Nuclear NAD⁺ deficiency by NMNAT1 deletion impaired hepatic insulin signaling, mitochondrial function and hepatokine expression in mice fed a high-fat diet

Abstract

Metabolic syndrome (MetS) is a worldwide challenge that is closely associated with obesity, nonalcoholic liver disease, insulin resistance, and type 2 diabetes. Boosting nicotinamide adenine dinucleotide (NAD⁺) presents a great potential in preventing MetS. However, the function of nuclear NAD⁺ in the development of MetS remains poorly understood. In this study, hepatocyte-specific Nmnat1 knockout (KO) mice were used to determine a possible link between nuclear NAD⁺ and high-fat diet (HFD)-induced MetS. We found that Nmnat1 knockout significantly reduced hepatic nuclear NAD⁺ levels but did not exacerbate high-fat diet (HFD)-induced obesity and hepatic triglycerides (TG) accumulation. Interestingly, loss of Nmnat1 caused insulin resistance. Further analysis revealed that Nmnat1 deletion promoted gluconeogenesis but inhibited glycogen synthesis in the liver. Moreover, Nmnat1 deficiency induced mitochondrial dysfunction by decreasing mitochondrial DNA (mtDNA)-encoded complexes I and IV, suppressing mtDNA replication and mtRNA transcription as well as reducing mtDNA copy number. In addition, Nmnat1 depletion affected the expression of hepatokines in the liver, particularly downregulating the expression of follistatin (Fst). These findings highlight the importance of nuclear NAD⁺ in maintaining insulin sensitivity and provide insights into the mechanisms underlying HFD-induced insulin resistance.

Introduction:

In recent decades, the prevalence of metabolic syndrome has risen alarmingly, emerging as a significant public health concern worldwide¹. Characterized by a cluster of interrelated metabolic abnormalities, this complex medical condition poses a substantial risk factor for the development of life-threatening diseases such as type 2 diabetes, cardiovascular diseases, stroke, and cancer^2,3. Metabolic syndrome represents a cluster of factors, including obesity, fatty liver, insulin resistance, hypertension, and dyslipidemia, which together create a perfect storm for chronic health issues⁴. As our modern lifestyles continue to evolve with unhealthy dietary choices, understanding MetS becomes increasingly vital. Although MetS has been intensively studied in recent years through numerous experimental and clinical investigations, its pathophysiology and underlying mechanisms are still not fully understood.

Nicotinamide adenine dinucleotide (NAD⁺) is a coenzyme found in all living cells which plays a crucial role in various biological processes, particularly in energy metabolism and cellular signaling^5,6. A number of studies have demonstrated that NAD levels decline with age and obesity^7–10. In recent years, increasing interest has been given to the role of NAD⁺ in the development and progression of MetS. Changing NAD⁺ levels and the balance between its oxidized (NAD⁺) and reduced (NADH) forms may significantly influence the pathogenesis of MetS¹¹. One key aspect is the role of NAD⁺ in regulating mitochondrial function and energy metabolism. NAD⁺ is a crucial component of the electron transport chain, which is responsible for generating energy in the form of ATP (adenosine triphosphate). Another key aspect is that NAD⁺ affects the activity of a series of nuclear enzymes such as sirtuins, which are NAD⁺ dependent histone deacetylases, involved in many biological processes such as cell survival, senescence, proliferation, apoptosis, DNA repair, and cell metabolism^12–15. Several studies have reported that disrupted NAD⁺ metabolism and reduced NAD⁺ levels may be associated with NAFLD^16,17, diabetes^7,18, and hypertension^19,20. However, a recent study reported that the loss of nuclear NAD⁺ does not affect the development of NAFLD²¹, indicating a functional difference between nuclear NAD⁺ and cytoplasmic NAD⁺ in the development of MetS.

NAD⁺ is highly compartmentalized by forming NAD⁺ pools in mitochondria, cytoplasm, and nucleus^22,23. Interestingly, most previous studies focused on the effect of cytosolic/mitochondrial NAD⁺ rather than nuclear NAD⁺. Thus, the role of nuclear NAD⁺ in MetS remains largely unknown. The aim of this study is to investigate the potential role of nuclear NAD⁺ in the development and progression of MetS using a hepatocyte-specific Nmnat1 knockout (KO) mouse model.

