Collection of serum and tissue specimens

We collected serum and abdominal adipose tissue samples from normal weight individuals (n = 6) and people with obesity (n = 6) aged 40–60 years from September 2021 to June 2022 in the Shihezi region of Xinjiang, China (According to China’s BMI classification: Normal weight: 18.5 kg/m2 ≤ BMI < 24 kg/m2; Obese: BMI ≥ 28 kg/m2). The group of normal weight individuals consisted of three males and three females, with a mean age of 55.7 years, mean weight of 56.7 kg, 18.5 kg/m2 ≤ BMI < 24 kg/m2, and fasting glucose <7.1 mmol/L. The group of people with obesity consisted of three males and three females, with a mean age of 57.5 years, mean weight of 95.0 kg, BMI ≥ 28 kg/m2, and fasting blood glucose ≥7.1 mmol/L.

Serum samples

A total of 3 ml of venous blood was collected from individuals who had fasted for 12 h. The serum was separated by centrifugation at 1788×g for 5 min. The upper serum layer was collected and frozen in an ultralow temperature freezer before RNA extraction.

Adipose tissue samples

In this study, adipose tissues within the abdominal omentum of normal weight individuals and individuals with obesity according to body mass index (BMI) were collected. The adipose tissue samples were obtained from inpatients of the Department of General Surgery of the First Affiliated Hospital of Shihezi University, all of whom required abdominal surgery. On the day of surgery, strict aseptic operation was performed, and liquid nitrogen cryopreservation was used to obtain approximately 3 cm × 3 cm of omental adipose tissue.

Biochemical indicator test

Triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured by a TG assay kit (A110-1-1, Nanjing Jiancheng Bioengineering Institute, China), a TC assay kit (A111-1-1, Nanjing Jiancheng Bioengineering Institute, China), an HDL assay kit (A112-1-1, Nanjing Jiancheng Bioengineering Institute, China), and an LDL assay kit (A113-1-1, Nanjing Jiancheng Bioengineering Institute, China), respectively. Plasma PA was detected by a human and mouse PA ELISA Kit (A042-2, Nanjing Jiancheng Bioengineering Institute, China; A185255, Shanghai Fusheng Bioengineering Institute, China).

Cell culture

Preadipocyte 3T3-L1 cells were purchased from the cell bank of the Typical Culture Preservation Committee of the Chinese Academy of Sciences. When the cells in a 25T culture bottle grow to 80–100%, they can be gone down to posterity. Inoculating cells in a six well plate for 2–3 days, and using cell differentiation induction medium I (DMEM high sugar medium, 10% foetal bovine serum, 10 μg/ml insulin, 1 μM/L dexamethasone and 0.5 mmol/L IBMX) was added for 48 h. Cell differentiation induction medium II (DMEM high sugar medium, 10% foetal bovine serum and 10 μg/ml insulin) was added for 48 h, and half of the medium was exchanged for mixed medium containing DMEM high sugar medium and 10% foetal bovine serum for 24 h. All the media were replaced with a mixed medium of DMEM high sugar medium and 10% foetal bovine serum.

Cell treatment

A 40 mM PA solution:PA (Sigma-Aldrich, St. Louis, USA, 0.0614 g) was added to 3 ml NaOH solution (0.1 mol/l),placed in a 75 °C full saponification water bath for 30 min until the PA particles were completely dissolved and the liquid is colourless and transparent. Then, the liquid was immediately added to 3 ml of BSA (40%, free of fatty acid) solution with sufficient mixing. A 200 mM TUDCA solution was prepared; TUDCA (MCE, USA, 5 mg) was dissolved in 1 ml of DMSO. A 10 mM Bay11-7082 solution was prepared; Bay11-7082 (MCE, America, 5 mg) was dissolved in 2.41 ml of DMSO. The experiment will be conducted in groups: the 200 μM PA group, the 500 μM PA group, the 200 μM PA plus 5 μM Bay11-7082 (to block p-p65) group, and 200 μM PA plus 200 μM TUDCA (to block ER stress) group.

miRNAs and RNA isolation and quantitative real-time PCR (qRT-PCR)

miRNAs were extracted using a miRcute miRNA isolation kit (cat# DP503; TianGen, Beijing, China). The miRcute Plus microRNA first strand cDNA kit (cat# KR211; TianGen, Beijing, China) was used for reverse transcription of miRNA first strand cDNA, and the miRcute Plus microRNA SYBR Green qPCR Kit (cat# FP401; TianGen) was used to detect the expression of microRNA. Relative miRNA expression levels were calculated as the ratio of the target gene to an internal reference (the U6 transcript). The primer sequences are shown in Supplementary Table 1.

Total RNA was extracted from cells using TRIzol reagent (cat# 15596-026; Life Technologies, California, USA), and reverse transcription was performed at 42 °C for 60 min and then at 70 °C for 15 min. PCR amplification was performed using a qRT‒PCR instrument (Qiagen, Hilden, Germany) with the following programme settings: 95 °C for 3–5 min, 40–45 cycles at 95 °C for 10 s, 50–60 °C for 30 s, and 72 °C for 40 s. GAPDH was used for normalisation. Relative gene mRNA expression levels were calculated as the ratio of the target gene to an internal reference (glyceraldehyde 3-phosphate dehydrogenase).

