Metabolic tracing of NAD+ precursors using strategically labelled isotopes of NMN

Abstract

Nicotinamide adenine dinucleotide (NAD+) is an important cofactor and substrate for hundreds of cellular processes involved in redox homeostasis, DNA damage repair and the stress response. NAD+ declines with biological ageing and in age-related diseases such as diabetes and strategies to restore intracellular NAD+ levels are emerging as a promising strategy to protect against metabolic dysfunction, treat age-related conditions and promote healthspan and longevity. One of the most effective ways to increase NAD+ is through pharmacological supplementation with NAD+ precursors such as nicotinamide mononucleotide (NMN) which can be orally delivered. Long term administration of NMN in mice mitigates age-related physiological decline and alleviates the pathophysiologies associated with a high fat diet- and age-induced diabetes. Despite such efforts, there are certain aspects of NMN metabolism that are poorly understood. In this thesis, the mechanisms involved in the utilisation and transport of orally administered NMN were investigated using strategically labelled isotopes of NMN and mass spectrometry. A mass spectrometry method was developed to trace the incorporation of labelled NMN moieties in NAD+ metabolites following supplementation with labelled NMN compounds. This was validated in biologically relevant models such as mammalian cell lines (Chapter 3) and bacteria (Chapter 4), the latter serving as a proof-of-concept model to investigate NMN metabolism through bacterial routes before investigating its metabolic fate in vivo (Chapter 5). Following oral administration with labelled NMN compounds in mice, labelled NAD+ metabolites were detected in abundance in the peripheral tissues of mice treated with antibiotics but were largely absent in control mice. This suggests the majority of orally administered NMN is consumed by gut bacteria, limiting its availability to host peripheral tissues and insinuates host-microbe competition for NAD+ precursors. Interestingly, an abundance of nicotinamide riboside (NR) was observed both in vitro and in vivo following supplementation with NMN supporting the indirect NMN transport mechanism whereby it is dephosphorylated to NR prior to entering the cell. Overall, these findings have therapeutic implications in the dosing and route of administration of NMN as an NAD+-boosting strategy to treat conditions related to metabolic dysfunction and age-related diseases and further to promote healthy ageing and longevity.