Materials and methods

Animals

Nmnat1 floxed mice were a gift from Pro. Chen at Baylor College of Medicine, Houston. In this mouse, the exon 2 of Nmnat1 gene is flanked by LoxP sequence²⁴. To induce hepatic nuclear NAD⁺ deficiency mice hepatocyte-specific Nmnat1 knockout mice were generated by breeding Nmnat1 floxed mice with Alb-cre mice (Strain #: 003574, Jackson Laboratory). All experiments were conducted in male mice with Nmnat1 floxed mice (Flox) used as control. Mice were maintained under controlled temperature and humidity (25 °C , 50%) with standard light conditions (12:12 h light-dark cycle) and were allowed ad libitum to food and water. All experiments were performed following the protocol approved by the North Carolina Research Campus Institutional Animal Care and Use Committee.

Diet-induced MetS model

MetS model was produced by using a rodent High-fat diet (HFD) with 45% kcal fat (Research Diet, D12451) and low-fat diet with 10 kcal% fat (Research Diet, D12450H) as control²⁵. Six-week-old male Flox mice and KO mice were fed the control or HFD for 13 weeks.

Primary hepatocyte isolation and culture

Primary hepatocytes were isolated from normal diet fed Flox mice and Nmnat1 knockout mice according to a two-step perfusion method²⁶. After isolation, the primary hepatocytes were cultured overnight in a 6-well cell culture plate (4 × 10⁵/well) and then treated with 100 nM insulin (Sigma, I2767–100mg) for 30 minutes. For NMNAT1 knockdown, the isolated hepatocytes were seeded at a density of 1×10⁵ in 1 ml standard growth medium per well in a 12-well tissue culture plate. After overnight incubation, cells were transfected with Nmnat1 siRNA (Santa Cruz Biotechnology, sc-45503) or control siRNA (Santa Cruz Biotechnology, sc-36869) using lipofectamine 3000 (Thermo Fisher Scientific, L3000015) for 48 h.

RNA isolation and real-time PCR

Total RNA was isolated from mouse liver tissue using TRIzol Reagent (Invitrogen, Oregon, USA). qRT-PCR was performed using SYBR Green. Primers used for real-time PCR are depicted in Supplementary Key Resources Table 1. The mRNA levels of genes were normalized to that of RPS17 and expressed relative to the control.

Protein isolation and Western blot

Whole protein lysates of the liver were extracted using a lysis buffer supplemented with the protease inhibitor and phosphatase inhibitor (Sigma-Aldrich). Aliquots containing 40 μg of proteins were loaded following standard procedures, all antibodies used are summarized in Supplementary Key Resources Table 2.

Nuclear NAD⁺ measurement

Levels of NAD⁺ in the liver were determined by Colorimetric Assay Kit (ab65348) according to the manufacturer’s instructions²⁷. To measure nuclear NAD⁺, fresh liver tissues were incubated with Triton X-100 extraction buffer [PBS containing 0.5% Triton X-100 (v/v), 2 mM PMSF, 0.02% NaN3 (w/v), 1 mM DDT, and 1× Halt Protease and Phosphatase inhibitor cocktail (at a cell density of 10⁷ cells/ml)] homogenized on ice for 10 mins, followed by centrifugation at 1000g for 10 mins at 4°C to isolate pelleted nuclear fractions. The nuclear fractions were incubated with NADH/NAD extraction buffer and homogenized. After centrifuged at 15,000 rpm for 5 min at 4°C, the supernatants were incubated with NAD⁺/NADH reaction mixture and measured at 450 nm absorbance. Data were normalized based on protein concentration and expressed as picomole NAD⁺ per milligram of protein or as a ratio of NAD⁺/NADH.

Oral glucose tolerance tests (OGTT)

At 11 weeks, mice were fasted for 6 h, and then the OGTT was carried out by oral administration of glucose (2 mg/g). Blood samples were collected from the tail vein at 0, 30, 60, 90, and 120 mins post glucose administration. The blood glucose levels were measured using a Glucometer (Nova Biomedical).

