Inhibition of NAMPT aggravates high fat diet-induced hepatic steatosis in mice through regulating Sirt1/AMPKα/SREBP1 signaling pathway
Nonalcoholic fatty liver disease is one of the most common liver diseases in the world and is a typical hepatic manifestation of metabolic syndrome which is characterized with lipid accumulation in liver. Nicotinamide phosphoribosyltransferase (NAMPT) has been recently identified as an enzyme involved in nicotinamide adenine dinucleotide (NAD+) biosynthesis and plays an important role in cellular metabolism in variety of organs in mammals. The aim of this study was to investigate the effects of NAMPT on high fat diet-induced hepatic steatosis.
Hepatic steatosis model was induced by high fat diet (HFD) in C57BL/6 mice in vivo. HepG2 and Hep1-6 hepatocytes were transfected with NAMPT vector plasmid or treated with NAMPT inhibitor FK866 and then incubated with oleic acid. Lipids accumulation was examined by HE staining or oil red staining. Quantitative RT-PCR and Western blot were used to measure expressions of the genes involved in lipogenic synthesis.
FK866 significantly promoted liver steatosis in the mice fed with HFD and hepatic lipid accumulation in vitro, accompanied by the increases of the expressions of lipogenic genes such as sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FASN). Nicotinamide mononucleotide (NMN) and NAD+ significantly rescued the actions of FK866 in vitro. In contrast, overexpression of NAMPT in HepG2 and Hep1-6 hepatocytes ameliorated hepatic lipid accumulation. In addition, FK866 decreased the protein levels of Sirt1 and phospho-AMPKα in liver of the HFD fed mice. Furthermore, Resveratrol, a Sirt1 activator, significantly reduced lipogenic gene expressions, while EX-527, a Sirt1 specific inhibitor, had the opposite effects.
Our results demonstrated that inhibition of NAMPT aggravated the HFD- or oleic acid-induced hepatic steatosis through suppressing Sirt1-mediated signaling pathway. On the one hand, the inhibition of NAMPT reduced the production of NAD+ through inhibiting the NAD+ salvage pathway, resulting in the decrease of Sirt1 activity, and then attenuated the deacetylation of SREBP1 in which the inhibition of SREBP1 activity promoted the expressions of FASN and ACC. On the other hand, the reduced Sirt1 activity alleviated the activation of AMPKα to further enhance SREBP1 activities.
KeywordsNampt Nad+ Nafld FK866 Sirt1 AMPKα Mouse
AMP-activated protein kinase
Fatty acid synthase
High fat diet
Nicotinamide adenine dinucleotide
Nonalcoholic fatty liver disease
Sterol regulatory element-binding protein 1
Over the past decade, the prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing globally, and it has become the predominant cause of chronic liver disease in the world . The morbidity of NAFLD varies between 20% and 50% in the western countries  and NAFLD is associated with many diseases such as obesity, type 2 diabetes and hepatocellular carcinoma [3, 4, 5]. Although abnormal liver lipid accumulation is considered to be one of the main causes of NAFLD, the molecular mechanisms of NAFLD are not fully elucidated.
Hepatic lipid accumulation results from an imbalance between lipid deposition and removal, which is associated with increased hepatic lipogenesis, augmented lipid uptake and/or decreased triglyceride export or β-oxidation [6, 7]. Hepatic lipid synthesis is regulated by many important transcription factors such as liver X receptor (LXR), carbohydrate response element binding protein (ChREBP) and sterol regulatory element–binding protein 1C (SREBP1C) [8, 9, 10]. As a major transcription factor, SREBP1 has been reported to widely regulate the key enzymes of synthesizing fatty acids including fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and stearoyl-CoA desaturase (SCD1) [9, 11, 12]. Moreover, it has been found that the phosphorylation of AMPKα at its Ser372 suppressed the cleavage and nuclear translocation of SREBP-1c and then further repressed the expressions of the SREBP1C-mediated target genes in hepatocytes when the cells were treated with high glucose, leading to reduction of lipogenesis and lipid accumulation .
