Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration

Abstract

The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR.

Conclusion: NAD availability is limiting during liver regeneration, and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. (Hepatology 2017;65:616-630).

Figure 1. Nicotinamide riboside promotes liver regeneration

10–14 week old male C57BL6/J mice were treated with nicotinamide riboside (NR) at a dose of ~500mg/kg/day. After 14 days, animals were subjected to 2/3 partial hepatectomy and analyzed 24, 48 and 192 hours later (n = 6 per group). (A) Left: Photographs of regenerating livers. Right: EdU incorporation in regenerating livers. Inset shows an enlarged view from an NR-treated liver at 48h post PHx. (B) Quantification of EdU positive hepatocytes. (C) Liver to body weight ratio. (D) Liver NAD content pre and post PHx. (E) Left: Representative liver sections stained with H&E at 40X showing mitotic figures and micro and macro-vesicular fatty changes. Right: Quantification of mitotic figures across multiple high power fields. (F) Left: Oil Red O staining to detect neutral lipids. Right: Hepatic triglyceride content in pre and post hepatectomized NR and H2O treated mice. Error bars represent S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2. Hepatocyte-specific overexpression of Nampt promotes liver regeneration

Mice carrying a Cre-inducible allele of Nampt in addition to Alb-Cre (denoted as N^Albcre) and littermate controls (N) were subjected to 2/3 partial hepatectomy and analyzed 48 hours later. (A) Nampt overexpression is driven by the CAGGS promoter, separated from the NAMPT transgene by a STOP cassette flanked with loxP sites. Under the influence of Cre recombinase, the STOP cassette is removed, resulting in overexpression of Nampt. The construct was inserted at the ColA1 locus. Left: Nampt protein and mRNA expression in liver. Right: Immunofluorescence showing overexpression of Nampt in peri-portal and peri-central venous areas of N^Albcre mice. Inset shows an enlarged view of N^Albcre. (B) Left: Photographs of regenerating livers. Right: Proliferating hepatocytes identified by EdU detected by immunofluorescence (green) and counterstained with DAPI (blue) with quantification of EdU positive hepatocytes (n=4–5/group). Inset shows an enlarged view of N^Albcre. (C) Left: Liver to body weight ratios. Right: Fasting blood glucose. (D) Liver NAD content before and after PHx. (E) Mitotic index as determined by counting mitotic figures in hepatocytes under high power in H&E stained sections. (F) Left: Representative liver sections stained with H&E. Right: Hepatic lipid content as determined by Oil Red O and triglyceride assay. Error bars represent S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 3. Adult onset Nampt over-expression is sufficient for regenerative phenotypes

Mice carrying a Cre-inducible allele of Nampt infected with AAV-Cre (denoted as N^AAVcre) and littermate controls (N^AAVgfp or WT^AAVcre) were subjected to 2/3 partial hepatectomy and analyzed 36 hours later. (A) Immunofluorescence showing over-expression of Nampt in peri-portal and peri-central venous areas of N^AAVcre mice. Inset shows an enlarged view of N^AAVcre. (B) Fasting blood glucose. (C) Left: Photographs of regenerating livers. Right: Liver to body weight ratios. (D) Left: Proliferating hepatocytes identified by EdU detected by immunofluorescence (green) and counterstained with DAPI (blue). Inset shows an enlarged view of N^AAVcre. Right: Quantification of EdU-positive hepatocytes (n=5/group). (E) Hepatic triglyceride content. (F) Left panels: representative liver sections stained with H&E. Right panels: lipid accumulation by Oil Red O. Error bars represent S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4. Hepatocyte-specific loss of Nampt impairs regeneration, and is rescued by nicotinamide riboside (NR)

Hepatocyte specific Nampt deficient mice were generated by injecting animals bearing two floxed alleles with an AAV expressing Cre recombinase (AAV-Cre). Littermates infected with AAV-Gfp served as controls. Treatment with NR (~500 mg/kg/day) began 5 days after infection and 2 weeks prior to PHx. Analyses were performed 48 hours after PHx. (A) Protein and mRNA expression of Nampt in livers. (B) Photographs of regenerating livers. (C) Left: Proliferating hepatocytes identified by EdU detected by immunofluorescence (red) and counterstained with DAPI (blue). Middle: Quantification of EdU positive hepatocytes (n=4–5/group). Left: Liver to body weight ratios. (D) NAD content in livers before and after PHx. (E) Mitotic index as determined by counting mitotic figures in hepatocytes under high power in H&E stained sections (n= 5–6/group). (F) Left: Representative liver sections stained with H&E (left panels) and Oil Red O (right panels). Right: Hepatic triglyceride content. Error bars represent S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5. Hepatocyte proliferation exhibits a strong correlation with liver NAD concentration and an inverse relationship with hepatic lipid content

Data from individual animals were plotted to study the correlations between NAD, lipid metabolism and hepatocyte proliferation. (A) Positive correlation between NAD concentration and hepatocyte proliferation. (B) Inverse correlation between hepatocyte proliferation and triglyceride content. (C) Liver ATP content assessed pre or post hepatectomy in NR treated mice (n=6/group). (D,E) ATP correlates positively with NAD concentration and hepatocyte proliferation. (F) Proposed model: Hepatic NAD promotes fatty acid oxidation, thereby generating ATP necessary for hepatocellular growth and regeneration. NAD is directly required in the 3-hydroxyacyl-CoA dehydrogenase (HADH)-catalyzed step of fatty acid oxidation as well as the tricarboxylic acid cycle (TCA), but may also act indirectly via signaling enzymes such as Sirt1, which use NAD as a cosubstrate. NAD concentration can be modulated based on the expression of Nampt, which catalyzes the formation of nicotinamide mononucleotide (NMN) from nicotinamide and phosphoribosylpyrophosphate. Alternatively, NMN can be generated from NR by the action of NR kinase. Error bars represent S.E.M. *, p < 0.05; **, p < 0.01.

Figure 6. Increased expression of lipolytic genes in NR treated mice

10–14 week old male C57BL6/J mice were treated with NR at a dose of ~500mg/kg/day. After 2 weeks, mice were subjected to 2/3 partial hepatectomy. Hepatic gene expression was determined either pre PHx or 24h post PHx. (A) Expression of genes related to the unfolded protein response in the mitochondria (UPR^mito) or endoplasmic reticulum (UPR^er), or to mitochondrial biogenesis. (B) Expression of genes related to lipid metabolism. Expression of Atgl, Hsl, Mcad, Acox1, Fasn, Srebp1c, Acc1, Acc2, Atf4, Xbp1m, Hspd1, Atp5a1, Sdhb and Cox5b were significantly different (p < 0.05) in regenerating liver as compared to pre-PHx livers. Error bars represent S.E.M. #, p < 0.1; *, p < 0.05; **, p < 0.01; ***, p < 0.001.