NRK1 deficiency impairs hepatic gluconeogenic capacity

NRK1 is essential and rate-limiting for NAD+ synthesis from exogenous NR and NMN in primary hepatocytes13. Furthermore, NRK1 is highly expressed in mouse liver13. Therefore, we aimed to investigate the effect of NRK1 deletion – i.e., the inability to utilize NR for NAD+ synthesis – on metabolic homeostasis and hepatic function in vivo. Given the critical role of the liver in glycemic control, we subjected NRK1 KO mice to intraperitoneal glucose, insulin and pyruvate tolerance tests. NRK1 KO mice exhibited similar glucose excursion curves to wild-type (WT) mice when challenged with glucose or insulin (Supplementary Fig. 1a, b). However, when injected with pyruvate, the glycemia in NRK1 KO mice rose to a lesser degree compared to control mice as shown by the difference in AUC between the two genotypes (Fig. 1a), suggesting an impaired gluconeogenic capacity. While basal glycemia after an overnight fast (Fig. 1a) or 24-h fasting (Fig. 1b) was similar between genotypes, we noticed that NRK1 KO mice exhibited lower glucose levels after 6 h of fasting, further supporting the concept of defective gluconeogenic capacity in NRK1 KO mice. The protein levels of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and the mRNA expression of the PPARγ coactivator 1α (Pgc1α), a key driver of the gluconeogenic gene program14,15, were similar between WT and NRK1 KO mice during the fasting time course (Fig. 1c, d). However, glucose-6-phosphatase (G6Pase) mRNA levels were lower in NRK1 KO mice at early fasting stages (Fig. 1d).

Fig. 1 NRK1 KO mice display lower gluconeogenic capacity. a Blood glucose levels during intraperitoneal pyruvate tolerance tests in NRK1 KO (n = 7) and control (WT) (n = 6) mice. b Blood glucose levels in NRK1 KO (n = 13) and WT (n = 12) mice subjected to a fasting time course. c Livers were snap frozen at the indicated time-points during a fasting time course. A piece of liver was then homogenized for protein extraction. Then, 20 μg of protein were used to evaluate NRK1, PEPCK, and GAPDH protein levels through western blot. d Livers were snap frozen at the indicated time points during fasting time course. Then, RNA was extracted to evaluate mRNA levels of genes involved in gluconeogenesis (n = 4 mice per group). e Blood glucose level during intraperitoneal glycerol tolerance test (n = 6 for WT mice, n = 7 for NRK1 KO mice). f Mitochondrial respiratory capacity properties were evaluated in liver homogenates from NRK1 KO and WT mice in fed and 24h-fasted conditions (n = 6 for WT mice; n = 5 for NRK1 KO mice). Results shown are mean ± SEM; * and *** indicate statistical difference between genotypes at p < 0.05 and p < 0.001, respectively. The individual values and statistical tests used for each panel can be found in the Source Data file Full size image

The initial steps of pyruvate driven gluconeogenesis occur in the mitochondria where pyruvate carboxylase (PCB) converts pyruvate into oxaloacetate. In contrast, glycerol enters the gluconeogenic pathway in the cytosol, where it is converted to glycerol 3-phosphate (G3P) via glycerol kinase (GK). Hence, we next used glycerol as the gluconeogenic precursor and observed similar glucose excursion curves between genotypes (Fig. 1e), indicating that the impaired glucose production from pyruvate could result from mitochondrial defects. Furthermore, it also suggests that the decrease in G6Pase expression we detected is not the limiting factor for defective glucose production from pyruvate in NRK1 KO mice. In this sense, we did not detect any change in the protein level of PCB (Supplementary Fig. 1c), suggesting that other aspects of mitochondrial function may be accountable for the defects observed. Thus, we examined mitochondrial respiration by high-resolution respirometry analysis. NRK1 KO mice manifested marked defects in mitochondrial respiratory capacity both in the fed or fasted state (Fig. 1f). Mitochondrial respiration through Complex I + II and maximal electron transport system (ETS) capacity were lower in the liver preparations from NRK1 KO mice. In contrast, we did not detect differences in skeletal muscle respiratory capacity (Supplementary Fig. 1d), most likely due to the relatively low protein levels of NRK1 in this tissue13. Taken together, these results validate the role of NRK1 and endogenous NR metabolism in the maintenance of hepatic mitochondrial function, in turn affecting gluconeogenic capacity.

