Abstract
Nicotinamide adenine dinucleotide (NAD+), the essential cofactor derived from vitamin B3, is both a coenzyme in redox enzymatic processes and substrate in non-redox events; processes that are intimately implicated in all essential bioenergetics. A decrease in intracellular NAD+ levels is known to cause multiple metabolic complications and age-related disorders. One NAD+ precursor is dihydronicotinamide riboside (NRH), which increases NAD+ levels more potently in both cultured cells and mice than current supplementation strategies with nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) or vitamin B3 (nicotinamide and niacin). However, the consequences of extreme boosts in NAD+ levels are not fully understood. Here, we demonstrate the cell-specific effects of acute NRH exposure in mammalian cells. Hepatocellular carcinoma (HepG3) cells show dose-dependent cytotoxicity when supplemented with 100–1000 μM NRH. Cytotoxicity was not observed in human embryonic kidney (HEK293T) cells over the same dose range of NRH. PUMA and BAX mediate the cell-specific cytotoxicity of NRH in HepG3. When supplementing HepG3 with 100 μM NRH, a significant increase in ROS was observed concurrent with changes in the NAD(P)H and GSH/GSSG pools. NRH altered mitochondrial membrane potential, increased mitochondrial superoxide formation, and induced mitochondrial DNA damage in those cells. NRH also caused metabolic dysregulation, altering mitochondrial respiration. Altogether, we demonstrated the detrimental consequences of an extreme boost of the total NAD (NAD+ + NADH) pool through NRH supplementation in HepG3. The cell-specific effects are likely mediated through the different metabolic fate of NRH in these cells, which warrants further study in other systemic models.
Highlights
To examine the mechanism by which NAD+ precursor is dihydronicotinamide riboside (NRH) exposure induced cell death, HEK293T and HepG3 cells were exposed to NRH at IC90 concentration (i.e., 500 μM) for 24 h, assessed the protein expression levels of the apoptotic markers after 24, 48, and 72 h using immunoblot (Fig 3)
We further demonstrated that this BAX-induced apoptosis is mediated by p53 upregulated modulator of apoptosis (PUMA), which showed significantly increased expression levels at 48 h (140 ± 41.2%) and 72 h (150 ± 2.5%) of NRH exposure (Fig 3D and 3F)
Most supplementation studies have examined nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid, or nicotinamide, but NRH has the highest potency in increasing total NAD concentrations in cells [31, 32]
Summary
Dihydronicotinamide riboside (NRH), the reduced form of NR, a recently characterized NAD precursor [26, 27], increases NAD levels. Recent investigations have demonstrated that NRH increased NAD+ levels more significantly in both cultured cells and mouse models than current supplementation strategies with NR or vitamin B3 (nicotinamide and niacin) [31, 32]. The consequences of the substantial increase in the total intracellular NAD pools fueled by NADH, instead of NAD+, are still unknown These consequences may be critical to cell health since NADH is not a substrate for sirtuins or ARTs. NADH can only contribute to sirtuin or ART regulatory processes once it is oxidized to NAD+ with the concomitant reduction of endogenous species (e.g., O2, pyruvate, etc.) [3, 33]. We describe our investigations to show the cell-specific effects of acute NRH exposure in mammalian cells (S1 Graphical abstract)
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