Liver triglyceride (TG) assay

The hepatic levels of TG were measured with colorimetric assay kits (BioVision). Briefly, lipids were extracted using chloroform/methanol (2:1), vacuumed, and redissolved in 5% Triton X-100/methyl alcohol mixture (1:1 vol/vol), then FFA and TG contents were determined according to the manufacturer’s instructions.

Histology and periodic acid schiff (PAS) staining of glycogen

Liver tissue paraffin sections were stained using Mayer’s Hematoxylin and Eosin (H&E) staining protocol and PAS staining protocol.

Statistical analysis

Data met with normal distribution were analyzed using the independent-samples t-test or one-way analysis of variance followed by LSD’s multiple comparison. Data that do not conform to a normal distribution were analyzed using Nonparametric tests followed by the Median test (K samples). Statistical analyses were performed using SPSS 21. Data were expressed as mean ± SD. In all tests, P < 0.05 was taken as significant.

Results

Hepatocyte-specific Nmnat1 deletion reduces nuclear NAD⁺ in the liver

To elucidate the role of nuclear NAD⁺ in the liver, the hepatic Nmnat1 knockout (KO) mice were generated (Fig. 1A, B). Furthermore, the qRT-PCR revealed a specific deletion of Nmnat1 while the other two isoforms Nmnat2 and Nmnat3, responsible for NAD⁺ biogenesis in cytosol or mitochondria respectively, did not change in the liver (Fig. 1C). Consequently, nuclear NAD⁺ levels in the liver were significantly decreased in KO mice compared with Flox mice (Fig. 1D). However, the Nmnat1 deletion did not affect body, liver, or fat weight (Fig. 1E–G).

Fig. 1: Nmnat1 deletion reduced nuclear NAD⁺ levels in the liver.

A Schematic representation of the generation of hepatic Nmnat1 knockout (KO) mice by crossing Nmnat1 flox (Flox) mice with Alb-Cre mice. B Genotyping PCR analysis confirming the deletion of Nmnat1 in the liver. C qRT-PCR analysis of expression of Nmnat1, Nmnat2 and Nmnat3 in the livers of 16-week-old male mice fed a normal chow diet. D Quantification of nuclear NAD⁺ levels in the livers of 16-week-old male mice fed a normal chow diet. E-G Body weight (E), liver weight (F), and fat weight (G), showing no significant differences between KO mice and Flox mice in 16-week-old male mice fed a normal chow diet. The data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 200 mm.

Hepatocyte-specific Nmnat1 deletion does not exacerbate lipid accumulation in the liver

Previous studies indicate that NAD⁺ deficiency decreases fatty acid oxidation, which results in development of steatosis²⁸. Thus, we investigated the association between low nuclear NAD⁺ levels and HFD-induced fatty liver in KO mice. We observed that there were no significant differences in body weight and liver weight between KO mice and Flox mice after HFD feeding (Fig. 2A, B) although the levels of nuclear NAD⁺ significantly decreased by 50% (Fig. 2C). To determine the effect of decreased nuclear NAD⁺ on hepatic lipid accumulation, we examined the liver gross morphology and H&E staining, but there was no significant difference between the two groups (Fig. 2D, E). Furthermore, the hepatic triglyceride (TG) and expression of genes involved in fatty acid synthesis (Pparα, Pparγ, Scd1, Acc, Fasn) and transport (Fabp1, Cd36), and TG synthesis (Dgat1, Dgat2) in the liver were analyzed. Similarly, we observed no significant differences in the hepatic TG accumulation and synthesis between these two groups. Although Pparγ was upregulated in HFD-fed mice after Nmnat1 deletion, the genes related to TG synthesis were not changed. Moreover, genes related to fatty acids synthesis including Scd1, Acc and Fasn were decreased after Nmnat1 deletion in the control group (Fig. 2F, G).

Fig. 2: Nmnat1 deletion did not aggravate HFD-induced lipid accumulation in the liver.

A Bodyweight curvature of mice. B Liver weight. C Quantification of nuclear NAD⁺ levels in the liver. D-E Gross morphology analysis and H&E staining of the liver in HFD-fed mice. F Liver triglyceride (TG) levels. G qRT-PCR analysis of gene expression related to lipid metabolism in the liver. All mice were 6-week-old male Flox and KO mice fed a control（Ctrl） or high-fat diet (HFD) for 13 weeks. The data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 50 μm.