NRK1 deletion aggravates hepatic insulin-resistance

To address whether the hepatic phenotype of NRK1 KO mice is tissue autonomous, we generated NRK1 liver-specific knockout mice (NRK1 LKO) by crossing NRK1loxP/loxP mice with mice expressing Cre recombinase under the albumin promoter. This led to a complete ablation of NRK1 expression in the liver (Fig. 2a), without affecting NRK1 levels in other tissues (Supplementary Fig. 2a). NRK1 deficiency was neither compensated by NRK2, which was undetectable in the liver, nor by changes in NAMPT protein levels (Fig. 2a).

Fig. 2 Liver-specific NRK1 deficiency exacerbates diet-induced insulin resistance. a Validation of NRK1 deletion and protein level of NAMPT and GAPDH in liver homogenates from NRK1 LKO and control (Ctrl) mice. b Body weight and body composition of NRK1 LKO (n = 10) and control (n = 9) mice on high-fat diet (HFD) at the indicated ages. c, d Food intake c and locomotor activity d of NRK1 LKO (n = 9) and control (n = 8) mice on HFD at 18 weeks of age. e Glycemia levels after 6h-fasting in NRK1 LKO (n = 9) and control (n = 7) mice on HFD at 24 weeks of age. f, g Blood glucose level during intraperitoneal glucose (n = 8 mice in the control group, n = 10 mice in the NRK1 LKO group) f and insulin g tolerance test in NRK1 LKO and control mice on HFD (n = 9 mice for the control group, n = 10 mice for the NRK1 LKO group) at 20 and 22 weeks of age, respectively. h Protein level of Akt, P-Akt, GAPDH, and NRK1 in the liver of NRK1 LKO and Ctrl mice on HFD 15 min after insulin injection (1U kg−1) (n = 3 mice per group). i Protein levels of Akt, P-Akt, GAPDH and NRK1 in primary hepatocytes from NRK1 KO and WT mice stimulated with insulin at the indicated concentration. Results shown are mean ± SEM, * indicates statistical difference vs the respective control value at p < 0.05. The individual values and statistical tests used for each panel can be found in the Source Data file Full size image

When mice were fed with a low-fat diet, the body weight and composition of NRK1 LKO mice were indistinguishable from control littermates (Supplementary Fig. 2b). Glucose excursion was similar between control and NRK1 LKO mice in response to a glucose or insulin challenge (Supplementary Fig. 2c, d). However, as in the whole body NRK1 KO mice, NRK1 LKO mice displayed impaired glucose production after a pyruvate bolus (Supplementary Fig. 2e), even if no differences were observed in the expression of gluconeogenic-related genes such as Pepck or G6Pase, or the transcriptional regulator Pgc1α (Supplementary Fig. 2f). These observations indicate that the impaired response to pyruvate observed in NRK1 KO mice is liver autonomous. Furthermore, it confirms that the reduced G6Pase expression observed in whole body NRK1 KO mice is not at the root of the gluconeogenic defects.

We next explored the response of NRK1 LKO mice to diet-induced metabolic damage by placing them on a high fat diet (HFD). Body weight gain and composition were comparable between control and NRK1 LKO mice (Fig. 2b), as well as food intake and daily activity (Fig. 2c, d). While, as expected, chronic HFD exposure increased glycemia in control mice fasted for 6 h, this increase was largely prevented in NRK1 LKO animals (Fig. 2e). NRK1 LKO mice on HFD developed glucose intolerance (Fig. 2f) and insulin resistance (Fig. 2g) as demonstrated by increased AUC and a reduced AAC, respectively. The altered glucose profile in NRK1 LKO mice during the ipGTT did not come in parallel to alterations of gluconeogenic gene expression (Supplementary Fig. 2g) nor variations in insulinemia, which were comparable between genotypes during the test (Supplementary Fig. 2h). Further testifying for hepatic insulin resistance, insulin-induced Akt phosphorylation was compromised in the livers of high-fat fed NRK1 LKO mice (Fig. 2h). In order to rule out the potential contribution of systemic factors, we performed a similar experiment in primary hepatocytes exposed to a lipid rich medium containing 0.2 mM oleic acid and 0.2 mM palmitate to mimic dietary lipid overload. Remarkably, after insulin stimulation, Akt phosphorylation was lower in NRK1 null hepatocytes compared to those from WT mice (Fig. 2i). These observations argue for a dramatic exacerbation of diet-induced glucose intolerance and hepatic insulin resistance, thereby pointing towards a crucial role of NRK1 and endogenous NR to maintain hepatic function upon diet-induced metabolic damage.

