(P30-018-23) Nicotinamide Riboside, a NAD+ Precursor, Attenuates Ethanol-Induced Oxidative Stress, Inflammation, and Mitochondrial Unfolded Protein Response in Hepatocytes

Objectives: Nicotinamide adenine dinucleotide (NAD⁺) is consumed during ethanol oxidation, depleting the cellular NAD⁺ pool in hepatocytes, which can alter mitochondrial biogenesis and functions. Therefore, this study determined whether nicotinamide riboside (NR), a bioavailable NAD⁺ precursor, can mitigate ethanol-induced hepatocyte dysfunctions by repleting the NAD⁺ pool. In particular, we focused on the mitochondrial unfolded protein response (UPR^mt) pathway. This mitochondrial-to-nuclear signaling pathway increases the synthesis of mitochondrial chaperones and proteases that help maintain the mitochondrial proteome integrity to protect cells from mitochondrial stresses, such as reactive oxygen species (ROS).

Methods: We used VL-17A cells, a HepG2 cell line expressing alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1), as our cell model. The cells were pre-treated with 1 mM of NR for 6 h, and then incubated with 80 mM of ethanol for 72 h for mRNA and protein analysis as well as cellular ROS measurement.

Results: Ethanol increased mRNA levels of ethanol metabolizing enzymes, including ADH and CYP2E1, which were significantly attenuated by NR. Also, NR significantly inhibited ethanol-triggered cellular ROS accumulation and the expression of inflammatory genes, such as interleukin 1β, interleukin 6, and tumor necrosis factor a. Concomitantly, ethanol increased the expression of UPR^mt-related genes, including transcription factors activating transcription factor 4 (ATF4), ATF5 and C/EBP homologous protein, mitochondrial chaperones heat shock protein 10 (HSP10) and HSP60, and proteases lon peptidase 1 and caseinolytic mitochondrial matrix peptidase proteolytic subunit. However, when the cells were co-incubated with ethanol and NR, UPR^mt gene expression was not induced.

Conclusions: This study indicates that ethanol increased inflammatory gene expression and cellular ROS accumulation, which might elevate the expression of UPR^mt genes for self-defense in hepatocytes. However, NR, likely by repleting NAD⁺, prevented ROS accumulation, making the activation of UPR^mt unnecessary. The findings suggest the potential role of NR as a therapeutic agent for alcohol-induced hepatic injury.

Funding Sources: Supported by the funds from the College of Agriculture, Health and Natural Resources at the University of Connecticut