Animal model establishment and experimental design
Pregnant ICR mice were purchased from Vital River Laboratory Animal Technology (Beijing, China). Mice were adapted to laboratory condition for 1 week before experiment. On the day of birth, the newborn mice were randomly divided into two groups. On postnatal day 2, the MSG-treated pups were injected subcutaneously (s.c.) with MSG (4 g/kg/d, Sigma-Aldrich, USA) for 7 consecutive days. Pups in the control group were s.c. injected with an equivalent volume of 0.9% physiological saline solution [16, 27]. Weaning after 21 days, MSG and control mice were separated by gender. Biochemical and physiological determinations were performed in these two groups at 1, 2, 4 and 6 months of age. The mice were allowed free access to water and a chow diet. All mice were kept in the Laboratory Animal Center of Qingdao University at an ambient temperature of 25 ± 2°C, 12 h light/dark cycles and humidity of 40–60%. After 24 weeks, compared with the control mice, the MSG mice showed significant centripetal obesity, accompanied by elevated serum TC, TG, and insulin levels. Insulin resistance was increased as well. Based on the body weight, Lee’s index, serum TC, TG and FBG, the 6-month-old MSG mice were divided into three groups: (i) the Model group, which was administered an equal volume of saline. (ii) the Piperine group and Metformin group, which were treated with 40 mg piperine/kg/day (Sigma-Aldrich, USA) and 150 mg metformin/kg/day (Sigma-Aldrich, USA) by gavage for 10 weeks, respectively. Meanwhile, the 6-month-old normal mice without MSG treatment were selected as normal controls. There were 8 mice per group. The food intake and body weight of the mice were recorded weekly during the 10-week period of the treatment. The experimental protocols are outlined in Fig. 1. All animal protocols were performed according to the Guidelines for the Care and Use of Laboratory Animals prepared and approved by the Animal Care and Use Committee of the Affiliated Hospital, Qingdao University and the Animal Experimental Ethical Committee of Affiliated Hospital, Qingdao University, Shandong Province, China.
Measurement of visceral organ indices and the collection of serum
The mice were euthanized by opening the heart under anesthesia with pentobarbital (50 mg/kg, Sigma-Aldrich, USA). Then, the abdominal adipose, pancreas, liver, and kidney were collected and weighted before frozen in liquid nitrogen until further analysis. RNA extraction and real-time PCR analysis were performed using these tissue samples. Blood samples were prepared by centrifuge at 4000 rpm for 10 min at 4°C. The samples were stored at -80°C for later analyses. Throughout the experiments, all mice were provided with housing that allows the expression of species-specific behaviors. Appropriate anesthesia was used to minimize pain, and the mice were trained to cooperate with procedures to minimize any distress. Thus, the experimentally induced stress was minimized. All experimental procedures conformed to the European Guidelines for the care and use of Laboratory Animals (directive 2010/63/EU).
Oral glucose tolerance test (OGTT)
Seven days prior to the experiments, 4-h-fasted mice received glucose (2 g/kg body weight) by gavage. A total of 3.5 μL blood was collected from the tip of the tail vein at different time points (0, 30, 60, and 120 min) after glucose load, which was used for blood glucose determination with a one Touch Ultra glucose meter (ACCU-CHEK Performa Nano, Roche Diabetes Care GmbH). The area under the curve (AUC) was calculated according to the following formula, AUC=1/4(PG0min+PG30min)+1/4(PG30min+PG60min)+1/2(PG60min+PG120min), where, PG0min, PG30min, PG60min, and PG120min are the blood glucose level at 0, 30, 60, and 120 min after glucose load, respectively.
Hyperglycemic clamp experiment
At the last week of the experiment, the mice fasted for 4 h were anesthetized with 50 mg/kg (i.p.) of pentobarbital, and the tracheotomy procedure was performed. When the animals were stabilized after 30 min post the operation, the hyperglycemic clamp experiment was performed. First, an initial dose of glucose (250 mg/kg B.W.) was injected through a jugular vein tube to quickly increase blood glucose to a higher level within 5 min, and then a variable rate of glucose (20%, w/v) was infused. The blood glucose levels were measured at 5-min intervals until the blood glucose level reached a steady state (between 13.5 and 14.5 mmol/L). After the steady state, the glucose infusion rate measured at 5 time points was averaged as the glucose infusion rate (GIR) at the steady state.