Hepatic NRK1 deletion impairs fatty acid oxidation capacity

The liver weight in NRK1 LKO mice on HFD was higher than in control animals, while no differences were observed on LFD (Fig. 3a). Although plasma cholesterol, triglycerides (TG) or free fatty acids (FFA) levels did not show significant changes (Supplementary Fig. 3a), hepatic TG levels increased in NRK1 LKO mice on HFD (Fig. 3b) suggesting an increased hepatic lipid accumulation, which was corroborated by Oil Red-O staining (Fig. 3c).

Fig. 3 NRK1 deficiency promotes hepatic steatosis by impairing fatty acid oxidation. a, b Liver weight a and triglycerides (TG) levels b in liver of NRK1 LKO and control mice on LFD and HFD (n = 7 mice for the control group; n = 10 mice for the NRK1 LKO group; 24 weeks of age). c Oil-Red O staining of liver sections from NRK1 LKO and control mice on LFD and HFD (scale bars: 200 μm). d mRNA level of genes involved in lipogenesis and lipoprotein metabolism in the liver of HFD-fed NRK1 LKO and control mice (n = 8 mice for the control group; n = 10 mice in the NRK1LKO group). e Respiratory exchange ratio (RER) in NRK1 LKO and control mice on HFD (n = 9 mice for the control group; n = 10 mice for the NRK1LKO group). f Lipid oxidation of NRK1 LKO and control mice on HFD. Data calculated from indirect calorimetry measurements: lipid oxidation = (1.67 x VO 2 ) – (1.67 x VCO 2 )43 (n = 9 mice for the control group; n = 10 mice for the NRK1 LKO group). g Fatty acid oxidation in primary hepatocytes isolated from 10 to 20 week-old WT and NRK1 KO mice. 3H-Palmitate oxidation was measured in conditions of low and high glucose (n = 12 for all groups). h Fatty acid oxidation in primary hepatocytes isolated from 8 to 12 week-old WT and NRK1 KO mice. 3H-Palmitate oxidation was measured in condition of lipid overload. i. mRNA level of genes involved in β-oxidation in the liver of HFD-fed NRK1 LKO and control mice (n = 8 for WT hepatocytes; n = 7 for KO hepatocytes). Results shown are mean ± SEM, *, **, and *** indicate statistical difference vs. respective control group at p < 0.05, p < 0.01 and p < 0.001, respectively. The individual values and statistical tests used for each panel can be found in the Source Data file Full size image

We next aimed to understand the factors influencing hepatic steatosis in NRK1 LKO mice. The mRNA levels of lipogenic genes such as Fas or Scd1 and related transcriptional regulators were similar between genotypes (Fig. 3d). Similarly, the levels of the AMP-activated protein kinase (AMPK), a key cellular controller of lipogenesis and lipid metabolism, and its phosphorylation state were similar between genotypes (Supplementary Fig. 3b). In contrast, we detected increased mRNA levels of Cd36, Ldlr, Lrp and ApoE (Fig. 3d), which suggest higher lipid incorporation from the circulation. This higher influx could prompt hepatic lipid accumulation if NRK1 LKO mice had impaired hepatic fatty acid catabolism. Supporting this possibility, indirect calorimetry studies indicated that, despite similar daily energy expenditure (Supplementary Fig. 3c), the respiratory exchange ratio (RER) was higher in HFD-fed NRK1 LKO mice during the dark phase (Fig. 3e). There was no change in whole body glucose oxidation between genotypes (Supplementary Fig. 3d), and lipid oxidation rates tended to be lower in NRK1 LKO mice compared to control littermates, albeit not significantly (Fig. 3f). These changes, however, were detectable only when mice were challenged by HFD, as mice on LFD did not exhibit changes in RER or energy substrates oxidation (Supplementary Fig. 3e–g).