Nicotinamide phosphoribosyltransferase (NAMPT) is a highly conserved 52 kDa protein which is expressed in nearly all tissues/cells . NAMPT has both intra- and extracellular forms in mammals. It is an important regulator of the intracellular nicotinamide adenine dinucleotide (NAD+) pool through regulating the rate-limiting step in the mammalian NAD+ salvage pathway from NAM . The intracellular NAMPT (iNAMPT) has been proposed to have cell protective benefits via influencing the activity of NAD-dependent enzymes, such as Sirtuins due to its boosting NAD+ level . The extracellular NAMPT (eNAMPT) in addition to its enzymatic function, it has cytokine-like activity. Although there are some debates, several reports suggest that circulating levels of eNampt may be closely related to obesity, NAFLD, atherosclerosis and diabetes mellitus [17, 18, 19, 20]. However, it has been recently reported that iNampt was downregulated in NAFLD and had anti-apoptosis effects . Moreover, other studies have found that aging-associated NAD+ deficiency was a critical risk factor for NAFLD, which resulted from NAMPT-controlled NAD+ salvage compromised in liver . Although these results indicated that NAMPT could play a role in NAFLD, the effects of NAMPT on the pathogenesis of these disorders, especially in hepatic steatosis were largely unknown. Recently, we observed that inhibition of NAMPT significantly aggravated the high fat diet-induced obesity in mice (unpublished data). In the present study, the potential role of intracellular NAMPT in regulating hepatic lipid metabolism was explored. Our results showed that FK866, an NAMPT inhibitor, significantly promoted hepatic lipid accumulation in vitro and in vivo by promoting the transcriptional activity of SREBP1, which functions as an upstream regulator of FASN and ACC expressions. In addition, we observed that the effects could be rescued by NMN and NAD+, or overexpression of NAMPT. Furthermore, we demonstrated that the inhibition of NAMPT-mediated the production of intracellular NAD+ promoted hepatic lipid synthesis through inhibiting Sirt1 signaling pathway, in turn, activating AMP-activated protein kinase (AMPKα) and then inhibiting SREBP1 activity.
FK866 (Cat.No: F8557), Resveratrol (Cat.No: R5010), EX527 (Cat.No: E7034), NAD+ (Cat.No: N5755), NMN (Cat.No: N3501), Oleic acid (O1383) and Oil Red O (Cat. No: O0625) were purchased from Sigma.
Animals and diets
Male C57BL/6 mice with 8 week old age were housed in an animal room with a 12-h:12-h light/dark cycle under controlled environment (22 ± 3 °C, 50–60% relative humidity) and initially fed with standard diet chow for 1 week to adapt the housing condition. Thereafter, the animals were randomly divided into four groups (n = 5) as follows: 1) normal diet (ND), 2) ND + FK866, 3) High Fat diet (HFD), and 4) HFD + FK866. The mice were first fed either ND or HFD (60% kcal as fat, D12492; Research Diets Inc.) with or without FK866 (2 mg/kg/day, IP) for 1 week. Then, the mice were continually fed with ND or HFD for 12 weeks. All the experimental procedures were approved by Nanchang University Institutional Animal Research Committee and were curried out in accordance with Jiangxi Province Laboratory Animal Care Guidelines for the use of animals in research.
Cell culture and treatment
HepG2 and Hep1-6 cells were cultured in 6-well plates in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco) in a 5% humidified CO2 incubator at 37 °C. The cells were used for the experiments when they were in the exponential phase of growth. At about 80% confluence, the cells were treated with the fresh media containing NAMPT inhibitor FK866, or Sirt1 activator/inhibitor, respectively. After 24 h treatment, the cells were harvested for further experiments.
The Myc-DDK-tagged NAMPT plasmid was purchased from Origene (MR207867) and the cells were transfected with NAMPT vector plasmid and empty vector plasmid using lipo3000 transfection reagent (Invitrogen). Briely, 1.0 × 106 cells were seeded in 6-well plates in DMEM. The plasmid DNA-lipid complexes were prepared according to the instructions and the complexes were incubated for 5 min at room temperature. The DNA-lipid complexes were added into the cells with 70%-80% of confluence, and incubated with the complex alone or plus oleic acid (0.5 mM) for 24 h.
Oil red O staining
HepG2 and Hep1-6 cells were cultured in 24-well plates. Briefly, At 70–80% confluence, the cells were given the appropriate treatment for 24 h, then washed 3-4 times with PBS, and fixed with 4% paraformaldehyde for 1 h, lastly staining with Oil-Red O for 1 h, and then washed 3 times with PBS. Finally, the cells were examined by light microscopy (magnification, 40×). For quantitative analysis of lipid accumulation, isopropanol was added to the stained culture plate and the absorbance was recorded at 490 nm.
