The animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, China). A total of 40 healthy male Sprague-Dawley (SD) rats (age, 3 weeks; weight, 40–60 g) were purchased from the Experimental Animal Centre of Sichuan University and raised in the Experimental Animal Centre of West China Hospital, Sichuan University. All rats were caged (n=4 per cage) under standard conditions with controlled humidity (45 to 65%) and temperature (20 to 25°C), 12/12 h light/dark cycles and access to food and water ad libitum. Rats were maintained for 7 days on a standard diet before they were divided into the normal diet group (320 kcal/100 g body weight, 4.65% calories derived from fat; n=12) and the HFD group (500 kcal/100 g body weight, 60% calories derived from fat; n=28). The feed for these diets were purchased from Trophic Animal Feed High-Tech Co., Ltd. (Jiangsu, China). Body weight, body length and tail length were measured every week for 24 weeks, and Lee's index was calculated using the following formula: [Body weight (g)^1/3 × 1,000]/body length (cm) (12). Following 24 weeks, rats with a mean body weight that reached ≥1.4-fold higher than that of controls were selected for further experiments. A total of 24 eligible rats were separated from the HFD group at random and divided into the HFD-control group (n=12) and the octreotide-treated group (n=12). These groups were continuously fed a HFD for 8 days, and rats in the octreotide-treated group were subcutaneously injected with octreotide (Chengdu Tiantai Mountain Pharmaceutical Co., Ltd., Chengdu, China) at a dose of 40 µg/kg body weight every 12 h for 8 days. During the octreotide administration period, body weight and food intake were monitored daily. At the end of the experiment, all rats underwent a 12-h starvation period and were sacrificed with 2% sodium pentobarbital (45 mg/kg body weight; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) administered intraperitoneally. Following sacrifice, blood samples were collected and centrifuged at 860 × g for 15 min at 4°C, and the supernatant was stored at −80°C for further analysis. The liver tissues were isolated, and one sample was rapidly frozen in liquid nitrogen prior to storage at −80°C for total RNA and protein extraction or Oil Red O staining. A second sample was fixed in 4% paraformaldehyde for 48 h at room temperature for histological examination. Abdominal fat was also collected and weighed.

Following fasting for 12 h, rats were subjected to the ipGTT and ipITT assays by intraperitoneal administration of glucose at 2.0 g/kg body weight or insulin at 7.5 U/kg body weight, respectively. Blood samples were drawn from the tail vein at 0, 15, 30, 60 and 120 min post-injection, and the glucose levels were measured using an Accu-Chek Active Glucometer (ACCU-CHEK Active; Roche Applied Science, Penzberg, Germany).

GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) was used to calculate the area under the curve (AUC) of ipGTT and ipITT, and the results are expressed as the mean ± standard deviation.

Fasting plasma glucose (FPG), serum triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined using the following reagent kits according to the manufacturer's protocol: GLU assay kit (cat. no. A031), TG assay kit (cat. no. A027), T-CHO assay kit (cat. no. A030), GPT assay kit (cat. no. A001) and a GOT assay kit (cat. no. A002), obtained from Changchun Huili Biotechnology Co., Ltd., Changchun, China. FFA levels were determined using a Non-Esterified Free Fatty Acids assay kit (cat. no. FA115; Randox Laboratories, Ltd., Crumlin, UK) and serum insulin levels were detected using a rat insulin enzyme-linked immunosorbent assay kit (cat. no. EZRMI-13K; EMD Millipore, Billerica, MA, USA), according to the manufacturer's instructions. The homeostatic model assessment (HOMA) index, which is an assessment of insulin sensitivity, was calculated to assess the insulin sensitivity of obese rats using the following equation: [FPG (mmol/l) × fasting serum insulin (µU/ml)]/22.5.

To assess hepatic TG levels, ~50 mg liver tissue from rats in all experimental groups was homogenized in a 2:1 chloroform-methanol mixture (v/v). The homogenized solution was mixed on a shaker for 20 min at room temperature, centrifuged at 310 × g for 10 min at 4°C, and the supernatant was collected. A total of 0.2x the supernatant volume of NaCl solution (0.9%) was added to the supernatant. The mixture was then centrifuged at 550 × g for 20 min at 4°C, and the resulting supernatant was collected and dried under a stream of nitrogen gas. To the residue, 0.5 ml Triton X-100 solution (3%) was added to re-dissolve the residue. The TG level of the 0.5 ml Triton X-100 solution was measured using a TG assay kit (cat. no. A027; Changchun Huili Biotechnology Co., Ltd.).