In order to further ascertain the critical impact of hepatic NRK1 on FAO capacity, we directly evaluated palmitate oxidation rates in primary hepatocytes. Hepatocytes lacking NRK1 showed no alterations in palmitate oxidation rates when incubated in regular high-glucose concentration (25 mM) medium but failed to increase lipid oxidation rates when they were shifted to a lower glucose concentration medium (Fig. 3g). Similarly, when incubated with high lipid containing medium to mimic the HFD intervention, NRK1-null hepatocytes were unable to sustain comparable palmitate oxidation rates as WT hepatocytes (Fig. 3h). In line with these observations, we detected a lower expression of β-oxidation-related genes, including medium-chain specific acyl-Coenzyme A dehydrogenase (Mcad) and carnitine palmitoyltransferase 2 (Cpt2) (Fig. 3i). Taken together, these data show that NRK1 deficient hepatocytes fail to properly increase lipid oxidation rates when metabolically challenged.

A critical element to sustain lipid oxidation is mitochondrial respiratory capacity. In line with the observations in whole body NRK1 knockout mice (Fig. 1f), mitochondrial respiratory capacity in liver homogenates from NRK1 LKO mice was impaired compared to control littermates (Supplementary Fig. 4a), and these defects were further accentuated by HFD feeding (Supplementary Fig. 4b). Similarly, mitochondrial respiratory capacity defects were also more pronounced in the whole body NRK1 KO mice (Supplementary Fig. 4c), supporting the idea that this is a genuine effect of NRK1 deletion. Strikingly, mitochondrial DNA content, citrate synthase activity and mitochondrial-related genes expression were similar between NRK1 LKO mice and control littermates (Supplementary Fig. 4d–f). In addition, the protein levels of respiratory complexes were similar between genotypes (Supplementary Fig. 4g, h). These results demonstrate that NRK1 deficiency leads to impaired mitochondrial respiratory capacity, which stems from intrinsic mitochondrial dysfunction rather than a lower mitochondrial content.

Collectively, we demonstrated that NRK1 LKO mice on LFD display only minor metabolic abnormalities. However, when prompted towards lipid utilization, by either HFD in vivo or by lipid overload in vitro, NRK1 ablated hepatocytes displayed an inability to oxidize lipids efficiently, promoting the development of insulin resistance and hepatic steatosis.

Exacerbated diet-induced hepatic damage in NRK1 LKO mice

Dysregulation of hepatic lipid metabolism has been largely associated with liver damage16. Accordingly, we detected significantly increased plasma levels of alanine transaminase (ALT) and aspartate transaminase (AST), two circulating markers of liver damage, in HFD-fed NRK1 LKO mice compared to control mice (Fig. 4a). Hematoxylin and eosin (H&E) staining in liver sections revealed increased infiltration of inflammatory cells in NRK1 deficient specimens (Fig. 4b top). This was also corroborated by a ~ 4-fold increase in CD45 positive cells, which is a leukocyte-specific marker (Fig. 4b bottom), as well as by an increased expression of inflammation markers such as Mcp1, Il-1β or Tnfα (Fig. 4c). Collectively, the exacerbated steatosis and insulin resistance along with the enhanced inflammatory profile suggest that NRK1 LKO progressed towards a more advanced stage of NAFLD. Solidifying this point, we observed a higher ratio of apoptotic cells by 20% and Ki-67 positive proliferating cells by 50% in the liver of NRK1 LKO mice on HFD (Fig. 4d). In addition, the fibrotic areas increased by 2.5-fold of in NRK1 LKO mice both in pericellular and perivascular structures (Fig. 4d), aligned with an increase in the mRNA levels of bona fide fibrosis markers including αSma and Pai-1 (Fig. 4c). Altogether, our results suggest that NRK1 deletion aggravated the effect of the HFD on the liver, promoting the progression from steatosis towards steatohepatitis characterized by liver steatosis, inflammation, and fibrosis.

Fig. 4 NRK1 liver-specific deletion fosters the development of NAFLD. a Plasma levels of liver damage markers, alanine transaminase (ALT) and aspartate transaminase (AST) in NRK1 LKO and control mice on LFD and HFD (n = 7 for all groups except NRK1 LKO on HFD, n = 8). b Representative H&E (top) and CD45 (bottom) staining on liver sections from NRK1 LKO and control mice on HFD showing immune cells infiltration (black arrows) (×20 magnification, scale bars: 100 μm). c Gene expression of markers of inflammation (left) and fibrosis (right) in the liver of NRK1 LKO mice and control mice on HFD (n = 8 for the control group; n = 10 for the NRK1 LKO group). d Immunohistochemistry on liver sections from NRK1 LKO and control mice on HFD. Representative staining (top) and quantification (bottom) for Apoptag (left), Ki-67 (center) (DAPI counterstaining) (×10 magnification, scale bars: 200 μm) and Sirius Red (right) (×20 magnification, scale bars: 100 μm) (n = 4 mice per group). Results shown are mean ± SEM, * and *** indicate p < 0.05 and p < 0.001, respectively, vs. the control group. The individual values and statistical tests used for each panel can be found in the Source Data file Full size image

