Twenty-four-week-old, male type 2 diabetic model OLETF rats were kindly provided by Otsuka Pharmaceuticals (Tokushima, Japan). The rats were individually housed and allowed free access to food and tap water under strictly controlled and pathogen-free conditions (room temperature, 23°C ± 1°C; relative humidity, 50% ± 10%; light cycle, 7:00 AM to 7:00 PM). The rats were fed a standard rodent pellet chow and acclimatized to their environment for 2 weeks prior to the commencement of the experiments. Thirty-two rats were randomly assigned to four subgroups and then treated daily with CK, metformin, or vehicle for 12 weeks: (1) control (n = 6) received vehicle (saline); (2) CK 10 mg/kg (CK10; n = 10); (3) CK 25 mg/kg (CK25; n = 10); and (4) metformin 300 mg/kg (n = 6) as a positive control. CK in the form of a dried powder was dissolved in saline and was orally administered by gavage daily. All treatments were orally administered. To induce hepatic steatosis, all rats were fed 60% HFD (Joongang Animal Laboratory, Seoul, Korea). The experimental procedures were conducted according to the animal care guidelines of the United States National Institutes of Health (NIH) and approved by the Animal Care and Use Committee of Kyung Hee University (IACUC approval No.2012-0002). The use of animals in the study was approved by the institution’s Animal Ethics Committee in accordance with Article 14 of the Korean Animal Protection Law or its equivalent. The use of animals in the study complied with Article 13 of the Korean Animal Protection Law (the principles for animal use) and relevant institutional policies.

The body weight of each rat was measured at the beginning of the experiment and every week thereafter. Food intake was determined by measuring the difference between the pre-weighed chows and the weight of the food that remained every 24 hours during the first and last week of the 3-month diet period. Blood samples were collected from the tail vein every week of treatment and the serum was stored at –20°C until analysis. The fasting blood glucose level was assessed using an Accu-Chek active glucometer (Roche, Basel, Switzerland). After the 12- week treatment, whole blood was collected by inferior vena cava puncture from all rats. Each blood sample was held at room temperature for 1 hour. Serum was then collected by centrifugation at 4,000 rpm for 10 minutes and stored at –70°C until analysis. Glycated hemoglobin (HbA1c) levels were measured using a DCA Vantage Analyzer (Siemens, Tarrytown, NY, USA). Serum insulin was assayed by enzyme-linked immunosorbent assay (Millipore, Billerica, MA, USA). The oral glucose tolerance test (OGTT) was performed with rats that were fasted for 16 hours at 0 weeks and at 12 weeks. Glucose (2 g/kg body weight) was orally administered to the rats and blood samples were collected 0, 30, 60, 90, and 120 minutes after glucose loading.

The livers were obtained from each rat after perfusion of phosphate buffered saline (PBS). They were then fixed in 10% buffered formalin, embedded in paraffin, stained with hematoxylin and eosin, and blindly scored for hepatic lipid accumulation by microscopy examination by the same pathologist. The score of each component of NAFLD (steatosis [0–3], lobular inflammation [0–3], ballooning [0–2], and fibrosis [0, 1a, 1b, 1c, 2, 3]) was quantitatively evaluated according to the standard criteria of grading and staging for NASH [8].

Hepatic tissues were isolated after pump-mediated perfusion of PBS, and whole cell lysates were prepared. Protein concentrations were determined and an equal amount of each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Following the transfer, the membranes were reversibly stained with a Ponceau S solution (Amresco, Solon, OH, USA) to confirm sample loading and gel transfer equivalence. After blocking with 5% nonfat milk for 1 hour, the membranes were individually incubated with AMPK antibodies, followed by antibodies to phospho-AMPK (1:1,000; Cell Signaling, Danvers, MA, USA), SREBP-1c (1:1,000; BD Biosciences, San Jose, CA, USA), fatty acid synthase (FAS; 1:1,000; Cell Signaling), PPAR-α (1:1,000; Cell Signaling), CPT-1 (1:1,000; Cell Signaling), and actin (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. This was followed by incubation with goat-anti-rabbit immunoglobulin G secondary antibodies (1:2,000; Santa Cruz Biotechnology) for 1 hour. Antibody binding was detected by enhanced chemiluminescence (ECL, Pierce, Thermo Fisher Scientific, San Jose, CA, USA). X-ray film (Kodak, Rochester, NY, USA) was used to visualize the bands. Densitometry quantification values were measured by the Image J program from NIH and expressed as fold-differences from the control at each time point.