Hepatocyte-specific Nmnat1 deletion impairs insulin signaling in the liver

Previous studies reported that NAD⁺ deficiency is associated with insulin resistance, which is a major factor in the pathogenesis of type 2 diabetes^7,29. To understand whether nuclear NAD⁺ contributes to HFD-induced insulin resistance, we performed OGTT in HFD-fed Flox and KO mice. We observed that Nmnat1 deletion diminished glucose tolerance and increased fasted blood glucose levels under HFD conditions (Fig. 3A, B). Insulin resistance relates to hepatic glucose production³⁰. Next, the liver glycogen and glucose metabolism-related genes and proteins were examined. PAS staining revealed that Nmnat1 deletion significantly decreased glycogen contents in the liver (Fig. 3C). Furthermore, qRT-PCR and Western blot analysis revealed that Nmnat1 deficiency significantly decreased the expression of gluconeogenesis-related genes and proteins including G6PC and PEPCK as well as protein expression of GSK3β, a key protein involved in liver glycogen synthesis (Fig. 3D–F). In parallel, we found that hepatic Nmnat1 deletion inhibited the insulin signaling pathway, as indicated by a reduction of phosphorylation of IRS1 and AKT (Fig. 3E, G). To further illustrate the function of NMNAT1 in insulin signaling, the isolated primary hepatocytes were treated with insulin. We found that insulin stimulated protein phosphorylation of AKT and inhibited gluconeogenesis in hepatocytes. However, these phenomena were abolished in NMNAT1 knockout cells (Supplementary Fig. S1A, B). These data supported that Nmnat1 knockout impaired hepatic insulin signaling.

Fig. 3: Nmnat1 deletion aggravated HFD-induced insulin resistance.

A Oral glucose tolerance test (OGTT) and quantification, OGTT was performed at 11 weeks in 13 weeks HFD feeding procedure. B Fasting blood glucose levels at 11 weeks in 13 weeks HFD feeding procedure. C Glycogen detected by periodic acid schiff (PAS) staining on formalin-fixed paraffin-embedded liver tissue sections. D qRT-PCR analysis of gene expression related to gluconeogenesis. E Western blot analysis of protein levels related to glucose metabolism and insulin resistance signaling pathway. F Quantification of protein levels of glucose metabolism in the liver. G Quantification of protein levels of insulin resistance in the liver. All mice were 6-week-old male Flox and KO mice fed a control（Ctrl）or high-fat diet (HFD) for 13 weeks. The data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 50 μm.

Hepatocyte-specific Nmnat1 deletion decreases mitochondrial-encoded OXPHOS components and induces hepatic mitochondrial dysfunction

Mitochondrial dysfunction resulting from alterations in mitochondrial oxidative phosphorylation (OXPHOS) has been shown to contribute to insulin resistance³¹. Nevertheless, whether nuclear NAD⁺ deficiency leads to alterations in OXPHOS in the liver remains unclear. To investigate that, we examined the expression of nuclear DNA (nDNA)-encoded OXPHOS genes and mitochondrial DNA (mtDNA)-encoded OXPHOS genes. We found that Nmnat1 deletion did not change genes expression of nDNA-encoded OXPHOS complexes I, II (Sdha, Sdhb), III, IV and V in HFD mice (Fig. 4A). Interestingly, Nmnat1 deletion significantly decreased genes expression of mtDNA-encoded OXPHOS complexes I and IV (Fig. 4B), which are key subunits for making up the first enzyme and the last rate-limiting enzyme of the mitochondrial electron transport chain (ETC), respectively³². However, the complexes III and V remained unchanged. Furthermore, we found that the expression of genes involved in mitochondrial DNA replication (Plog) and RNA transcription (Polrmt, Mtif2) was significantly decreased by Nmnat1 deletion but not protein translation (Mrpl45) in HFD fed mice (Fig. 4C). It’s consistent with the downregulation of mtDNA copy numbers in KO mice (Fig. 4D). In addition, to further confirm the effect of NMNAT1 on mitochondrial OXPHOS and function, the protein level of mtDNA-encoded MTCO1 (complex IV subunit), nDNA-encoded NDUFB8 (complex IV subunit) and ATP5A1 (complex V) were tested. As expected, Nmnat1 deletion decreased mtDNA-encoded MTCO1 but not nDNA-encoded NDUFB8. Intriguingly, although we did not observe that Nmnat1 deletion altered mitochondrial complex V gene expression in either nDNA-encoded or mtDNA-encoded genes, we found that ATP5A1, a subunit of mitochondrial ATP synthase was decreased in KO mice (Supplementary Fig. S2A, B). These data suggested that loss of Nmnat1 could cause mitochondrial dysfunction.