NRK1 deletion limits PARP activity and enhances DNA damage

One of the reasons why overt physiological phenotypes were only observed in NRK1 LKO mice on HFD may come from the fact that hepatic NAD+ content was not affected in NRK1 LKO mice on LFD (Fig. 5a). However, when exposed to HFD, NRK1 LKO mice failed to sustain hepatic intracellular NAD+ levels (Fig. 5a). This decrease can be fully attributed to a reduction in NAD+ levels in the nucleo-cytoplasmic fraction, as mitochondrial NAD+ levels were similar between genotypes (Supplementary Fig. 5a). We also detected lower NAD+ content in isolated NRK1 null primary hepatocytes when subjected to lipid overload (Supplementary Fig. 5b). The decline of hepatic NAD+ levels in HFD-fed NRK1 LKO mice occurred in parallel to an accumulation of NR, while NMN levels significantly decreased and NAM levels remained unaffected (Fig. 5b). Moreover, NAMPT mRNA and protein levels were comparable between HFD-fed control and NRK1 LKO mice (Fig. 5c, d). We also analyzed the hepatic mRNA level for multiple enzymes involved in the de novo and Preiss–Handler routes for NAD+ biosynthesis which were unaffected by NRK1 deletion, with the exception of a 20% decline in Nmnat3 expression (Fig. 5c). Considering that even full deletion of Nmnat3 does not alter hepatic NAD+ content17, it seems likely that impaired NR utilization is at the root of the NAD+ decline observed in HFD-fed NRK1 LKO mice. Therefore, NRK1 LKO mice on HFD are deficitary in NAD+ levels even when NAM is available and NRK1-independent NAD+ synthesis paths are unaltered.

Fig. 5 NRK1 deletion curbs PARP activity and exacerbates DNA damage. a NAD+ levels in the liver of NRK1 LKO mice upon LFD and HFD (n = 8 for all groups except NRK1 LKO on HFD, n = 10; 24 weeks of age). b NR (left), NMN (center) and NAM (right) levels in the liver of NRK1 LKO and control mice upon HFD (n = 6 mice per group). c mRNA level of genes involved in the NAD+ biosynthesis pathways (n = 8 mice for the control group, n = 10 mice for the NRK1 LKO group). d Levels of poly(ADP-ribose) protein modification, PARP1, SIRT1, NAMPT, and GAPDH in the liver of NRK1 LKO and control mice on HFD. e Representative staining (top) and quantification (bottom) of γH2AX (left) and 53BP1 (right) immunofluorescence on liver sections from NRK1 LKO and control mice on HFD (n = 4 mice per group, ×10 magnification, DAPI counterstaining, scale bars: 200 μm). Results shown are mean ± SEM, * and *** indicate statistical difference vs. control group at p < 0.05 and p < 0.001, respectively. The individual values and statistical tests used for each panel can be found in the Source Data file Full size image

It must be noted that HFD feeding did not influence hepatic NAD+ levels in control mice (Fig. 5a). Nevertheless, total NAD+ levels do not testify for NAD+ turnover rates. Indeed, HFD feeding led to a marked increase in N-methyl-X-pyridone-5-carboxamide (MeXP, being Me2P or/and Me4P) in control mice (Supplementary Fig. 5c), one of the products of NAM metabolism, indicative of increased NAD+-consuming enzymatic activity. This could be due, in part, to the increased expression level of the nicotinamide N-methyltransferase enzyme (Nnmt) which initiates the routing of NAM towards methylation and oxidation (Supplementary Fig. 5c). Of note, NRK1 protein levels are not affected by HFD in control mice (Supplementary Fig. 5d), suggesting that endogenous NRK1 levels can sustain the higher demand for NAD+ biosynthesis generated by HFD in control mice. Altogether, these observations illustrate that in situations of high NAD+ turnover the utilization of NR might be critical to sustain NAD+ levels.

