Insulin

The TFA was provided by Chengdu Pusi Biotechnology, Co., Ltd. (Chengdu, Sichuan, China). Metformin was purchased from GBCBIO technology (Guangzhou, Guangdong, China). Insulin was provided by Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Lipopolysaccharide (LPS) was purchased from Sigma (St. Louis, MO, USA). ELISA kits for measuring total bile acid (TBA), total cholesterol (TCHO), total triglyceride (TG), glutamic oxalacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), high-density lipoprotein (HDL-C), and low-density lipoprotein (HDL-C) were purchased from Jiancheng (Nanjing, Jiangsu, China). Primary antibodies for apical sodium-dependent bile acid transporter (ASBT) were purchased from PL Laboratories (Richmond, Canada). CYP7A1 and FXR were purchased from Santa Cruz (Dallas, TX, USA). TGR5 was provided by Abcam (Cambridge, MA, USA). GLUT2 was derived from Bioss (Beijing, China). Other reagents are from commercial sources.

Lysed NETs (1:300) or insulin (1:200, Yuanye, Shanghai, China, 1 mg/ml) was diluted in buffer (0.1 M Na2CO3, 0.1 M NaHCO3, pH=9.6) and incubated at 4°C overnight in 96-well plates. After blocking these two plates with 5% BSA (37°C, 2 h), the serum samples were diluted 1:200 in 5% BSA and incubated with NET protein at 37°C for 2 h. Another uncoated plate was also blocked with 5% BSA (37°C, 2 h) and then incubated with the same diluted samples at the same time. Next, all samples from NET-coated plates or uncoated plates were collected and added to insulin-coated plates, followed by 2 h of incubation at 37°C. After subsequent routines, the differences in absorbance were compared between pretreated subjects and untreated subjects of the same sample. The cross-reaction ratio =(OD _{(uncoated+insulin)} - OD _{(NET protein+insulin)})/OD _{(uncoated+insulin)}

Immortalized human chorionic trophoblast line HTR-8/SVneo was purchased from Procell (Wuhan, Hubei, China), and cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS) (Biological industries, Kibbutz Beit-Haemek, Israel) at 37°C with 5% CO₂.
The transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) with serum-free medium.
The cells were incubated with insulin (Solarbio) of 10^-6 mol/l for 48 h to induce IR, and insulin of 10^-7 mol/l was used to stimulate cells to verify the IR.
JNK signaling agonist Anisomycin (1 μg/ml) (Yuanye, Shanghai, China) and antagonist SP600125 (20 μM) (Aladdin, Shanghai, China) was used to treat cells for 30 min to activate or inactivate the JNK signaling.