C57BL/6 mice (male, 8–10-weeks old) obtained from the Animal Experimental Center at Tsinghua University (Beijing, China) was housed with standard cages at constant room temperature (22±2°C) and relative humidity of 45±15%, with free access to food and water on a 12/12 h light/dark cycle. Then, these mouse were fed with standard food as controls or the HFD (fat 60%, carbohydrate 20.6%, protein 19.4%; Research Diets, Inc., New Brunswick, NJ, USA) for 8 weeks. All mice were then sacrificed. The liver wet weight and body weight were measured, and alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were detected using an automatic biochemical analyzer. The histopathological changes of the liver were evaluated by hematoxylin and eosin (H&E) staining. Furthermore, the level of SIRT1 in the serum was determined using the ELISA method.

To investigate the functional roles of QKI 5 in NAFLD, 24 C57BL/6 mice were randomized into four groups as follows (n=6 per group): Control group, mice fed with a HFD (model group), mice fed with a HFD and treated with 100 mg/kg/day per os of SRT1720 (SRT1720 group) or vehicle (1% DMSO in 20% cyclodextrin; vehicle group) (21). Following 8 weeks of continuous administration, the mice were sacrificed. Blood samples were collected for the determination of serum SIRT1, ALT, AST, total cholesterol (TC) and triglyceride (TG) levels. Additionally, liver tissues were subjected to pathological H&E staining, determination of NAFLD activity score (NAS), and an assay of the synthesis of TG. All animal experiments were approved by the Animal Ethics Committee of Tsinghua University.

The mouse body composition, including fat mass, was assessed with non-invasive quantitative magnetic resonance using an EchoMRI700 instrument. Values are expressed as a percentage of body weight (BW). All experiments were performed at Tsinghua University. Homogenization and protein extraction from the liver tissue-homogenization and extraction of individual liver sections were performed in NP-40 lysis buffer.

To observe the liver histo-pathological changes, standard H&E staining was performed. Briefly, following dehydration with an ethanol gradient, the liver tissues fixed using formalin (4%) were embedded in paraffin, then cut into 5-µm sections. The sections were then stained with H&E or Oil Red O staining, and immunostaining with anti-SIRT1 or anti-QKI 5 was performed. The slides were mounted with aqueous mountant and viewed with a fluorescent microscope (IX71; Olympus Corporation, Tokyo, Japan). Hepatic TG and TC or TG in cells were measured using the infinity TG and TC reagent kits (cat. nos. TR13421 and TR-22421; Thermo Fisher Scientific, Inc., Waltham, MA, USA), respectively.

The serum levels of TG, TC, ALT and AST were assessed with an automatic biochemical analyzer (Olympus Corporation).

Based on the manufacturer's protocol, the serum SIRT1 concentration was measured by ELISA using an SIRT1 mouse ELISA kit (Abbexa, Cambridge, UK).

For liver biopsies of NAFLD, the novel NAS system is suggested for the grading of steatosis, hepatocellular ballooning and inflammatory activity, referring to a guide on the diagnosis and treatment for NAFLD (22).

Mouse hepatocytes were dissociated from the tissues of the C57BL/6 mice using an in situ recirculating collagenase perfusion method (23). The liver tissues were transferred into Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) on ice and filtered through a multi-layer gauze to obtain a hepatocyte suspension. Following washing with PBS, the supernatant was discarded. The primary hepatocytes were maintained in DMEM with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) for cell adherence with 5% CO₂ at 37°C for 4–6 h.

The obtained cells were rinsed with fresh medium to remove the dead cells and cell fragments, and then washed with PBS and re-suspended in serum-free medium containing 50 ng/ml SRT1720 or 10 µM niacinamide for 48 h at 37°C, respectively. The cells were collected for synthesis of TG at each time point using a TG assay kit for quantification (cat. no. ab65336; Abcam, Cambridge, MA, USA).

To initially investigate the effect of SRT1720 treatment on the FOXO1 and PPARα signaling pathway, the mouse hepatocytes (5×10⁵ cells/well) were cultured in a 12-well plate at 37°C, and treated with either DMSO (control), SRT1720 (50 ng/ml), FOXO1 inhibitor AS1842856 (100 nM; cat. no. 344355; Merck KGaA, Darmstadt, Germany) or SRT1720 (50 ng/ml) + FOXO1 inhibitor AS1842856 (10 nM). Following 48 h of treatment, the protein expression levels of FOXO1 and PPARα were determined by western blotting. The synthesis of TGs in the mouse hepatocytes was detected.

Recombinant adenoviruses (Ads) containing SIRT1 were produced with an AdEasy (Ad) Vector system (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA). The concentration of 10 plaque-forming units/hepatocyte was used to infect cells. AdGFP was used as the infection control.