Fig. 4: Nmnat1 deletion downregulated mitochondrial DNA-encoded genes in the liver.

A qRT-PCR analysis of nuclear DNA (nDNA)-encoded mitochondrial oxidative phosphorylation (OXPHOS) genes. B qRT-PCR analysis of mitochondrial DNA (mtDNA)-encoded mitochondrial OXPHOS genes. C qRT-PCR analyzed genes related to mitochondrial DNA replication, RNA transcription, and protein translation. D Mitochondrial DNA contents were measured by qRT-PCR of mitochondrial Nd4 DNA levels using the nuclear gene RPS17 as a control. All mice were 6-week-old male Flox and KO mice fed a control（Ctrl）or high-fat diet (HFD) for 13 weeks. The data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Hepatocyte-specific Nmnat1 deletion alters the expression of hepatokines in the liver

Hepatokines are hepatocyte-secreted hormone-like proteins, and accumulating evidence indicates that hepatokine is a key link between metabolic dysfunction and insulin resistance^33–35. To explore whether nuclear NAD⁺ affects the expression of hepatokines, we analyzed the expression of related genes by qRT-PCR and found that Nmnat1 deletion pe se increased expression of Angptl4, Fetuin-A, Igf1 and Smoc1, with no further impact by HFD (Figure 5A). Conversely, we found that the expression of follistatin (Fst) was significantly decreased in the liver in HFD-fed mice and it was exacerbated by Nmnat1 deletion (Figure 5A). Fst-315 and Fst-288 are the two major isoforms of Fst in mice³⁶. Next, we analyzed these two isoforms and found that both Fst-315 and Fst-288 were decreased in HFD-fed mice, and Nmnat1 deletion exacerbated HFD-induced downregulation of Fst-315 (Figure 5B). At the protein level, we also found that FST was significantly decreased in HFD-fed mice and further decreased in KO mice (Figure 5C, D). To further confirm the downregulation of FST was caused by Nmnat1 deficiency, the small interfering RNA (siRNA) of Nmnat1 was employed. As expected, NMNAT1 knockdown significantly reduced FST expression in primary hepatocytes in vitro (Figure 5 E–G). These data suggested that Nmnat1/nuclear NAD⁺ is involved in expression of hepatokines.

Fig. 5: Nmnat1 deletion disrupted expression of hepatokines in the liver.

A qRT-PCR analyzed genes related to hepatokines in the liver. B qRT-PCR analyzed two isoforms of follistatin (Fst, Fst-315 and Fst-288) in the liver. C Western blot analysis of protein levels of FST in the liver. D Quantification of protein levels of FST in the liver. E qRT-PCR analysis of Nmnat1 in primary hepatocytes with siRNAs transfection for 48 h. F Western blot analysis of NMNAT1 in primary hepatocytes with siRNAs transfection for 48 h. G Quantification of protein levels of FST in primary hepatocytes with siRNAs transfection for 48 h. The data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

Recent studies have highlighted the potential role of NAD⁺ in the pathogenesis of MetS^11,37. However, the role of nuclear NAD⁺ in the development of MetS remains unclear. The present study demonstrates that the deletion of hepatic Nmnat1, a key regulator of nuclear NAD⁺ levels, neither affects body, fat, or liver weight nor hepatic lipid accumulation, indicating that nuclear NAD⁺ depletion alone may not contribute to the development of HFD-induced fatty liver or obesity. However, loss of nuclear NAD⁺ exacerbated insulin resistance, which is associated with impaired hepatic glucose metabolism, mitochondrial dysfunction, and hepatokine dysregulation. These findings shed light on the impact of nuclear NAD⁺ depletion on liver function and its association with insulin resistance in the context of HFD-induced MetS.

