Endogenous nicotinamide riboside metabolism protects against diet-induced liver damage

Supplementation with the NAD+ precursor nicotinamide riboside (NR) ameliorates and prevents a broad array of metabolic and aging disorders in mice. However, little is known about the physiological role of endogenous NR metabolism. We have previously shown that NR kinase 1 (NRK1) is rate-limiting and essential for NR-induced NAD+ synthesis in hepatic cells. To understand the relevance of hepatic NR metabolism, we generated whole body and liver-specific NRK1 knockout mice. Here, we show that NRK1 deficiency leads to decreased gluconeogenic potential and impaired mitochondrial function. Upon high-fat feeding, NRK1 deficient mice develop glucose intolerance, insulin resistance and hepatosteatosis. Furthermore, they are more susceptible to diet-induced liver DNA damage, due to compromised PARP1 activity. Our results demonstrate that endogenous NR metabolism is critical to sustain hepatic NAD+ levels and hinder diet-induced metabolic damage, highlighting the relevance of NRK1 as a therapeutic target for metabolic disorders.

The individual values and statistical tests used for each panel can be found in the Data Source file. 7 AMPK, P-AMPK, and GAPDH in the liver of NRK1 LKO and Ctrl mice on HFD. c. Energy expenditure in NRK1 LKO (n=10) and control (n=9) mice on LFD. d. Glucose

Western blotting
Protein extraction, quantification and western blotting procedures were performed as described previously (Ratajczak et al., 2016). All primary antibodies used are listed in Table S2. Primary antibodies were used in 1:5000 dilution for GAPDH and 1:1000 dilution for others. Antibody detection reactions were developed by enhanced chemiluminescence (Amersham). For quantification, the intensity of each band was determined by densitometry using ImageJ software, normalized to GAPDH unless otherwise stated and relative to control condition.

RNA extraction and qPCR
Total mRNA from all studied tissues or cells was extracted using TRIzol (Life Technologies) according to the manufacturer's instructions. Reverse transcription was performed using SuperScript II (Life Technologies) according to the manufacturer's protocol. Quantification of mRNA expression was performed using SYBR Green real time PCR technology (Roche) using a Light Cycler (Roche). Gene expression was normalized with b2-microglobulin and cyclophillin as housekeeping genes. Relative gene expression between genotypes was assessed using the ΔΔCt method. The primers used are provided in the Table S1.

Mitochondrial Isolation
Mitochondria were isolated from mouse livers, which were immediately rinsed in ice-cold mitochondrial isolation buffer (IB) (250 mM sucrose, 10 mM Tris-MOPS and 0.1 mM EGTA/Tris; adjusted to pH=7.4). Pieces of tissue were placed in 5 ml of ice-cold fresh IB, transferred to a glass potter and homogenized at 4°C using a Teflon pestle rotating at 1600 rpm (4 strokes). The homogenate was centrifuged at 600g for 10 min at 4°C and the supernatant was then centrifuged at 7'000g for 10 min at 4°C. After this step, the supernatant containing cytosolic fraction was discarded and the pellet was washed once more with 5 ml of ice-cold IB and centrifuged for 10 min at 4°C. The pellet, containing mitochondria, was resuspended in 100 µl of buffer. Mitochondrial protein concentration was then determined using the BCA assay (Pierce).

Primary hepatocyte isolation
Hepatocytes were isolated from WT and NRK1 KO mice by continuous recirculating perfusion of the mouse liver in situ with collagenase digestion as described previously 1

Triglycerides content
Triglycerides were measured in liver tissue using the Bioassay System Assay Kit (ETGA-200) according to manufacturer's instructions.

NAD + assay
NAD + was extracted from tissues and measured by EnzyChrom NAD/NADH Assay Kit (BioAssay Systems) according to manufacturer's instructions.

Citrate synthase activity assay
Citrate synthase activity was assessed in liver tissue homogenates using Citrate Synthase Assay kit (Sigma, CS0720) following manufacturer's instructions.

Mitochondrial DNA content
Genomic DNA was extracted from liver tissue using DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's instructions. Mitochondrial and nuclear DNA content were