Whether SIRT1 inhibited the activation of FOXO1 was then determined, in addition to the PPARα signaling pathway, in mouse hepatocytes. The mouse hepatocytes were transfected using Invitrogen™ Lipofectamine^® 2000 Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) with either scrambled small interfering (si)RNA or siRNA of SIRT1, followed by treatment with or without SRT1720 (50 ng/ml). Western blotting was used to quantify the protein levels of SIRT1, and the synthesis of TGs was measured. The sequences of scrambled siRNA and siRNA of SIRT1 were as follows: Sense, 5′-ACUUUGCUGUAACCCUGUAdTdT-3′ and antisense, 5′-UACAGGGUUACAGCAAAGUdTdT-3′ (scrambled siRNA); sense, 5′-CCUACGCCACCAAUUUCGU-3′ and antisense, 5′-ACGAAAUUGGUGGCGUAGG-3′ (siRNA of SIRT1).

A lysis buffer (150 mM NaCl, 0.1% SDS, 0.02% NaN₃, 1% NP-40, and 50 mM pH 8.0 Tris) containing a cocktail of inhibitors for proteases and phenylmethylsulfonyl fluoride (1 mM) was used to lyse cells. Western blotting was performed using the Bio-Rad Protean II minigel system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The concentrations of total proteins were quantified by bicinchoninic acid assay. A 50 µg protein sample was injected into every gel (12%) well, and then transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Bedford, MA, USA) following electrophoresis. Subsequently, 1× Tween-20 TBS (5% dry skimmed milk) was used to block the non-specific binding. The membranes were then sequentially incubated with primary at 4°C overnight, and secondary antibodies for 1 h at room temperature. The membranes were visualized with ECL chemiluminescence reagent using the Hyperfilm ECL kit, and exposed using x-ray film. The primary antibodies included anti-SIRT1 (D739; cat. no. CST 2493; Cell Signaling Technology, Inc., Danvers, MA, USA), QKI 5 (cat. no. BL 1041; Bethyl Laboratories, Inc., Montgomery, TX, USA), FOXO1 (L27; cat. no. CST 9454; Cell Signaling Technology, Inc.), PPARα (467D1a; cat. no. sc-130640; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) with dilution 1:1,000, and β-actin (13E5; cat. no. CST 4970S; 1:4,000; Cell Signaling Technology, Inc.). The secondary antibody was horseradish peroxidase-conjugated secondary antibody (cat. no. CST 4410S; Cell Signaling Technology, Inc.).

Mouse primary hepatocytes were seeded into a 12-well cell culture plate, following treatment with SRT1720 for 48 h as described above. The supernatant was discarded, then 3×10⁵ cells were collected and lysed. The cell lysate was transferred into a 1.5-ml centrifuge tube and a sample was obtained to be analyzed. The standard protein concentration gradient was performed according to the kit protocol. A total of 50 µl of gradient standard and 10 µl of the sample were added into the 96-well microtiter plate. Following the addition of lipase into the main reaction system, the absorbance at 570 nm was measured with a microplate reader, and the absorbance of the sample was calculated according to the standard curve. The concentration of TG was normalized by the quantity of protein per unit (µg).

The local tool of KA-predictor, which can be freely downloaded (http://sourceforge.net/p/ka-predictor), was used to predict the acetylation modification site of QKI 5.

The cells/tissues were fully lysed with IP lysate and then centrifuged at 6,720 × g for 10 min at 4°C to remove the precipitate. The protein (1 mg) solution was added to 20 µl agarose cross-linked acetylated lysine antibody (acetyl lysine antibody, agarose) (cat. no. CST 9441S; Cell Signaling Technology, Inc.; dilution, 1:800). Following mixing and incubation overnight at 4°C, the supernatant was discarded. The sediment was washed five times with ice pre-cooled PBS, following which 50 µl of elution buffer was used to elute the proteins which were mixed gently. Following centrifugation at 336 × g for 1 min at 4°C, the IP product was assessed using the aforementioned western blotting protocol. The primary antibody of QKI 5 (cat. no. BL 1041; Bethyl Laboratories, Inc.; dilution, 1:500) was used to detect QKI 5 content in the IP products.

