Sixty male Sprague-Dawley rats (6 weeks of age, bred at Hebei Medical University, China) were used for the experiments. All procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals. The protocol was approved by the Hebei Medical University Animal Care Research Committee. All rats were kept at 23–24°C on a 12/12-h light/dark cycle. Animals were randomly divided into 4 groups: i) the control group (n=15, fed standard laboratory chow); ii) the HF-4W group [n=15, fed a high-fat (HF) diet for 4 weeks (HF diet consisting of 5% fat, 55% carbohydrates, 22% protein, 7% ash and 5% fiber); iii) the HF-8W group (n=15, fed a HF diet for 8 weeks); and iv) the T2DM group (12–15) [n=12, after 8 weeks of the HF diet, 15 rats were administered a streptozotocin (STZ) injection (35 mg/kg); 10 days later, 12 rats with fasting blood glucose (FBG) above 200 mg/dl were enrolled in the T2DM group].

The body weight of the rats was monitored once every week. FBG, total cholesterol (TC) and triglycerides (TG) were determined by an automatic biochemistry analyzer (Beckman ×20; Beckman Coulter, Miami, FL, USA).

After an overnight fast for over 12 h, the rats were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally), and the left jugular vein and left femoral artery were catheterized for infusion and blood sampling, respectively. The glucose and insulin solutions were stored in 2 digital syringe pumps and were joined by a ‘Y’ connector to the jugular catheter. Human insulin (Novolin R, Novo Nordisk, Bagsvaerd, Denmark) was continuously infused at 10 mU/kg/min for 2 h. The blood glucose concentration was clamped at the basal level by estimating blood glucose concentration at 5 min intervals in samples taken from the femoral artery and adjusting the rate of infusion of a 20% glucose solution. Clamping was achieved by 60 min and was maintained for 60 min. The glucose infusion rate (GIR) during the second hour of the clamp was taken as a response parameter, indicating insulin sensitivity.

At 8:00 a.m. after overnight fasting for over 12 h, blood was obtained from the rats and was transferred into chilled tubes with 1 mg/ml EDTA-2Na and 500 U/ml aprotinin. Blood samples were immediately centrifuged at 4°C (1,600 rpm for 15 min) and were stored at −80°C until assay. After the rats were sacrificed, the stomach was quickly removed, immediately frozen in liquid nitrogen and stored at −80°C. To quantify plasma and stomach ghrelin levels, a commercial ghrelin enzyme-linked immunosorbent assay (ELISA) kit (Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) was used according to the manufacturer’s instructions. Inter- and intra-assay coefficients of variation were <14 and <5%, respectively.

After overnight fasting for over 12 h, blood was collected in tubes with gel and clot activator and centrifuged at 4°C (1,600 rpm for 15 min) to obtain serum. Blood serum was stored at −80°C until assay. Epididymal fat pads were separated from the left side of each rat during experiments, and they were immediately frozen in liquid nitrogen and stored at −80°C. Cold normal saline (1 ml) was added to each 200 mg epididymal fat pad sample; samples were homogenized at 20,000 rpm for 30 sec at 4°C, repeated twice at an interval of 10 sec. The homogenate was centrifuged at 12,000 rpm for 20 min at 4°C; the supernatant was collected and stored at −20°C. Serum and adipose leptin and serum insulin were determined by radioimmunology assay (RIA) with leptin and insulin RIA kits (Beijing Hi-Tech Atomic Technology, Inc., Beijing, China) according to the manufacturer’s instructions.

The stomach, left epididymal fat pad and hypothalamus were rapidly excised, snap-frozen on dry ice and immediately processed for RNA isolation. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Single-stranded complementary DNA (cDNA) was synthesized using Moloney murine leukemia virus (MMLV) reverse transcriptase with random hexamer primers. Quantitative real-time PCR was performed using SYBR^® Premix Ex Taq™ (Takara, Shiga, Japan) on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s instructions. The mRNA level was normalized to the housekeeping gene β-actin. The fold-change for an mRNA from control to HF (or DM) was calculated as 2^(−ΔΔCt), where ΔΔCt = ΔCt control - ΔCt HF (or ΔCt DM) and ΔCt = Ct mRNA - CT-actin mRNA. The primers and thermal cycle parameters are shown in Table I.

