Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice

Abstract

With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high-fat high-sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD(+) ) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD(+) repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD(+) biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1- and SIRT3-dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β-oxidation and mitochondrial complex content and activity. The cell-autonomous beneficial component of NR treatment was revealed in liver-specific Sirt1 knockout mice (Sirt1(hep-/-) ), whereas apolipoprotein E-deficient mice (Apoe(-/-) ) challenged with a high-fat high-cholesterol diet affirmed the use of NR in other independent models of NAFLD.

Conclusion: Our data warrant the future evaluation of NAD(+) boosting strategies to manage the development or progression of NAFLD.

Figure 1

NAD⁺ regulating enzymes and NAD⁺ levels correlate with lipid metabolism in both humans and mice. Transcripts from NAD⁺‐synthesis genes (shown in blue font), in contrast to NAD⁺‐consuming genes (shown in red font), were positively correlated with regulators of β‐oxidation using custom‐designed data sets derived from (A) two human data sets, including 42728 and 22027 human liver samples or (B) two mouse data sets, including 42 BXD strains fed either chow or high‐fat diets.9, 26 As seen on a correlogram, blue correlations are positive (red correlations are negative—intensity of the colors correlates with level of significance). NRK1 and NAMPT transcripts, in contrast to Parp1, were positively correlated to mitochondrial β‐oxidation genes, ACADL, ACADM, and HADH, in data sets from 42728 and 22027 human liver samples (C) or 42 BXD strains fed either chow or high‐fat diets.9, 26 (D) Consistent with these findings in mice and humans, we see reductions in (E) hepatic NAD⁺ levels, followed by increases in (F) hepatic TG levels, in mice fed 9‐18 weeks with a HFHS diet (n = 5‐10). **P < 0.001; ***P < 0.0001 compared to the CD cohort. Data are expressed as mean ± SEM. One‐way ANOVA with a post‐hoc Bonferroni test was used for all statistical analyses.

Figure 2

Glucose intolerance and NAFLD‐induced insulin sensitivity are reversed with NAD⁺ repletion. Phenotyping was performed on CD, HFHS, NR‐Prev, and NR‐Ther cohorts after 5‐18 weeks of treatment (NR dose: 400 mg/kg/day). (A) Schematic illustrating the three experimental groups; animals starting on an HFHS diet at 7 weeks of age are given NR in a preventive (NR‐Prev) mode for 18 weeks or 9 weeks in a therapeutic approach (NR‐Ther). Mice were sacrificed after a 4‐hour fast. The control chow‐diet regimen is not shown on the schematic. NR improved (B) glucose handling and (C) plasma insulin levels after an oral glucose tolerance test (OGTT; insets show the area under the curve [AUC]), measured after 15 weeks of diet (n = 5; chow‐diet regimen is not shown). NR also (D) improved insulin sensitivity during an insulin tolerance test (ITT), measured after 17 weeks of diet (n = 5; chow‐diet regimen is not shown) [Correction added February 3, 2016, after first online publication: “(mg/ml)” in Y axis of Fig. 2D was changed to “(mg/dl).”] and (E) reduced body weight (n = 8‐10). Both cohorts treated with NR exhibited lower (F) whole‐body fat mass, as measured by echo magnetic resonance imaging after 18 weeks of diet, and epididymal fat mass at sacrifice, compared to the HFHS cohort (n = 8‐10). *P < 0.05; **P < 0.001; ***P < 0.0001 compared to the HFHS cohort. Data are expressed as mean ± SEM. One‐way ANOVA with a post‐hoc Bonferroni test was used for all statistical analyses. Male mice were used for these experiments.

Figure 3

NAD⁺ repletion protects and reverses the development of NAFLD in HFHS‐fed mice. Phenotyping was performed on CD, HFHS, NR‐Prev, and NR‐Ther cohorts following 9‐18 weeks of treatment (NR dose: 400 mg/kg/day). (A) After sacrifice, we noted that NR elevated hepatic whole‐tissue and mitochondrial NAD⁺ levels, with each cohort having consumed similar amounts of food (n = 8‐10). (B) NR reduced circulating ALT and AST, indicators for liver damage (n = 8‐10), and improved liver TG and cholesterol accumulation and LPO (n = 5). (C) NR lowered liver weight (n = 8‐10) and blinded H&E scores for severity and extension of steatosis. (D) Matching images of representative livers and liver sections stained with H&E, Oil Red O (lipid content appears red), and picrosirius red (liver fibrosis represented by collagen stained red) (n = 4‐5) are also represented. (E) These functional and morphological changes are supported by the relative expression of genes associated with fibrosis, lipogenesis, lipid metabolism, and inflammation (n = 6). (F) Preventive and therapeutic treatments also attenuated HFHS‐induced levels of plasma TNF‐α. *P < 0.05; **P < 0.001; ***P < 0.0001 compared to the HFHS cohort. Data are expressed as mean ± SEM. One‐way ANOVA with a post‐hoc Bonferroni test was used for all statistical analyses. Male mice were used for these experiments.

