

NAD is pivotal for cell life, first as a reusable redox coenzyme for energy production, second as a consumable substrate in enzymatic reactions regulating crucial biological processes, including gene expression, DNA repair, cell death and lifespan, calcium signaling, glucose homeostasis, and circadian rhythms [1,2,3,4,5]. The NAD biosynthetic pathway operating in mammals [11,12,13], as summarized, is composed by a de novo route starting from tryptophan, and three alternative recycling routes starting from pre-formed pyridine moieties, i.e. nicotinic acid (Na), nicotinamide (Nam), and nicotinamide riboside (NR). These NAD precursors, collectively referred to as niacin or vitamin B3 [6], may arise from dietary supply and/or intracellular NAD catabolism. NAD biosynthesis in mammals may occur via distinct ‘‘amidated’’ and ‘‘deamidated’’ routes, that connect to each other only after the dinucleotide NaAD has been formed, and show no other cross-talk at any intermediate level (Figure 1)

Methods

Results

Conclusion