Animals and Study Design
Six to seven-week-old C57BL/6J mice were purchased from Vital River Laboratory Animal Technology Co., Ltd., (China) and acclimatized to the animal facility for 1 week. Mice were maintained on a 12-h light-dark cycle with free access to food and water. All experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986 and approved by the Ethics Committee of Women’s Hospital of Nanjing Medical University (No.2018-49).
Female mice were fed normal chow diet (NCD group, Research Diets AIN-93G, consisting of 20.3 % protein, 63.9 % carbohydrate, and 15.8 % fat), a high-fat/sucrose diet (HFD) (GDM group, Research Diets D12451, consisting of 19.8% protein, 35.2 % carbohydrate, and 45% fat), high-dose inulin supplemented a high-fat/sucrose diet (Inulin-H group, 3.33g/kg/d via oral gavage ) or low-dose inulin supplemented a high-fat/sucrose diet (Inulin-L group, 1.67g/kg/d via oral gavage) for 4 weeks before being mated with age-matched male mice. Upon identification of a copulatory plug, considered to be day 0 of pregnancy (GD0). Mice were euthanized by CO2 inhalation on GD18 (or equivalent) after fasting for 6 hours from 8 AM and blood and tissues were collected. Liver and inguinal fat was quickly collected and kept at -80 ◦C.
Measurement of Body Weight, Blood Glucose and Serum Insulin
Body weight, blood glucose and serum insulin were monitored at different time points, including before dietary intervention, after 4 weeks of HFD, and on GD 0, 10, 14 and 18. Blood glucose and insulin levels were determined from tail venipuncture blood samples. Blood glucose concentration was measured immediately using a blood glucose meter and strips (Roche Accu-Chek Active, Mannheim, Germany). The blood samples were then centrifuged at low speed (4°C, 3000 rpm, 15 mins) within 1 hour, the supernatant was harvested and stored at -80°C for measuring serum insulin level by enzyme linked immune sorbent assay (ELISA; NJJCBIO Co., Ltd, Nanjing, China) according to the manufacturer’s instructions. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by the following formula: fasting blood glucose (FBG, mmol/L) × fasting serum insulin (µIU/mL)/ 22.5. In addition, urine volume and fluid intake were observed daily.
Oral Glucose Tolerance Test
Glucose tolerance was determined by an oral glucose tolerance test (OGTT) on GD 14. Mice were fasted for 6 hours with free access to water and received an oral gavage of 20% D-glucose (2 g/kg body weight). Blood samples were collected from the tail vein at 0, 30, 60, 90 and 120 mins after glucose administration. Blood glucose levels were measured instantly using methods as mentioned above. Meanwhile, area under the curve (AUC) of blood glucose was calculated .
Serum Lipid Measurement
Levels of serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were measured using commercial kits (NJJCBIO Co., Ltd, Nanjing, China).
Liver and inguinal fat tissues were fixed in 4% paraformaldehyde, decalcified, paraffin-embedded and stored at 4°C. After tissues were sliced into 4 µm sections, haematoxylin-eosin staining was performed. First, sections were stained with haematoxylin for 5–10 minutes, immersed in 70% ethanol for 30 minutes to remove cytoplasm colouring, alkalized with alkaline solution and washed with distilled water for 1 minute. Second, sections were stained with eosin for 30–60 seconds, dehydrated with gradient ethanol, cleared two times with xylene, dried and mounted. Finally, the morphological structures of the liver and inguinal fat tissues were observed under an optical microscope.
RT2 Profiler PCR Array Analysis
Total RNA was isolated from the liver samples of NCD group, GDM group and Inulin-H group using Qiagen RNeasy® Mini Kit (QIAGEN, Shanghai, China) according to the manufacturer’s instructions. Single-strand cDNA was synthesized from 1 µg of total RNA by reverse transcription reaction using Qiagen RT2 First Strand Kit (QIAGEN, Shanghai, China). The cDNA was mixed with Qiagen PCR RT2 SYBR Green Master Mix (QIAGEN, Shanghai, China).
To explore the underlying mechanisms of inulin induced-effects, the expression of 84 genes involved in Glycolipid metabolism including RETN were examined using RT2 profiler PCR array (PAMM-006Z-mouse glucose metabolism, QIAGEN, Shanghai, China). Relative quantification of mRNA levels was determined by real-time quantitative PCR using a ABI 7500 RT-PCR machine/Bio-Rad CFX96 Sequence Detector instrument. The quantitative expression of gene was calculated from the cycle threshold (CT) value of each sample in the linear part of the curve using the relative quantification method (2−ΔΔCT) . The samples were analyzed in triplicate and corrected for the selected internal standard which had the smallest standard deviation among the housekeeping genes. Candidate genes were selected from those whose expressions differed greater than 2-fold or less than 2-fold, or which differed significantly (p < 0.05) between the GDM group and Inulin-H treatment group.
Quantitative PCR (qPCR) Analysis for the Candidate genes
The five most differentially-expressed genes between GDM group and Inulin-H group were selected from the RT2 profiler PCR array as candidate genes and were analyzed the correlation with FBG further. Specific PCR primers were designed for further quantitative real-time PCR analysis (Takara, Dalian, China) as follows.
G6pc: 5’-CGACTCGCTATCTCCAAGTGA-3’ and 5’-GGGCGTTGTCCAAACAGAAT-3’
RETN: 5’-ACAAGACTTCAACTCCCTGTTT-3’Cand 5’-TTTCTTCACGAATGTCCCACG-3’
Igfbp5: 5’-CCCTGCGACGAGAAAGCTC-3’ and 5’-GCTCTTTTCGTTGAGGCAAACC-3’
Slc14a2: 5’-AAGGAGATGTCTGACAGCAACA-3’ and 5’-GGGCTGGGTGTGTATCCTG-3’
IL10: 5’-CCCATTCCTCGTCACGATCTC-3’and 5’-TCAGACTGGTTTGGGATAGGTTT-3’
Western Blot Analysis
Liver and inguinal fat tissues were harvested and were homogenized on ice in the presence of protease and phosphatase inhibitors. Homogenates were centrifuged at 12,000 × g at 4°C for 15 mins. Protein concentration in supernatants was quantified by the BCA method using bovine serum albumin (BSA) as the standard. Proteins were analyzed by 10% SDS-PAGE and transferred to PVDF membranes that were incubated in 5% non-fat milk at room temperature for 1 hr, then incubated with appropriate primary and secondary antibodies. Membranes were washed and proteins were detected by enhanced chemiluminescence (ECL) using a LAS-4000 lumino-image analyzer (Fuji Film, Tokyo, Japan). Bands were digitally scanned and analyzed using ImageJ software (NIH Image, National Institutes of Health, Bethesda, MD, USA).
All data were calculated as means ± SD and checked using the Kolmogorov-Smirnov (KS) test before further analysis. Statistical significance between two datasets was assessed using the Student’s t-test. Multiple groups were compared using one-way ANOVA followed by Tukey multiple comparison testing. A P value of < 0.05 was considered statistically significant. All statistical tests were performed using GraphPad Prism Version 7.0 (GraphPad Prism Software, Inc. CA, USA).