Figure 4

Treatment with NR increases liver tissue respiratory capacity ex vivo and β‐oxidation gene expression. Measurements were performed on liver samples from CD, HFHS, NR‐Prev, and NR‐Ther cohorts after 9‐18 weeks of treatment (NR dose: 400 mg/kg/day) and are represented as fold changes compared to the HFHS group. NR increased (A) citrate synthase activity, (B) basal OCR and CI‐coupled driven OCR, in the presence of glutamate, pyruvate, malate, and ADP, but was not significant for CII‐coupled driven OCR, in the presence of succinate, although a trend was noted (n = 6). (C) Histological sections show improvements in both COX and SDH activities in NR‐treated groups compared to HFHS‐fed mice (n = 4), culminating in the attenuation of the decline in (D) ATP levels (n = 5). (E) Increases were observed for mRNA transcript levels of genes regulating mitochondrial biogenesis, Pparα and Pparδ/β, and genes regulating β‐oxidation (n = 6). (F) Liver gene transcript expression measurements were performed for Sirt1, Pgc‐1, and genes associated with β‐oxidation and lipogenesis in mice given 2 weeks of an HFHS or HFHS‐NR diet (NR dose: 400 mg/kg/day). Starting at 8 am, mice were fasted for 24 hours or fasted for 18 hours and refed for 6 hours. Data are represented as fold changes compared to the fasted CD group. For panels A, B, D and E: *P < 0.05; **P < 0.001; ***P < 0.0001 compared to the HFHS cohort and ^ϵ P < 0.05 compared to the CD cohort. For panel F: ^ϵ P  < 0.05, overall effect of treatment versus control mice; ‡P < 0.05, interaction of each treatment versus control mice. Data in all panels are expressed as mean ± SEM. One‐way ANOVA with a post‐hoc Bonferroni test was used for statistical analyses of panels A, B, D, and E. Two‐way ANOVA with a post‐hoc Holm‐Sidak test was used for statistical analysis of panel F. Male mice were used for these experiments.

Figure 5

Improvements in mitochondrial function driven by NAD⁺ and UPR^mt induction and UPR^er attenuation in vivo. (A) Mitochondrial abundance was higher in livers from mice treated with NR. Results are expressed as mtDNA amount (Cox2) relative to nDNA (Hk2) (n = 6). (B) This was matched by increases in mitochondrial complexes and supercomplexes, as evidenced by blue native PAGE of isolated liver mitochondria. (C) NR induced a mitonuclear protein imbalance, indicated by the reduced ratio between SDHB (nuclear encoded complex II protein) and MTCO1 (mtDNA encoded complex IV protein) expression, from whole‐liver extracts. (D) Activation of UPR^mt by NR is demonstrated by increases in hepatic gene transcripts for Clpp and Hspe1 (HSP10), but not Hspd1 (HSP60) (n = 6). These are matched by elevations in CLPP and HSP10 protein levels and the mitochondrial proteins, ATP5A and UQCRC2, from isolated liver mitochondria, indicating higher expression of oxidative phosphorylation proteins per amount of mitochondria. (E) HFHS diet‐induced transcripts involved in the UPR^er, including atf4, chop, and grp78, were mostly reduced after NR treatments, as confirmed with ATF4 and GRP78 protein expression analysis. *P < 0.05; **P < 0.001, compared to the HFHS cohort, and ^ϵ P < 0.05 compared to the CD cohort. Data are expressed as mean ± SEM. One‐way ANOVA with a post‐hoc Bonferroni test was used for all statistical analyses. Male mice were used for these experiments.

Figure 6

NR‐mediated liver benefits arise from liver‐specific SIRT1‐dependent activation of UPR^mt retrograde signaling. Primary hepatocytes cells treated with NR (1 mM; 24 hours) show increased (A) cellular NAD⁺ levels and mitochondrial abundance. (B) NR treatment of Sirt1 ^L2/L2 hepatocytes infected with an adenovirus expressing Cre attenuated mitochondrial protein expression and mitonuclear imbalance (SDHB/MTCO1 ratio) upon Sirt1 LOF. Increases in HSP10 are also shown to be dependent on SIRT1. (C) Body‐weight change, liver weight, and (D) liver TG levels in liver‐specific Sirt1^hep−/− and SirT1^L2/L2 mice fed an HFHS diet with NR for 14 weeks. (E) Images of representative liver sections stained with Oil Red O (n = 5). (F) Relative changes in transcript levels of genes associated with mitochondrial biogenesis and β‐oxidation (n = 5) in mice of the indicated genotypes. *P < 0.05; **P < 0.001; ***P < 0.0001 compared to the HFHS cohort. ^ϵ P  < 0.05, overall effect of treatment versus control mice; ‡P < 0.05, interaction of each treatment versus control mice. Data are expressed as mean ± SEM. Two‐way ANOVA with a post‐hoc Holm‐Sidak test was used for statistical analyses of panels B, C, D and F. Student t test was used for the statistical analysis of panel A. Male mice were used for these experiments. Abbreviation: Veh, vehicle.

Figure 7

Liver damage in Apoe ^−/− mouse model of NAFLD was reversed by NAD⁺ repletion. Apoe ^−/− mice were fed either a low‐fat diet (Apoe ^−/−/LF) or given an HFC diet for 3 weeks that was either continued to induce NAFLD (Apoe ^−/−/HFC) or supplemented with NR (Apoe ^−/−/NR‐Ther) for another 7 weeks (NR: 500 mg/kg/day). Mice were sacrificed after overnight fasting. (A) NR elevated hepatic NAD⁺ levels resulting in (B) reduced body weight. Elevated NAD⁺ levels reduced (C) circulating ALT, an indicator for liver damage (n = 8‐10), and improved hepatic cholesterol and TG accumulation. (D) NR led to less steatosis, as observed by H&E‐stained liver sections (n = 8‐10), with similar food intake between HFC‐fed animals (n = 8‐10). (E) NR showed a trend to reduce liver and epididymal fat pad weights. This resulted in (F) a trend to increase mtDNA abundance and in a significant elevation of the relative expression of genes associated with mitochondrial biogenesis and β‐oxidation after NR (n = 8‐10). *P < 0.05; **P < 0.001; ***P < 0.0001 compared to the HFHS cohort. Data are expressed as mean ± SEM. One‐way ANOVA with a post‐hoc Bonferroni test was used for all statistical analyses. Male mice were used for these experiments.