IN VITRO EFFECTS OF NAD⁺ BOOSTERS ON VIABILITY, MITOCHONDRIAL BIOENERGETICS AND MIGRATION IN SAF-1 CELLS

Introduction

Nicotinamide adenine dinucleotide (NAD⁺) is a cofactor involved in metabolic processes and cellular functions. In fish, NAD⁺ helps manage oxidative stress, adapt to environmental changes, and mitigate the toxicity of pollutants. NAD⁺ can be synthesised via de novo and salvage pathways. Salvage pathways employ B3 derivatives, nicotinamide mononucleotide (NMN), and nicotinamide riboside (NR) to produce NAD⁺. Consequently, strategies to elevate intracellular NAD⁺ levels through precursor supplementation have gained significant interest because of their potential health benefits. In this context, in vitro cell models are useful for investigating the effects of NAD⁺ enhancers under controlled conditions.

Methods

This study investigated the effects of two NAD⁺ enhancers, NMN and NR, on in vitro viability (MTT assay), mitochondrial function (Agilent Seahorse XF), and migration (scratch assay) of the established cell line SAF-1 (ECACC n°00122301).

Results

NMN did not exhibit significant cytotoxicity at concentrations up to 0.5 mM. Conversely, NR either maintained or transiently enhanced viability at low to moderate concentrations, whereas a concentration of 5 mM resulted in a decrease in viability to approximately 60% at 96 h. Regarding bioenergetics, NMN induced only modest alterations, with a notable increase in maximal respiration observed only at 1 mM. In contrast, NR significantly increased basal respiration within the range of 0.16–0.75 mM, as well as ATP-linked respiration and coupling efficiency, without causing significant changes in proton leakage. However, NR elevated non-mitochondrial oxygen consumption, indicating the need to evaluate the role of reactive oxygen species. In the wound closure assay, both NMN and NR facilitated progressive closure only at their lowest concentrations.

Conclusions

NMN demonstrated an optimal promigratory effect on SAF-1 cells at low doses, with moderate bioenergetic impact. Conversely, NR reliably enhances mitochondrial activity and efficiency at intermediate doses. High doses and prolonged exposure to NMN and NR may compromise cell viability.