Total RNA was isolated from liver samples after pump-mediated perfusion with 5% PBS. The method was based on the phenol/guanidine isothiocyanate RNA zol B protocol (Cinna/Biotecx, Houston, TX, USA). Total RNA (1 μg) was reverse transcribed using MMLV (Moloney Murine Leukemia Virus) reverse transcriptase (Gibco, Grand Island, NJ, USA) with random hexamer priming. For semi-quantitative PCR, aliquots of cDNA were amplified in a 20-μL PCR mixture according to the protocol provided by the manufacturer (TaKaRa Bio, Kyoto, Japan). PCR primer sequences for genes regulating fatty acid synthesis and fatty acid oxidation are shown in Table 1. The primers were synthesized by Bioneer (Seoul, Korea). PCR products were separated by electrophoresis on 1.5% agarose gels containing ethidium bromide, and the bands were visualized under ultraviolet light. For real-time RT-PCR, the resultant cDNA was amplified using a LightCycler (Roche Diagnostics, Lewes, UK). Real-time PCR was carried out with SYBR Green I (Roche Diagnostics). PCR reactions with SYBR Green I and primers were performed in a 20 μL volume with 2 μL cDNA, 0.5 μM each primer, and 4 mM MgCl₂. Taq polymerase, PCR buffer, deoxynucleotides, and SYBR Green I dye were included in the LightCycler-Fast Start DNA Master SYBR Green I mix (Roche Diagnostics). The thermal cycling profile consisted of a pre-incubation step at 95ºC for 10 minutes followed by 40 or 50 cycles of a 95ºC denaturation step for 10 seconds, 59ºC annealing for 5 seconds, and a 72ºC extension step for 20 seconds. After each extension step, the temperature was raised to 88ºC to measure SYBR Green I fluorescence at a temperature 2°C below the product Tm and 2°C above the Tm of the primers-dimers. At the end of the PCR, melting curve analysis was performed by gradually increasing the temperature from 65°C to 95°C at a rate of 0.1oC/second to confirm the amplification specificity of the PCR products. The level of expression of each mRNA and their estimated crossing points (Cp) for each sample were determined relative to the standard preparation using LightCycler computer software (version 3.5). PCR amplification was performed with a series of standards prepared by successive dilutions and a linear standard curve was automatically generated. A standard curve was constructed for each PCR run. All samples to be compared were run in the same assay.

The results are presented as the mean ± SD. The effect of CK on each parameter was examined by one-way analysis of variance. Prism software version 5 (Graphpad Software, San Diego, CA, USA) was used for statistical analysis and graphing. A p < 0.05 was considered statically significant.

HFD-fed OLETF rats were treated for 12 weeks with saline (control), CK10, CK25, or metformin (as a positive control). The amount of food intake and the body weight were not significantly different among the four groups (Fig. 1A and 1B). Fasting plasma glucose levels of the CK25 and metformin groups were significantly lower than those of the control group during the 12-week study period (Fig. 1C). HbA1c was not significantly different among the four groups (Fig. 1D). Concerning OGTT, the glucose concentrations after glucose loading in the CK25 group were significantly lower than those of the control group (Fig. 2A). When the area under the curve (AUC) of glucose in the OGTT was analyzed, the AUC of the CK25 group was found to be significantly lower than that of the control group and decreased to the level of the metformin group (Fig. 2B). Fasting insulin levels of the CK25 and metformin groups were significantly lower than those of the control group were (Fig. 2C).

To demonstrate the effect of CK on hepatic steatosis, lipid accumulation was measured in HFD-fed OLETF rats. Histological examination showed that hepatocytes of HFD-fed rats were distended by large cytoplasmic lipid droplets. The quantitative grade of hepatosteatosis was significantly lower in the CK25 group than in the control group (1.5 ± 0.82 vs. 2.7 ± 0.23, p < 0.05). Advanced fibrosis was not found in all groups. This change in cellular morphology was significantly improved by CK25 treatment (Fig. 3).

To study the effect of CK on the phosphorylation of AMPK, which is a key regulator of glucose and lipid metabolism, the expression of phospho-AMPK in the liver of HFD-fed OLETF rats was examined by western blotting and compared among four groups. CK25 and metformin significantly increased hepatic AMPK phosphorylation (Fig. 4).

To investigate the mechanism by which CK improved hepatic steatosis in HFD-fed OLETF rats, the expression of genes related to fatty acid synthesis and β-oxidation was evaluated by real-time PCR and Western blotting. CK25 significantly inhibited the expression of SREBP-1c and FAS, which are involved in lipogenesis (Fig. 5). CK25 stimulated the mRNA expression of CPT-1, a rate-limiting enzyme for fatty acid oxidation (Fig. 5A). The mRNA expression of PPAR-α, a transcriptional factor that regulates fatty acid oxidation, was significantly increased by CK25 (Fig. 5A). In addition, western blotting showed that the expression of CPT-1 and PPAR-α in the CK25 and metformin groups were significantly higher than those of control groups (Fig. 5B).