Protein isolation from adipose tissue

Adipose tissue (0.5 cm × 0.5 cm) was removed and placed into a 1.5 ml EP tube. A total of 3–4 grinding beads were added, and 100 μl of mixed protein lysis buffer (RIPA buffer and PMSF mixed at a 100:1 ratio) was added to the tube. The sample was subsequently ground into powder. Then, 900 μl of protein lysate was added to the tube, which was placed on a shaker in a refrigerator at 4 °C. After 6–8 h of vigorous shaking, the extraction of tissue proteins was started; the sample was centrifuged at 12,000 r/min for 20 min at 4 °C. New 1.5 ml EP tubes were prepared in advance, and after centrifugation was completed, the bottom layer of the protein lysate was removed with a 1-ml syringe (at this time, the liquid in the EP tubes was divided into three layers). The liquid in the EP tubes was filtered through 0.22-μm filters into new 1.5 ml EP tubes (removing the protein lysates) after removal of the needle (to remove grease and tissue waste). Then, the extracted protein concentration was determined using a nucleic acid detector, and the EP tube was labelled clearly on the cap or the outer wall. Then, the sample was placed in a −20 °C refrigerator for freezing and preservation to prepare for subsequent experiments.

Western blot and antibody

The protein sample was prepared using 4× loading buffer at a 3:1 ratio. The sample was mixed well and centrifuged before boiling in a dry bath. After returning to room temperature, the sample was processed. Antibodies against β-actin (Cat#TA-09) were from ZSGB-BIO, antibodies against GRP78 (3177S), ATF6 (65880S), PERK(312S), PERK(3192S), IRE1α(3294S), p-p65(3033SS), p65(8242SSS) were from Cell Signalling Technology. Antibodies against TSG101(ab125011), HSP70 (ab2787) were from Abcam. The vertical electrophoresis apparatus and protein transfer apparatus used in the experiment were purchased from Bio-Rad Company (United States), and images were processed using Photoshop software.

Calculation of fold change for western blot results

All the target bands were scanned in grayscale using ImageJ software; subsequently, the grayscale value of each group of target genes was divided by the grayscale value of each group of internal references to obtain a ratio X. After the ratios of the three replicates of the experiments were averaged, the ratio Y was obtained by dividing the previously obtained ratio X of each group by the average, and finally, the Y value was used to make a quantification graph.

Extraction of exosomes

Three days before cell treatment, the serum was replaced with exosome-free serum. After the cell culture medium was collected, the exosomes were extracted with a Novazan Exosome Extraction Kit (cat# R601; Novazan, Nanjing, China) and stored at −80 °C.

Identification of exosomes

Then, 5–10 μl of the exosome suspension was adsorbed on copper mesh for 3–5 min and stained with phosphotungstic acid for 3 min. Next, the samples were infrared-baked for 20 min and observed under a transmission electron microscope. A 20 μl aliquot of the exosome suspension was diluted with sterile PBS to 1–1.5 ml. It was then thoroughly mixed and shaken, placed in a colorimetric dish, and analysed experimentally using the NanoSight system.

Animal experiment

Four-week-old C57BL/6 male mice were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., and raised at the SPF Experimental Animal Center at the Liyushan Campus of Xinjiang Medical University. In order to reduce the number of animal sacrifices and ensure the success rate of operation in experimental mice, we adopted a small sample size design. Before group feeding, all male mice were randomly assigned to test and control groups, and both groups were fed ad libitum in order to ensure normal survival of the mice. Mice were housed at 3–5 mice/cage. The food and water were changed once a day, and the weight and body length were measured weekly. After adaptive feeding for 1 week, the mice were randomly divided into a normal control diet feeding group (NCD, 10% calories from fat, cat# MD12031, purchased from Jiangsu Medison Biomedical Co., Ltd.; n = 6) and a high-fat diet feeding group (HFD + PA, 60% calories from fat and 5% PA, cat# MD12033, purchased from Jiangsu Medison Biomedical Co., Ltd.; n = 18). The body weight and Lee’s index of mice in the high-fat diet group increased significantly (P < 0.05) compared with those in the normal diet group; and the FBG, as well as the serum TG, TC, LDL-C, and PA content were significantly higher than those in the normal diet group (P < 0.05). It was suggested that the diet-induced obese mouse model was successfully constructed. After being fed for 11 weeks, the HFD-fed mice were divided into three groups: the HFD group (DMSO, n = 6), the group that received an intraperitoneal injection of Bay11-7082 (HFD+Bay11-7082, 2.5 mg/kg/week; n = 6) and the group fed a HFD and intraperitoneally injected with TUDCA (HFD + TUDCA, 0.25 g/kg, 4 times per week, n = 6).

Glucose tolerance test and insulin tolerance test

The glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted at the 12th week. For the GTT, the feed was removed, water was provided for 12 h. Then, 50% glucose solution (4 μl/g) was injected intraperitoneally, and blood glucose was measured at 0, 0.5, 1, 1.5, and 2 h. For the ITT, the feed was removed, and the mice were continuously supplied with water for 6 h. The mice were intraperitoneally injected with different doses of insulin (0.5 IU/g), and blood glucose was measured at 0 h, 0.5 h, 1 h, 1.5 h, and 2 h. After the GTT and ITT, adipose tissue, liver, and serum samples were collected for analysis.

Blinding

Four researchers were involved in this animal study. The first researcher was responsible for group feeding and numbering of the mice, the second researcher was responsible for injecting the relevant agents (grouping of mice unknown), the third researcher was responsible for taking gross photographs of the mice and surgical dissection of the tissues (grouping of mice unknown),and the fourth researcher was responsible for analysing the levels of expression of the factors in the tissues according to the numbering (grouping of mice unknown), and sending the data to the first researcher for Analysis.

Statistical analysis

The statistical software SPSS 18.0 was used for data analysis. For normally distributed data with similar variance, Student’s t tests were used when two groups of data were compared. For comparisons between three groups of data, one-way ANOVA-LSD analysis was conducted. When P < 0.05, the difference was considered statistically significant. The values are expressed as the means ± SEMs (Prism 7; GraphPad Software). In this study, no power analysis was performed to determine the sample size. The sample size was based on previous studies employing mice.