In order to determine hepatic FFA levels, ~50 mg liver tissue was homogenized in phosphate-buffered saline (PBS), centrifuged at 860 × g for 20 min at 4°C and the supernatant was collected. The protein concentrations of the supernatant were measured using a bicinchoninic (BCA) Protein assay kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the hepatic FFA concentration was detected using a Nonesterified Free Fatty Acid assay kit (cat. no. FA115; Randox Laboratories, Ltd.) according to the manufacturer's instructions. The FFA values are presented as mM/g protein.

In order to assess hepatic glycogen content, ~50 mg liver tissue was homogenized in 500 µl ice-cold PBS. The homogenates were centrifuged at 14,000 × g for 10 min at 4°C, and the supernatants were collected for analysis using glycogen content assays. The glycogen concentration in the liver was evaluated using an EnzyChrom™ Glycogen assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer's instructions. Glycogen concentration was normalized to the protein concentration of liver tissues.

Formalin-fixed liver tissues were embedded with paraffin and sectioned (3-µm). Sections subsequently underwent xylene gradient dewaxing twice for 10 min and gradient dehydration with 100% alcohol for 3 min, 100% alcohol for 3 min, 95% alcohol for 3 min and 90% alcohol for 3 min. Sections were rinsed with tap water three times and immersed in tap water for 15 min. Sections were then stained with 0.2% hematoxylin and eosin (H&E) for ~5 min at room temperature. White fat vacuoles and steatosis were visualized using a light microscope (Olympus BX41TF Microscope; Olympus Corporation, Tokyo, Japan), and images were captured at ×400 magnification. Evaluation of NAFLD was performed described previously (13).

Frozen liver tissue was embedded with Tissue-Tek O.C.T. Compound (Sakura Finetek USA, Inc., Torrance, CA, USA), and cryosections (5-µm in thickness) of liver tissue were prepared. The sections were then stained for 30 min in 0.3% freshly diluted Oil Red O solution at 37°C, followed by counterstaining with 0.2% H&E at 37°C for 30 sec following washing with tap water. Images of the sections were collected using a light microscope (Olympus Corporation) from five different fields at ×400 magnification. Image Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA) was used to analyze the integrated optical density (IOD) of the Oil Red O-stained areas. Quantitative analysis of liver tissue photomicrographs was performed as described previously (14).

The human hepatoblastoma HepG2 cell line, which was originally thought to be derived from a hepatocellular carcinoma, however was later determined to be derived from a human hepatoblastoma (15), was obtained from CoBioer Biosciences Co., Ltd. (Nanjing, China). HepG2 cells were cultured in RPMI-1640 medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (Biological Industries Israel, Beit Haemek Ltd., Beit Haemek, Israel), 100 U/ml penicillin and 100 µg/ml streptomycin (Hyclone; GE Healthcare Life Sciences). The cells were maintained at 37°C under humidified conditions with 95% O₂ and 5% CO₂. When cell confluence had reached 80%, cells were starved of glucose overnight in serum-free or 0.5% low-concentration fetal bovine serum (Biological Industries Israel, Beit Haemek Ltd., Beit Haemek, Israel) RPMI-1640 medium and then divided into the following 3 groups: The standard control group, where cells were incubated with 0.5% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) for 24 h; the palmitate (PA)-treated group, where cells were cultured with 125 µM PA (Sigma-Aldrich; Merck KGaA) plus 0.5% BSA for 24 h; and the PA plus octreotide-treated group, where cells were cultured with 125 µM PA plus 0.5% BSA for 24 h prior to treatment with 10⁻⁸ mmol/l octreotide for 6 h. Subsequently, cells in all groups were incubated with 100 µM insulin for 20 min prior to cell collection. A KeyGEN Whole Cell assay kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used to isolate total protein. TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China) was used to extract the total mRNA, according to the manufacturer's protocol.

