The synthesis of O^2',O^3',O^5'-tri-acetyl-N₆-(3-hydroxylaniline)adenosine, named as WS070117 (Figure 1A), began with acetylation of inosine using commercially available acetic anhydride, chlorination with SOCl₂, and substitution with 3-aminophenol. Briefly, acetic anhydride was added to a suspension of inosine in dry pyridine at 0°C and stirred at room temperature for 6 h to yield the pure product O^2',O^3',O^5'-tri-acetylinosine (Compound 1). Compound 1 was then dissolved in dry CH₂Cl₂:DMF (50:1) and a solution of SOCl₂ in CH₂Cl₂ was added in a drop-wise manner. After dilution with CH₂Cl₂, the organic layer of the reaction mixture was washed with saturated NaHCO₃ solution and brine and dried with anhydrous sodium sulfate. To a solution of O^2',O ^3',O^5'-triacetyl-6-chloroadenosine in absolute ethanol was added 3-aminophenol and dry triethylamine. The mixture was then refluxed for 8 h at 60°C. The solution was concentrated in vacuum and the residue was chromatographed (ethyl acetate:petroleum ether, 2:1) to give WS070117 as a white solid.

Golden hamsters (12-month-old) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). All animal treatment procedures described in this study were approved by the Animal Care and Use Committee at the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). Animals were housed under well-controlled conditions of temperature (22 ± 2°C), humidity (55 ± 5%) and a 12 h light-dark cycle with access to food and water ad libitum.

Except the control hamsters (n = 5), other hamsters (n = 40) were fed high fat diet (HFD) accustomedly for 2 weeks, blood was collected from orbit vein for assaying the content of serum total cholesterol and triglyceride for grouping. Then, The HFD fed hamsters were divided into the following groups with eight animals per group respectively, accounted into their body weight and the levels of serum total cholesterol and triglyceride, HFD model group, HFD+Simvastatin (2 mg/kg) as a positive group, HFD+WS070117 low dose group (2 mg/kg), HFD+WS070117 middle dose group (6 mg/kg) and HFD+WS070117 high dose group (18 mg/kg). The animals in normal control group were fed ordinary diet, and the other groups were still fed HFD ad libitum for 8 weeks with the indicated drugs oral administration. WS070117 was dissolved in distilled water prior to administration. All drugs were given once daily by oral gavage for 8 weeks. The animals in the normal control and in the HFD model control group received the same volum of distilled water as that of the drug-treated groups.

The hepatic cell line HepG2 was purchased from the Cell Resource Center(IBMS, CAMS/PUMC, Beijing China) was grown in Dulbecco's modified Eagle's medium (DMEM, GIBCO, NY, USA) supplemented with 10% fetal bovine serum (FBS, MDgenics, St. Louis, MO, USA) and 1% penicillin-streptomycin under a humidified atmosphere of 5% CO₂ in air. After growth to 70% to 80% confluence, cells were rendered quiescent by incubating with serum-free medium for 12 h before fatty acid treatment. For OLA-induced lipid deposition, the medium for cells at 75% confluence was exchanged for new medium with or without 0.25 mmol/L OLA (Sigma, St. Louis, MO, USA) [⁸]. Meanwhile, different concentration of WS070117 was added into medium and treated with OLA for 12h.

Blood samples were collected from the retro-orbital plexus 1 h after drug treatments. Serum was isolated at room temperature. Standard enzymatic methods were used to determine TC, TAG, LDL-c and high density lipoprotein cholesterol (HDL-C) levels with commercially available kits purchased from Bio Sino Bio-technology and Science Inc. (Beijing, China). Each sample was assayed in duplicate.

100 mg of hamster liver tissue was taken for lipid extraction. Briefly, the frozen tissue was thawed and homogenized with a motor driven homogenizer in 2 ml chloroform/methanol (2:1). After homogenization, lipids were further extracted by rocking samples for 1 h at room temperature, followed by centrifugation at 5,000 rpm for 10 min. The liquid phase was washed with 0.2 volume of 0.9% saline. The mixture was centrifuged again at 2,000 rpm for 5 min to separate the two phases. The lower phase containing lipids was evaporated and lipids were dissolved in 0.5 ml isopropanol containing 10% Triton X-100 for TAG and TC measurements.

For histological examination, pieces of liver tissues were fixed in 4% paraformaldehyde and dehydrated in 30% sucrose solution at room temperature. Tissues were then immersed in Optimal Cutting Temperature (OCT) solution on dry ice for Oil Red O staining. Three hamster livers were randomly picked from each group and three different tissue sections from each liver were processed and stained using routine laboratory procedures. For fat accumulation examination in HepG2 cells, culture dishes were washed with cold phosphate-buffered saline and fixed in 4% paraformaldehyde. After 2 changes of 60% isopropanol, Oil-Red-O was added with agitation for 10 minutes, followed by washing in 50% isopropanol. For each dish, 3 images were photographed, and a representative image is shown.

AMPK activity assays were performed on lysates of HepG2 cells cultured in 6-well plates with 10% FBS/DMEM supplemented with 1,6,12 hours time course (10 μM) and 1,10,100 μM dosage (12 hours) of WS070117 by measurement of [γ-³²P]ATP (Perkin-Elmer, Boston, MA, USA) radioactivity phosphorylated by a synthetic peptide substrate, SAMS [⁹]. AMPK activity was expressed as nmol of phosphate incorporated into the peptide substrate per min per mg of lysate (nmol/min/mg).

