A113 1 1

At weaning and 9 weeks of age, blood samples of offspring were centrifuged at 4,000 g for 15 min and stored at –80°C. Enzyme-linked immunosorbent assay (ELISA) kits were used to quantify the concentration of serum insulin (90080; Crystal Chem, Downers Grove, IL, United States), leptin (MOB00B, R&D Systems, Minneapolis, MN, United States) and adiponectin (MRP300; R&D Systems, Minneapolis, MN, United States). Serum total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were detected using commercial kits (A111-1, A112-1-1, A113-1-1, Jiancheng Bioengineering Institute, Nanjing, China). All samples were measured in duplicate.

Commercially available kits for the measurement of total-cholesterol (TC) (A113-1-1), low-density lipoprotein-cholesterol (LDL-C) (A113-1-1), high-density lipoprotein-cholesterol (HDL-C) (A112-2-1) and triglyceride (TG) (A110-2-1) were purchased from Nanjing Jiancheng Bio-Engineering Institute Co., Ltd. (Nanjing, China). TC-sensitivity: 6.5 mM; linearity: 0 mM–25.9 mM; inter-assay CV: 3.0%; intra-assay CV: 6.0%. LDL-C-sensitivity: 3.1 mM; linearity: 0.01 mM–10.34 mM; inter-assay CV: 3.0%; intra-assay CV: 8.0%. HDL-C-sensitivity: 1.3 mM; linearity: 0.04 mM–2.59 mM; inter-assay CV: 3.0%; intra-assay CV: 6.0%. TG-sensitivity: 1.7 mM; linearity: 0.01 mM–11.3 mM; inter-assay CV: 3.0%; intra-assay CV: 6.0%.
Serum TC, LDL-C, TG, and the levels of TC, TG in liver were measured by the biochemical kits according to the manufacturer’s instructions. Briefly, 10–20 mg of liver tissue was homogenized in 300 μL isopropyl alcohol and centrifuged supernatants were harvested. The liver extracts and serum were the enzymatically measured with their respective kits.

Blood samples were centrifuged at 4500× g and 4 °C for 10 min to collect the serum. Then, the serum concentrations of alanine aminotransferase (ALT) (BC1555, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), aspartate aminotransferase (AST) (BC1565, Solarbio), glucose (GLU) (BC2505, Solarbio), total cholesterol (TC) (BC1985, Solarbio), high-density lipoprotein (HDL-C) (A112-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and low-density lipoprotein (LDL-C) (A113-1-1, Jiancheng) were detected following the manufacturer’s protocols.

After the 8-week experiment concluded, animals that had fasted overnight were sedated with 100% isoflurane (2 mg/kg body weight or equivalent to 2% inhaled concentration) and dissected. Using a dry Eppendorf tube, blood was collected via cardiac puncture, allowed to coagulate for 30 min at room temperature, and then centrifuged for 10 to 15 min at 3000 rpm.
Nonhemolyzed serum samples were promptly collected and stored at −20 °C for the evaluation of various parameters, including total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), fasting blood glucose (FBG), insulin, homeostatic model assessment for insulin resistance (HOMA-IR), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels. The levels of TC, TG, HDL-C, LDL-C, AST, and ALT were determined using biochemical assays following the instructions provided with the corresponding kits (Cat. No. A110-1-1, A111-1-1, A112-1-1, A113-1-1, C009-3-1, and C010-3-1, respectively, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Additionally, FBG levels were measured using a kit according to the manufacturer’s instructions (Cat. No. YC0713; Shanghai YaJi Biotechnology Co., Ltd., Shanghai, China). These assays were conducted by using an automatic biochemical analyzer (AU480 from Beckman Coulter, Miami, FL, USA).

Serum was obtained by centrifugation (4 °C, 1,500 g, 20 min) of blood collected from the eye sockets of the mice and stored at −80 °C. The serum fasting insulin (FINS) levels were examined using an enzyme-linked immunosorbent assay (CSB-E05071m, Cusabio, Wuhan, Hubei, China). The serum TC, TG, LDL-c, and HDL-c levels were examined using commercial reagent kits (A111-1-1, A110-1-1, A113-1-1 and A1122-1-1, Jiancheng, Nanjing, Jiangsu, China).