Cells (1×10⁶) were washed in PBS three times and then fixed for 30 min with 4% formaldehyde solution at room temperature. Following two washes with 60% isopropyl alcohol, the cells were stained with a working solution of 0.3% Oil Red O for 30 min at 37°C. The reagent was then removed, cells were washed several times with tap water and cell nuclei were counterstained with 0.2% hematoxylin for ~20 sec at room temperature. Photomicrographs of stained cells were captured using a light microscope (Olympus Corporation) at ×400 magnification and the results were analyzed using the aforementioned methods.

TRIzol reagent was used to extract total RNA from liver tissues or HepG2 cells, according to the manufacturer's instructions. RNA (2 µg) was reverse transcribed to cDNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. qPCR was performed using 2X SYBR-Green Master Mix (Biotool, LLC, Houston, TX, USA) and the primers listed in Table I (TSINGKE Biological Technology, Co., Ltd., Beijing, China). Samples were analyzed in triplicate and the thermal cycling parameters were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) was used for qPCR analysis. Target gene mRNA expression levels were normalized to β-actin mRNA in the same sample. The relative method of quantification was applied to calculate ΔΔC_q values in each sample, and the results are expressed as 2^−ΔΔCq (16).

Total protein was isolated from liver tissues or HepG2 cells using a KeyGEN Whole Cell assay kit (Nanjing KeyGen Biotech Co., Ltd.), and the Enhanced BCA Protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to measure protein concentrations, according to the manufacturer's instructions. Protein samples (40 µg) from each group were electrophoresed on 10% SDS-PAGE gels and then blotted onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked with 5% non-fat milk in Tris-buffered saline-Tween-20 (TBST; 0.1%) at room temperature for 2 h, prior to incubation with anti-phosphorylated (p)-Akt rabbit monoclonal antibody (mAb; cat. no. 4060; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p-GSK3β rabbit mAb (cat. no. 9323; dilution, 1:1,000; Cell Signaling Technology, Inc.), anti-Akt rabbit polyclonal antibody (cat. no. STJ91543; dilution, 1:500; St. John's Laboratory Ltd., London, UK), anti-GSK3β rabbit polyclonal antibody (cat. no. STJ93448; dilution, 1:500; St John's Laboratory Ltd.), anti-β-actin mouse mAb (cat. no. EM21002; dilution, 1:5,000; Epitomics, Burlingame, CA, USA) at 4°C overnight. This was followed by incubation with a homologous horseradish peroxidase-conjugated secondary antibody (cat. no. ZB2301; dilution, 1:10,000; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 2 h at room temperature following washing with TBST. Protein bands were immunodetected using enhanced chemiluminescence reagent (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), according to the manufacturer's instructions. The intensity of the bands was analyzed using Quantity One software (version 4.6.2; Bio-Rad Laboratories). The expression levels of all proteins in each sample were normalized to β-actin levels, and the experiment was repeated three times.

Data were analyzed using SPSS software (version 20.0; IBM SPSS, Armonk, NY, USA) and the results are presented as the mean ± standard deviation. One-way analysis of variance followed by the Dunnett's test was performed to evaluate the significant differences among groups. The homogeneity of variances (Levene's test) was necessary, if there is heterogeneity of variance, that is P<0.05, then the method of the variable conversion such as Lg10 is required to meet the homogeneity of variance (P>0.05). P<0.05 was considered to indicate statistically significant differences.

At the end of the experiment, the body weight of rats in the HFD group had increased by 28.5% when compared with the control group (P<0.01; Table II). Lee's index, which provides an estimate of obesity in adult rats, was also increased by 5.0% in the HFD group when compared with the control group (P<0.01; Table II). The liver weight and quantity of abdominal fat in obese rats was higher than that of the control rats (both P<0.01; Table II), and the abdominal fat index in the obese rats was higher in the HFD group when compared with the control group (P<0.01; Table II). Apart from Lee's index values, octreotide treatment significantly reduced these parameters in rats fed on a HFD (Table II).

When compared with the control group, plasma glucose levels in rats from the HFD group were significantly elevated (P<0.01; Table III). Following administration of octreotide, the plasma glucose levels were significantly lower compared with those observed in the obese rats of the HFD group (P<0.01; Table III). Rats in the HFD group exhibited the highest serum insulin, TG, TC, FFA, ALT and AST levels among the three groups (Table III). Octreotide intervention significantly decreased the serum insulin concentration (P<0.05); however, there was no marked reduction in serum TG, TC, FFA, ALT and AST levels (P>0.05). The HOMA index, which provides an estimation of insulin resistance, significantly increased in rats from the HFD group when compared with the control rats (P<0.01), and was significantly inhibited by octreotide treatment (P<0.01; Table III).