Insulin tolerance test (ITT)
The mice were administered with insulin (0.4 unit/kg, novolin, Novo Nordisk, Danmark) 4 h after fasting via intraperitoneal injection. The blood glucose levels were calculated using blood samples collected at 0, 40 and 90 min after insulin injection. The percentage of blood glucose reduction at 40 min was calculated accordingly.
Routine blood test and biochemical analysis
The blood WBC counts were detected on a PE-6800VET Automated Animal Blood Cell Analyzer (Pukang Inc, Shenzhen, China). Serum total cholesterol (TC) and triglyceride (TG) were measured by an enzymatic colorimetric method using a commercially available kit (Jiancheng Bioengineering Institute, Nanjing, China). Fasting blood glucose was detected by a one Touch Ultra Meter (ACCU-CHEK Performa Nano, Roche Diabetes Care GmbH).
Enzyme-linked immunosorbent assays
Serum insulin was determined by using a mouse ultrasensitive insulin ELISA kit (American Laboratory Products Company, USA). LPS produced by the Gram-negative bacteria in the intestine, M1-like pro-inflammatory cytokines (IL-1β and Gal-3) and M2-like anti-inflammatory cytokine (IL-10) in the serum samples were measured by an ELISA kit (Mlbio, Shanghai, China).
Semiquantitative reverse transcriptase polymerase chain reaction
Total RNA was extracted from the adipose tissues or cultured cells homogenized in Trizol (CWBIO, Beijing, China), which was used for the synthesis of cDNA. qRT-PCR amplification (TaKaRa Bio, Shiga, Japan) was performed on a BioRad CFX96 detection system (BioRad, Hercules, CA, USA ) using the primers obtained from the GeneBank. The expression levels of the genes were measured relative to the β-actin level and evaluated using the 2-ΔΔCT method. The primer sequences are presented in Table 1.
Morphologic and immunohistochemical analysis
The abdominal adipose and liver were collected at the end of the experiment, fixed with 4% polyformalin in PBS solution for over 24 h and embedded in paraffin. Hepatic fat lesions and adipocyte diameters in abdominal adipose were observed by HE staining for morphologic analysis. Paraplast-embedded sections (5 μm) were subject to immunoperoxidase staining. The sections were incubated with anti-mouse CD11c (1:250, Affinity, USA) and Galectin-3 (1:250, Abcam, Cambridge, USA) antibody. Amplification and staining were performed using an avidin-biotin-complex and 3,3-diaminobenzidine method, respectively.
Cell culture and treatment
RAW 264.7 macrophages were obtained from ATCC, USA and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, USA) in a 5% CO2 humidified cell incubator at 37°C. Piperine was added to the cell culture to a final concentration of 0, 20, 40, and 80 μM, respectively, and the macrophages were incubated for 12 h. LPS (1 μg/mL, Sigma-Aldrich, USA) was added afterward to induce M1 macrophage polarization. After the treatment, cell-free supernatants were collected to determine the IL-1β level using an ELISA kit assay, and the cells were collected for RNA as described above. The piperine solution was prepared by dissolving piperine in DMSO. The final concentration of DMSO in the cell culture should not exceed 0.05%.
Western blot analysis
The stimulated cells were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors (Slarbio, Beijing, China) after being washed with phosphate buffered saline (PBS) twice. The homogenate was subject to 10-12% SDS-PAGE electrophoresis before transferred to a PVDF membrane, which was then incubated with the primary antibody CD11c, Toll like receptor-4 (TLR-4) (1:1000; Affinity, USA) and IL-1β (1:2000; Abcam Cambridge, MA, USA) overnight at 4°C, followed by incubation with the HRP-linked secondary antibody at 25°C for 2 h. An eECL western blot kit (CWBIO, Beijing, China) was used to detect the proteins.
The software GraphPad Prism 7.00 was used for data analyses. All values are reported as means ± SD. An unpaired t-test was used for comparisons between two groups. One-way ANOVA was used for comparisons among three or more groups. p<0.05, p<0.01, p<0.001 and p<0.0001 were used to represent the levels of significant difference.