OLA HepG2 cells was co-incubated with 0.1,1 and 10 μM WS070117 for 12h. Incorporation of [1^-14C] acetic acid (Perkin-Elmer, Boston, MA, USA) into lipid was performed as described previously [¹⁰]. After replacement with 1 ml of fresh medium, the cellular lipid was labeled by incubating for 2.5 h with [1^-14C] acetic acid (0.2 μCi/ml). Lipid was extracted with n-hexane:2-propanol (3:2, v/v) and dried under a nitrogen stream. Aliquots of lipid samples were loaded onto thin-layer chromatography plates and chromatographed in a solvent mixture containing hexane-diethylether-acetic acid (85:30:1, v/v/v). Bands corresponding to triglyceride and cholesteryl ester were scraped off, immersed in non-aqueous scintillation fluid, and counted for radioactivity.

The expression and phosphorylation of each protein were analyzed by Western blot analysis. Briefly, protein lysates were subjected to 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a nitrocellulose membrane and the membranes were blocked for 1 h at room temperature with 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20. Immunostaining to detect each protein was achieved after over-night incubation (4°C) with a 1:1000 dilution of anti-phospho (thr-172) AMPK (1:2000), AMPK (1:2000) and anti-beta-actin (1:5000). Proteins were visualized after subsequent incubation with a 1:5000 dilution of anti-mouse or rabbit IgG conjugated to horseradish peroxidase and use of a Super Signal Chemical luminescence detection procedure. All antibodies used in this study were from Cell Signaling Technology Inc. (Beverly, MA, USA). Protein concentrations were determined using a bicinchoninic acid assay (Applygen Inc., Beijing China). Three independent experiments were performed for each condition and the intensity of immune-reactive bands was analyzed with a Bio-Rad calibrated densitometer.

One-way ANOVA was used to determine significant differences among groups, after which the modified Student's t-test with the Bonferroni correction was used for comparison between individual groups. P < 0.05 was considered statistically significant.

AMPK has been proposed to act as a fuel gauge in mammalian cells and to play a crucial role in regulating fat metabolism in the liver. The major pathways that regulate intracellular lipid metabolism are evidently present in HepG2 cells. The change of AMPK activity in HepG2 cells is strongly associated with intracellular lipid metabolism. Because WS070117 is an adenosine analogue, we studied the possibility that WS070117 could activate AMPK. Incubation of HepG2 cells with 1 ~ 100 μM WS070117 for 12h significantly activated AMPK, as measured in cell lysates. WS070117 also activated AMPK in a time dependently manner within 12 hours treatment. AICAR, one of the potent AMPK activators, also increased AMPK activity (Figure 1B, C).

HFD with obese hamsters fed a diet containing 20% fat was used for producing the lipid metabolism dyslipidemia model. After 8 weeks, hamster body weights increased about 20% with no difference among the HFD treated groups. The liver/body weight ratio was markedly increased at the end of the experiment. The increases in the groups treated with WS070117 were significantly decreased the liver hypertrophy as compared with the HFD group (Table 1). WS070117 (2, 6 or 18 mg/kg/day) treatment resulted in significant decreases in serum TC, TAG and LDL-c as well as hepatic TAG and TC (Figure 2B).

Oil Red O staining of livers showed that the hepatocytes of animals in the HFD group were compressed and separated by bulks of fat, demonstrating the abnormally high levels of fat accumulation in liver (Figure 2A). 8 weeks treatment with 18 mg/kg WS070117 substantially repressed these changes in HFD hamsters. Therefore, WS070117 helped to keep normal fat accumulation and deposition under HFD feeding conditions.

Hepatic steatosis in humans is associated with accumulation of excess OLA and the end-product of de novo fatty acid synthesis [⁴]. AMPK-mediated phosphorylation of protein targets involved in cholesterol and fatty acids synthesis, however, lead to inhibition of cholesterol and TAG synthesis. In present study, Oil Red O staining showed that 10 μM WS070117 decreased the lipid accumulation in HepG2 cells induced by 250 μM OLA (Figure 3A). We then evaluated the impact of WS070117 on TAG and cholesterol synthesis by incubating HepG2 cells for 2 h with [¹⁴C]acetate in the presence of increasing concentrations of WS070117 (Figure 3B). Both cholesterol and TAG synthesis were dose-dependently inhibited by WS070117 (0.1 ~ 10 μM).

AMPK activation is thought to be a key proximal event in the cellular energy balance response, and AMPK phosphorylation levels are currently accepted as a marker of AMPK activity. Therefore, we determined the phosphorylation of AMPK in OLA overload HepG2 cells. After 12h treatment of WS070117, AMPK phosphorylation is activated at 0.1 10 and 1 μM. In HFD-fed hamster livers, AMPK phosphorylation was also increased by treatment with 6 or 18 mg/kg WS070117 (Figure 4).

Serum was separated after 8 weeks of WS070117 and Simvastatin treatment at the indicated doses from hamsters fed a high fat diet (HFD). The actual values of mean ± SD from 8 animals (control group, n = 5) are presented. ^##P < 0.01, ^#P < 0.05, as compared to vehicle control; ** P < 0.01, *P< 0.05 as compared to HFD model.