As shown in Fig. 1A, the blood glucose concentration of rats in the HFD group at 0, 15, 30, 60 and 120 min was higher than that observed in the control rats, and the relative AUC increased by 27.7% (P<0.05; Fig. 1B). Similarly, glucose levels in rats from the HFD group was higher when compared with the control group following the ipITT test (Fig. 1C), and the AUC of ipITT in the HFD group increased by 28.8% when compared with the control group (P<0.05; Fig. 1D). Compared with the HFD group, the ipGTT and ipITT AUCs decreased by 5.2 and 10.5% in the octreotide-treated group, respectively, which did not reach statistical significance (Fig. 1B and D).

In the HFD-fed rats, hepatic TG and FFA levels were significantly increased and the hepatic glycogen content was significantly decreased when compared with the control group (P<0.01; Table IV). Following octreotide treatment, the hepatic TG and FFA levels were significantly decreased when compared with the HFD group (P<0.01; Table IV). By contrast, hepatic glycogen content significantly increased in the octreotide-treated group when compared with the HFD group (P<0.05; Table IV).

Example livers collected from the rats are shown in Fig. 2A. Liver tissues from rats in the HFD group were slightly yellow in color and displayed marked steatosis. The livers of rats in the octreotide-treated and control groups were similar in macroscopic appearance (Fig. 2A). Hepatic histopathology analysis revealed that the obese rats displayed diffuse fat deposition, particularly near the central vein, which exhibited macrovesicular and microvesicular steatosis (Fig. 2B). No alterations in histopathological structure were observed in the livers collected from the control rats (Fig. 2B). Fat deposition was significantly improved by octreotide treatment (Fig. 2B). Oil Red O staining of liver tissues demonstrated marked lipid infiltration of hepatocytes in obese rats, as indicated by an increased number of red hepatocytes and higher IOD values when compared with the control rats (Fig. 2). Octreotide treatment significantly decreased lipid infiltration in obese rats (Fig. 2C). Similarly, octreotide intervention markedly alleviated PA-induced hepatocyte lipid droplet accumulation in HepG2 cells (Fig. 2D). In addition, the IOD values of the octreotide-treated group were lower when compared with the PA-treated group (Fig. 2).

The Akt/GSK3β signaling pathway serves an important role in regulating hepatic glycogen synthesis (17). Therefore, western blotting was used to detect the protein expression levels of p-Akt and p-GSK3β, and RT-qPCR analysis was performed to determine the expression levels of GS mRNA in octreotide-treated obese rats in the present study. The results demonstrated that the expression of p-Akt and p-GSK3β in the HFD group was significantly lower when compared with the control group (0.70±0.21 vs. 1.36±0.35 and 0.83±0.29 vs. 1.19±0.24, respectively; P<0.05; Fig. 3). Octreotide treatment significantly increased the level of p-Akt and p-GSK3β protein expression when compared with the HFD group (1.24±0.26 vs. 0.70±0.21 and 1.04±0.27 vs. 0.83±0.29, respectively; P<0.05; Fig. 3). In addition, the results indicated that octreotide significantly reversed the HFD-induced reduction in GS mRNA levels (P<0.05; Fig. 3).

The results presented thus far indicate that octreotide may improve hepatic glycogen synthesis in obese rats. To further elucidate the mechanisms underlying the effects of octreotide on glycogen synthesis, the expression of p-Akt and p-GSK3β was determined by western blotting, and GS mRNA levels were examined by RT-qPCR analysis in PA-treated HepG2 cells. The western blotting results revealed that the levels of p-Akt and p-GSK3β in the PA group decreased by 74.2 and 18.7%, respectively, when compared with the control group (P<0.05; Fig. 4A-D). Following octreotide treatment, p-Akt and p-GSK3β levels increased by 140.8 and 12.2% when compared with the PA group, respectively (P<0.05; Fig. 4A-D). RT-qPCR analysis demonstrated that the expression of GS mRNA in the PA group was the lowest among the three groups (Fig. 4E). However, following octreotide treatment, GS mRNA levels were significantly increased when compared with the PA group (0.940±0.07 vs. 0.60±0.08; P<0.05; Fig. 4E).