An interesting observation of this study is that loss of hepatic nuclear NAD⁺ did not exacerbate lipid accumulation, despite previous studies suggesting that NAD⁺ deficiency promotes steatosis^16,17. This discrepancy may indicate the presence of compensatory mechanisms or alternative pathways involved in lipid metabolism that counteract the effects of reduced nuclear NAD⁺. However, it is noteworthy that NAD⁺ is highly compartmentalized in cells and these animal models in which NAD⁺ deficiency was found to promote steatosis were based on reduced levels of cytoplasmic NAD⁺ rather than nuclear NAD⁺ levels^38,39. In this study, the expression of genes related to fatty acid synthesis, transport, and triglyceride synthesis did not show significant differences between KO and Flox mice, which is consistent with a recently published study²¹, suggesting that nuclear NAD⁺ depletion does not contribute to the development of HFD-induced fatty liver.

In contrast to the limited impact on lipid accumulation, loss of nuclear NAD⁺ was found to exacerbate insulin resistance in HFD-fed mice in the present study. This observation aligns with earlier evidence linking NAD⁺ deficiency to insulin resistance and the development of type 2 diabetes^7,40. Furthermore, impaired glucose tolerance, and increased fasting blood glucose levels were observed in the HFD-fed KO mice indicating compromised glucose metabolism. The downregulation of gluconeogenesis-related genes and proteins, along with reduced glycogen content in the liver, further supports the role of nuclear NAD⁺ in regulating hepatic glucose production and glycogen synthesis. Moreover, inhibition of the insulin signaling pathway, as evidenced by reduced phosphorylation of IRS1 and AKT, suggests that nuclear NAD⁺ depletion affects insulin sensitivity in hepatocytes. These findings highlight the important role of nuclear NAD⁺ in maintaining proper glucose metabolism and insulin sensitivity in the liver. However, further investigations are necessary to elucidate the underlying molecular mechanisms by which nuclear NAD⁺ depletion influences insulin signaling and glucose metabolism.

Mitochondrial dysfunction has been implicated in the pathogenesis of insulin resistance^41–43. In this study, we found that nuclear NAD⁺ depletion may be associated with this process. While the expression of nuclear DNA-encoded OXPHOS genes remained largely unaffected, depletion of nuclear NAD⁺ selectively influenced the expression of mitochondrial DNA-encoded OXPHOS genes, particularly complex I and complex IV. This observation suggests a potential role for nuclear NAD⁺ in regulating mtDNA replication or mtRNA transcription. The downregulation of key genes involved in mtDNA replication and mtRNA transcription, as well as the decrease in mtDNA copy numbers, further supports the notion that nuclear NAD⁺ depletion can lead to mitochondrial dysfunction in the liver.

In addition, nuclear NAD⁺ depletion has been found to affect the expression of hepatokines, which are known involved in the crosstalk between MetS and insulin resistance^33–35. The altered expression of hepatokines, such as Angptl4, Fetuin-A, Igf1, Smoc1, and Fst, suggests that nuclear NAD⁺ depletion may disrupt the delicate balance of hepatokine signaling, especially Fst, which was further decreased after Nmnat1 deletion in HFD-fed mice. Although more work is needed to investigate the regulatory mechanism of Fst, it has been reported that Fst is closely related to insulin resistance. Tao et al. found that reducing hepatic Fst increases systemic insulin sensitivity and improves glucose intolerance⁴⁴, whereas Han et al. reported that overexpression of Fst markedly increased insulin sensitivity in insulin-resistant mouse muscle⁴⁵. suggesting the function of Fst may be tissue-specific. However, our observation that decreased Fst along with impaired glucose tolerance was in contrast to the findings by Tao et al in liver⁴⁴.

Overall, this study provides valuable insights into the role of nuclear NAD⁺ depletion in hepatic function and its association with insulin resistance. The findings demonstrate that the loss of nuclear NAD⁺ leads to impaired glucose metabolism, induced mitochondrial dysfunction, and dysregulated hepatokines expression, suggesting a significant role of nuclear NAD⁺ in maintaining liver homeostasis. Further research is warranted to elucidate the precise molecular mechanisms underlying these effects and to explore strategies aimed at restoring nuclear NAD⁺ levels in the liver, which may hold promise for developing novel interventions for metabolic disorders.

Data Availability Statement

Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon reasonable request.