The differences between two groups were statistical analyzed using Student's t-test. Normally distributed continuous variables are presented as the mean ± standard deviation. Abnormally distributed data among groups was analyzed with the Kruskal-Wallis one-way analysis of variance method. SPSS (version 18.0) software (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

The mice fed the HFD became obese at 8 months of age. The BW (Fig. 1A) and the relative fat mass ratio (Fg. 1B) of the mice in the model group and vehicle group increased significantly, but were only enhanced ~20% in the mice treated with SRT1720. Furthermore, the model and vehicle mice developed hyperlipidemia, as measured by total plasma TG (Fig. 1C) and TC levels (Fg. 1D). Furthermore, these changes were reversed by SRT1720. Consistent with an increase in plasma lipids, the HFD model mice exhibited significantly enlarged fatty livers at 8 months of age (Fig. 1E and 1F), which was reduced by SRT1720.

The hepatic lipid content of the HFD model mice at 8 months of age was significantly increased compared with their litter-mate controls, as separately measured using H&E and Oil Red O staining of liver sections (Fig. 2A), and quantification of Oil Red O staining (Fig. 2B). These results indicated the typical pathology of liver steatosis. Furthermore, the TG (Fig. 2C) and TC (Fig. 2D) contents of the extracted liver tissues from the HFD model were significantly increased compared with those of the controls, but were reduced by SRT1720. This demonstrated that the developmental process of liver steatosis was reversed by SRT1720 (Fig. 2).

In addition, mildly increased plasma activities of ALT (Fig. 3A) and AST (Fig. 3B) were identified in the model mice. However, the mice treated with SRT1720 demonstrated no signs of liver damage or inflammatory changes in plasma ALT or AST activity. The serum level of SIRT1 in the model mice was significantly decreased compared with that in the control group, however, this was reversed by SRT1720 (Fig. 3C). Furthermore, the NAS was elevated in the model mice; the results revealed significant differences in the model and vehicle groups compared with the control group mice. The NAS of the group treated with SRT1720 was reduced compared with that in the model and vehicle groups (Fig. 3D), however, the NAS score of the SRT1720 group was increased compared with that in the control mice. These results suggested that the administration of SRT1720 improved the pathophysiological process of NAFLD in mice.

An inhibitor of FOXO1 was also used in the present study. The results, as shown in Fig. 4, demonstrated that the inhibitor of FOXO1 (AS1842856) suppressed FOXO1 and PPARα, however, this was reversed by SRT1720. AS1842856 did not inhibit the expression levels of SIRT1 or QKI 5. This demonstrated that hepatic SIRT1 regulated the expression of QKI 5 via the PPARα/FoxO1 signaling pathway.

In the liver tissues of the model mice, the acetylation level of QKI 5 increased, and this was reversed by SRT1720 (Fig. 5A). An increase in the acetylation level of QKI 5 was induced by the inhibitor of SIRT1 (niacinamide), whereas the acetylation of QKI 5 decreased in hepatocytes treated with SRT1720 (Fig. 5B). Furthermore, the increased acetylation level of QKI 5 was induced by siRNA of SIRT1, whereas the acetylation of QKI 5 decreased in hepatocytes treated with Ad-SIRT1 (Fig. 5C). The results of the protein interaction analysis confirmed the interaction between SIRT1 and QKI 5, and its level in the SRT1720 group was higher than in the model mice (Fig. 5D and E).

To evaluate the ability of hepatic SIRT1 to maintain lipid homeostasis, the agonist (SRT1720) and inhibitor (niacinamide) of SIRT1 was used in the present study. Furthermore, Ad-mediated gene repletion of SIRT1 was used in mice primary hepatocytes, and siRNA of SIRT1 was used to downregulate the expression of SIRT1.

In the primary hepatocytes, SRT1720 enhanced the expression of SIRT1, and the expression levels of QKI 5, FOXO1 and PPARα were increased. However, the expression level of SIRT1 was reduced by niacinamide, which also induced the downregulation of QKI 5, FOXO1 and PPARα (Fig. 6A). In addition, a decrease in intracellular TG content was induced by SRT1720, whereas niacinamide increased the content of TG in the primary hepatocytes (Fig. 6B). Additionally, in the primary hepatocytes, Ad-SIRT1 enhanced the expression of SIRT1 QKI 5, FOXO1 and PPARα, and this was inhibited by SIRT1 siRNA (Fig. 6C). SRT1720 reduced the TG content of the primary hepatocytes, which was promoted by SIRT1 siRNA (Fig. 6D). Therefore, SIRT1 mediated the synthesis of TGs in NAFLD mice, and this was associated with the QKI 5 and the PPARα/FoxO1 signaling pathway.

The results of the immunohistochemistry assay indicated that, the expression levels of SIRT1 and QKI 5 were downregulated in the NAFLD model mice, but were promoted by SRT1720 (Fig. 7A). In addition, the western blot assay demonstrated that the expression levels of SIRT1, QKI 5, FOXO1 and PPARα were decreased in the model mice, and this was also reversed by SRT1720 (Fig. 7B).