(P14-015-24) Nicotinamide Riboside Mitigates Ethanol-Induced Cell Stress in Kupffer Cells by Modulating the Mitochondrial Unfolded Protein Response

Objectives: Ethanol oxidation depletes cellular NAD⁺ levels, affecting energy metabolism and mitochondrial function. This study aimed to investigate whether nicotinamide riboside (NR), a bioavailable NAD⁺ precursor, could inhibit ethanol-induced cellular dysfunction in Kupffer cells, the resident macrophages in the liver. We focused on the potential regulation of the mitochondrial unfolded protein response (UPR^mt) pathway by NR to exert its role.

Methods: Immortalized mouse Kupffer cells (ImKCs) were pre-treated with 1 mM NR for 6 h and then exposed to 80 mM ethanol for 72 h. We then measured mRNA and protein expression, reactive oxygen species (ROS) levels, and mitochondrial function and structure. Also, we explored whether activating transcription factor 5 (ATF5) plays a role in mediating UPR^mt by knocking it down using siRNA and also determined the cellular localization of ATF5 for activation.

Results: Ethanol significantly upregulated the expression and secretion of pro-inflammatory mediators, but all of which were reduced by NR. NR also inhibited ethanol-triggered cellular and mitochondrial ROS accumulation, at least in part, by reversing the suppression of antioxidant genes by ethanol. Additionally, ethanol impaired mitochondrial respiration, reduced mitochondrial copy number, and disrupted mitochondrial cristae structure, which NR attenuated. The ethanol-induced upregulation of UPR^mt genes and proteins, including ATFs and, mitochondrial chaperones and proteases, was alleviated by NR. Notably, when Atf5 was knocked down, the induction of UPR^mt genes by ethanol was significantly diminished. Ethanol increased the nuclear localization of ATF5 while decreasing its mitochondrial content, and NR suppressed the ethanol-induced nuclear localization of ATF5, a crucial step for its activation.

Conclusions: This study highlights the inhibitory effect of NR on ethanol-induced oxidative stress and inflammation via the modulation of ATF5-dependent UPR^mt pathway in ImKCs, likely by repleting cellular NAD⁺. By modulating ATF5-dependent UPR^mt, NR emerges as a potential therapeutic agent for alcohol-induced hepatic injury, with implications for targeting ATF5 modulation.

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