Abstract
OBJECTIVES: Macrophages play an essential role in the development of alcohol-induced inflammation. The objective of this study was to investigate whether nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor naturally found in milk, can attenuate alcohol-induced inflammation and oxidative stress in macrophages with the elucidation of mechanisms of action. METHODS: RAW 264.7 macrophages and mouse bone marrow-derived macrophages (BMDMs) were stimulated with 80 mM ethanol with or without 1 mM of NR for 72 h. Expression of genes associated with inflammation and oxidative stress and cellular reactive oxygen species (ROS) accumulation were measured. Also, to evaluate the contribution of sirtuin 1 (SIRT1) to the NR's effect, cellular NAD + level (a cofactor of SIRT1), SIRT1 activity, and mitochondrial DNA copy number were measured. SIRT1 activity was inhibited or activated by sirtinol and resveratrol, respectively, to confirm SIRT1 functions further. Parameters related to mitochondrial respiration were determined using a Seahorse XFe24 Extracellular Flux analyzer. RESULTS: NR significantly decreased ethanol-induced inflammatory gene expression, with a concomitant decrease in nuclear translocation of nuclear factor kB p65 in macrophages. Increased cellular ROS levels by ethanol were also attenuated concomitantly with decreased expression of NADPH oxidase 2, a ROS-producing enzyme, by NR in both macrophage cell types. Ethanol decreased SIRT1 mRNA, protein and activity, cellular NAD + level, and mitochondrial DNA, all of which were markedly attenuated by NR. SIRT1 inhibition by sirtinol augmented the inflammatory effects of ethanol, while SIRT1 activation by resveratrol elicited the opposing results. Ethanol increased mitochondrial respiration, ATP production, and proton leak, but decreased maximal respiration and spare respiratory capacity. The ethanol-induced changes in mitochondrial respiration were abolished by NR. CONCLUSIONS: NR showed anti-inflammatory and antioxidant properties in ethanol-treated macrophages by counteracting the effect of ethanol on lowering SIRT1 expression and cellular NAD(+) levels. Therefore, NR may be a potential therapeutic agent for alcohol-induced inflammation and oxidative stress. FUNDING SOURCES: This work is supported by the NIH 3R01DK108254-04S1.