The content of intracellular triglycerides in the HepG2 and Hep1-6 cells with inhibition or overexpression of NAMPT was measured using triglyceride assay kit (PPLYGEN, Beijing, China) according to the manufacturer’s instruction, and normalized to total protein concentrations.
Protein extraction from liver tissues and cultured cells
Livers were isolated from eight-week-old mice which fed with ND or HFD for 12 weeks, and then washed with cold PBS followed by being lysed with RIPA buffer (0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-Cl, pH 7.5). After homogenization, the proteins were obtained by centrifugation at 4 °C for 5 min at 12000 rpm, then the supernatant was collected for further analysis and the pellet was discarded. And protein concentration was determined using the Bradford reagent (Bio-Rad) with BSA as a standard. The cultured cells were first washed twice with cold PBS, and then lysed with 2 × loading buffer (Bio Rad). Subsequently, scraped off the adherent cells, the lysates were collected for further analysis.
Western blot analysis
Total proteins were separated by SDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF) membrane and then the membranes were incubated overnight at 4 °C with various primary antibodies including anti-phospho-AMPKα (Thr172), anti-AMPKα, anti-ACC, anti-tubulin, anti-FASN, anti-SIRT1, anti-SREBP1 and anti-GAPDH, respectively. The membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature and visualized using enhanced chemiluminescence western blotting detection reagents.
Total RNA extraction and real-time RT-PCR
QPCR primers used in this study
Ethanol-dehydrated, xylene-treated, and paraffin-embedded tissue sections were sliced at 5 μm. All liver sections were stained with haematoxylin and eosin (H&E) according to standard protocol. The degree of hepatic steatosis was determined with light microscopy.
Data and results were reported as means ± SE. Statistical comparisons were performed with Student’s t test. Statistical significance was set at *p < 0.05, **p < 0.01, ***p < 0.001.
Inhibition of NAMPT aggravates hepatic lipid accumulation in vivo
Inhibition of NAMPT promotes lipid accumulation in HepG2 cells
Overexpression of NAMPT reverses oleic acid-induced lipid accumulation in hepatocytes
NAMPT inhibition-mediated lipid accumulation was associated with inhibition of Sirt1/SREBP1 signaling pathway
NAFLD is well demonstrated as the most common cause of liver disorders including simple steatosis, nonalcoholic steatohepatitis, liver cirrhosis and hepatocellular carcinoma . The most notable characteristic of NAFLD is the accumulation of triglycerides in liver. It has been reported that dysregulation of lipid synthesis is one of the main reasons for abnormal lipid accumulation in the liver . It was reported that NAFLD not only existed in mammals, also happened in vertebrates. It has been found that genetic ablation of solute carrier family 7a3a leaded to hepatic steatosis in zebrafish during fasting . Recently, we observed that FK866 significantly aggravated the high fat diet-induced obesity in mice, in which the NAMPT inhibition-induced obesity might be involved in suppression of Sirt1-SREBP1-FASN signaling pathway in adipose cells (unpublished data). In the present study, we found that the expression of NAMPT was decreased in the liver of the mice fed with HFD. Furthermore, the expression of NAMPT was also reduced in HepG2 cells treated with oleic acid, which was the end product of de novo fatty acid synthesis . In our study, an oleic acid-induced hepatic steatosis model was used to evaluate the effects of NAMPT on NAFLD in vitro since the accumulation of oleic acid played an important role in the development of hepatic steatosis in human beings. Our results suggested that NAMPT played a critical role in hepatic steatosis model in vivo and in vitro.
To further confirm the role of NAMPT in hepatic lipid metabolism, we first examined the effects of FK866, a highly specific NAMPT inhibitor, on HFD-induced hepatic steatosis. We observed that FK866 increased hepatic lipid deposition in mice fed with HFD, but not normal diet, indicating that FK866 promoted the synthesis of lipid in context of high fat diet in vivo. Besides, we found that FK866 significantly increased the expressions of lipid synthesis genes in HepG2 cells, suggesting that inhibition of NAMPT enhanced hepatic lipid synthesis in vivo and in vitro. In addition, we observed that NMN, an enzymatic product of NAMPT, could partially reverse the effects of FK866 in HepG2 cells. Previous studies showed that NMN supplementation improved diet and age-induced diabetes  and other damage such as vascular dysfunction, oxidative stress  and cognitive impairment . NAD+, served as substrate in various signaling conduction pathways, plays an important role in age and metabolism-associated diseases [30, 31]. Our results showed that NAD+ could completely reverse the effects of FK866, suggesting that NAD+ played a central role in the development of NAFLD.