Sirtuins are a family of enzymes closely related to metabolic sensing and whose activity can be rate-limited by NAD+, especially in the case of SIRT1 and SIRT32. Interestingly, the hepatic deficiency of SIRT1 as well as the whole body deletion of SIRT3 have been linked to increased susceptibility to hepatic steatosis and related metabolic complications18,19. Hence, we evaluated the activity of different sirtuins, i.e.,: SIRT1, SIRT3 and SIRT5, by measuring acetylation levels of NF-κB and the profile of acetylation and malonylation of mitochondrial proteins, respectively. However, we did not observe differences in these protein modifications between genotypes (Supplementary Fig. 5e–g).

NAD+ is also a mandatory co-substrate for poly(ADP-ribose) polymerases (PARPs), including PARP1, which is critical for DNA damage repair. Therefore, we asked whether reduction of intracellular NAD+ levels in the liver of NRK1 LKO mice affects PARP1 activity. We assessed PARP1 protein level, as well as global poly(ADP-ribose) (PAR) levels, which reflect PARP1 PARylation activity. PARP1 and PAR levels were lower by about 50% and 25% in NRK1 LKO mice on LFD and HFD, respectively (Fig. 5d and Supplementary Fig. 5h). The reduction in PARP1 protein levels might be explained by the parallel reduction in Parp1 gene expression (Supplementary Fig. 5i). Given the role of PARP1 in DNA damage repair, we then evaluated DNA damage at the molecular level by quantifying cells with γH2AX and 53BP1 positive foci in the nucleus. The results illustrated a ~2.5-fold increase in the abundance of foci for both markers in the liver of NRK1 LKO mice compared to control littermates (Fig. 5e). Therefore, the reduced PARP1 activity in NRK1 LKO mice is not due to lower DNA damage. Rather, our results imply that the increased hepatic DNA damage induced by HFD triggers PARP1 activity in the liver12,20, which generates a higher NAD+ demand that cannot be met in the NRK1 LKO mice. Therefore, this suggests that endogenous NR utilization is required to sustain NAD+ levels and PARP1 activity in the liver of HFD-fed mice and that the failure to do so aggravates NAFLD.

Finally, we aimed to test whether restoring NAD+ levels in NRK1 LKO mice by supplementing with NRK1-independent NAD+ precursors could rescue their phenotype. To that end, NRK1 LKO mice were exposed to HFD and supplemented with or without NAM in drinking water for 12 weeks, since NAM-induced NAD+ synthesis is independent of NRK1 (Supplementary Fig. 6a). Surprisingly, hepatic NAD+ and NAM levels remained similar between NAM and vehicle-treated groups (Supplementary Fig. 6b,c) indicating that NAM supplementation failed to increase absolute NAD+ levels in the liver of NRK1 LKO mice. In contrast, we observed a 2.2-fold increase in 1-methylnicotinamide (MeNAM) and a 2.4-fold increase in MeXP levels in NAM-supplemented mice (Supplementary Fig. 6c). NAM supplementation in control mice did not increase NAM levels, but also increased MeNAM and MeXP, albeit to a lower degree than in NRK1 LKO mice. In particular, NAM supplementation led to a 1.8-fold increase in MeNAM (0.019 ± 0.002 for the Control-Veh group vs. 0.034 ± 0.002 for the Control-NAM group, expressed as mean ± SEM of peak area couns; n = 7 and n = 6, respectively) and a 1.8-fold increase in MeXP (0.023 ± 0.002 for the Control-Veh group vs. 0.043 ± 0.001 for the Control-NAM group, expressed as mean ± SEM of peak area counts; n = 7 ad n = 6, respectively).

NAM supplementation also prompted an increase in the mRNA level of Nnmt catalyzing the methylation of NAM in those animals (Supplementary Fig. 6d). At the phenotypical level, NAM-supplemented NRK1-LKO mice displayed a slight decrease in body weight and a trend towards lower liver weight and hepatic TG content (Supplementary Fig. 6e–f). However, in line with the absence of effect on NAD+ levels, NAM supplementation failed to have any significant impact on glucose tolerance, gluconeogenic capacity or mitochondrial respiration (Supplementary Fig. 6g–i). Nevertheless, we observed a modest decrease in γH2AX positive cells in NAM-supplemented NRK1 LKO mice compared to vehicle-treated mice (Supplementary Fig. 6j), suggesting a mild rescue of the PARP activity. Altogether, NAM supplementation did not significantly improve NAD+ levels or mitochondrial dysfunction in NRK1 ablated livers, even if it conferred a modest protection against excessive DNA damage in NRK1 deficient mice.