Animals were anesthetized and intracardially perfused with 0.1 M PBS followed by freshly prepared 4% paraformaldehyde in PBS. The stomach and brain were removed, postfixed overnight, paraffin embedded and cut into 5-μM sections with a microtome. After the paraffin had been removed, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min at room temperature (RT). The slides were microwaved for 20 min in 0.01 M citrate buffer (pH 6.0). Slides were rinsed in PBS and then blocked with 10% goat serum (Sigma, St. Louis, MO, USA) for 30 min at RT. Tissue slices were incubated with primary antibodies (1:100) overnight at 4°C and were washed with PBS for 5 min 3 times before a reaction with secondary antibody for 1 h at RT. The slides were reacted with 3,30-diaminobenzidine tetrahydrochloride (DAB) for 1–3 min at RT for a color reaction, and then they were counterstained with Mayer’s hematoxylin and observed under a microscope. Negative controls were reacted with PBS instead of primary antibodies. The primary antibodies used were rabbit anti-mouse ghrelin, rabbit anti-mouse GHS-R1a (Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) and rabbit anti-mouse OB-Rb (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China). The secondary antibody used was a biotinylated goat anti-rabbit IgG. Image Pro Plus 6.0 software was used for imaging analysis.

All data are expressed as the means ± SEM. A normality and a homogeneity test for variance were conducted. One-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) pairwise multiple comparisons were used to assess differences in the means among groups. The Pearson correlation test was performed between 2 parameters. All analyses were performed using SPSS for Windows, version 10.0. P<0.01 was considered to indicate a statistically significant difference.

Comparing the HF-8W to the control group, the average body weight increased significantly (P<0.01); however, no significant difference was found between the HF-8W and T2DM groups (P>0.05). The TC and TG serum concentrations increased significantly (P<0.01) in the T2DM compared to the control group. The average FBG levels of the control, HF-4W and HF-8W groups were similar, but in the T2DM group, levels increased to 9.38±0.56 mmol/l (P<0.01, Table II).

The serum insulin levels increased from the control to the HF-8W group (P<0.01); however, compared with the HF-8W group, the levels decreased significantly in the T2DM group (P<0.01). Compared with the control rats, GIR was gradually reduced in the HF-4W, HF-8W and T2DM group rats (P<0.01, Table II).

The gastric ghrelin levels were reduced significantly in the HF-4W and HF-8W rats compared with the control rats (P<0.01), whereas no significant differences were found between the HF-8W and T2DM rats (P>0.05; 0.81±0.11 in the control rats, 0.67±0.11 in the HF-4W rats, 0.57±0.11 in the HF-8W rats and 0.59±0.09 in the T2DM group rats). The ghrelin mRNA levels and ghrelin-expressing cells showed similar results (Fig. 1A and B).

Compared with the control rats (2.03±0.22 ng/ml), the plasma ghrelin level was significantly suppressed in the HF-4W and HF-8W rats (1.81±0.20 and 1.63±0.11 ng/ml; P<0.01); however, there were no significant differences between the HF-8W and T2DM group rats (1.64±0.13 ng/ml; P>0.05).

Compared with the control rats, a significant reduction in the number of GHS-R1a-expressing cells was observed in the ARC from the control to the HF-8W rats (P<0.01, Fig. 2A); however, no significant differences were observed between the HF-8W and T2DM rats (P>0.05, Fig. 2A). Accordingly, total hypothalamic GHS-R1a mRNA showed similar results (P<0.01, Fig. 2B)

Compared with the control rats, there were significantly increased adipose leptin levels in the HF-4W, HF-8W and T2DM rats (P<0.01, Fig. 3A). Accordingly, the leptin mRNA levels in the adipose tissue showed similar results (P<0.01, Fig. 3B).

During the development of T2DM, the circulating concentrations of leptin gradually, yet significantly increased in the HF-4W, HF-8W and T2DM group rats (P<0.01, Fig. 4).

With the development of T2DM, in the ARC of the hypothalamus, the number of OB-Rb-expressing cells reduced significantly (P<0.01, Fig. 5A). Similarly, total hypothalamic OB-Rb mRNA levels decreased significantly when comparing the control to the T2DM group (P<0.01, Fig. 5B).

Pearson correlation analysis demonstrated a significant negative correlation between the plasma ghrelin and serum leptin levels (r=−0.813, r²=0.551, P=0.001). GIR positively correlated with plasma ghrelin levels (r=0.945, r²=0.893, P=0.0001) and negatively correlated with serum leptin levels (r=−0.973, r²=0.946, P=0.001).