In order to further confirm the roles of NAMPT in NAFLD, the effects of overexpressing NAMPT on lipid accumulation have pursued in liver cell lines. Because the amino acid sequence of the human NAMPT has 96% homologous identity of mouse, the mouse NAMPT was used for gain-of-function experiments in both human HepG2 and mouse Hep1-6 cells. As expected, overexpression of NAMPT significantly attenuated the lipid accumulation and the expressions of lipogenic genes such as SREBP1 and its target genes including FASN and ACC. These results further demonstrated that NAMPT played an important role in lipid synthesis in liver.
SREBP1 is a critical regulator of lipid metabolism, in which it promotes expressions of lipogenic genes such as FASN and ACC, and it plays an important role in nonalcoholic fatty liver disease [32, 33, 34]. It has been reported that Sirt1, an NAD+-dependent protein deacetylase, can directly deacetylate SREBP1 . Here, we found the expression of Sirt1 was reduced in FK866-treated mice fed with HFD. Furthermore, to further elucidate the mechanisms of Sirt1 in hepatic lipid metabolism, we examined the effects of EX527 (a specific inhibitor of Sirt1) and resveratrol (an activator of Sirt1) on lipogenesis in heptocytes and found that resveratrol significantly reduced lipogenic gene expressions, demonstrating that NAMPT-mediated inhibition of lipid metabolism was involved in NAD+-Sirt1 signaling pathway. Moreover, our results also showed that OA-induced expressions of FASN and ACC were remarkably downregulated by resveratrol and upregulated by EX527. These results indicated that NAMPT inhibited lipid synthesis through activating Sirt1 signaling pathway. Moreover, AMP-activated protein kinase (AMPK) is a major regulator of cellular energy homeostasis . Recently, Li et al. reported that SREBP1c was one of the target proteins directly phosphorylated by AMPK . In addition, Sirt1 was found to regulate AMPK activity via modulation of Lkb1, a major upstream kinase of AMPK in hepatic cells and animal models [37, 38], and in turn, AMPK enhanced Sirt1 activity by increasing cellular NAD+ levels . Here, we provided in vivo evidence that the phosphorylation of AMPKα was significantly attenuated by FK866 in the mice fed with HFD. NAFLD is now considered to be the hepatic manifestation of the metabolic syndrome and has insulin resistance as its hallmark [40, 41]. In addition to the influence of NAMPT on lipid metabolism, we also found that FK866 markedly reduced Akt phosphorylation, indicating that NAMPT also improve insulin resistance in NAFLD through promoting insulin signaling in hepatocytes.
The results from our present study demonstrated that NAMPT decreased hepatic lipid accumulation in vitro and in vivo, and the mechanisms of NAMPT-induced inhibition of hepatic lipid accumulation were mainly associated with its suppression of lipogenic genes including FASN and ACC expression through activating Sirt1 signaling pathway and then affecting the transcriptional activity of SREBP1 and activating AMPK. The findings of this study will broad our understanding of the roles of NAMPT in hepatic lipid metabolism and provide important insights in targeting NAMPT for treating liver steatosis and NAFLD.
We thank Professor Jian Li for providing Hep1-6 cell line (Beijing Institute of Geriatrics and Beijing Hospital). We are grateful to the members of the Transgenic Mouse Facility in the Institute of Translational Medicine of Nanchang University for animal housing.
This work was supported by the National Natural Science Foundation of China (91,639,106, 81,270,202 and 91,339,113 to Xin HB), the National Basic Research Program of China (2013CB531103 to Xin HB and Deng KY), Jiangxi Provincial Department of Science and Technology, China (20142BCB24001 to Deng KY).
Availability of data and materials
The datasets during and/or analysed during the current study are available from the corresponding author on reasonable request.
HBX and KYD conceived and designed the experiments; LFW, XNW performed the experiments. CCH, LH, YFX, XHG and YSQ analyzed the data. LFW wrote the draft. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All the experimental procedures were approved by Nanchang University Institutional Animal Research Committee and were curried out in accordance with Jiangxi Province Laboratory Animal Care Guidelines for the use of animals in research.
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