Animal care

C57BL/6 male mice were obtained from HUNAN SJA Laboratory Animal Co, LTD. (Hunan, China), and Leprdb/db, Leprdb/–male mice were obtained from the Model Animal Research Centre of Changzhou Cavens (Changzhou, China). The mice were housed in groups of 5 animals/cage on a 12 h light/dark cycle in an SPF facility at 22–24 °C and 40–50% humidity. 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. In male C57BL/6 mice raised on 60% high-fat diet, 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 weights of liver and various parts of adipose tissues (Epi, Sub, PAT, and MAT), FBG, as well as the serum TG, TC, LDL-C, and FFA 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.All animal care and handling were carried out according to the international laws and policies, and all animal experiments were approved by the animal ethics committee of the first affiliated Hospital of Shihezi University (A2019–087–01).

Cell culture

HepG2 and L02 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and were cultured in 25 mmol/L glucose Dulbecco’s modified Eagle’s medium (DMEM, Gbico) supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin (100 µg/mL) at 37 °C in a humidified atmosphere with 5% CO2. After transfection, RNA and protein were extracted or insulin sensitivity assay was performed. 293A cells were purchased from Procell. (Wuhan, China), and were cultured in 25 mmol/L glucose Dulbecco’s modified Eagle’s medium (DMEM, Gbico) supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin (100 µg/mL) at 37 °C in a humidified atmosphere with 5% CO2.

Adenovirus amplification and purification

The miRNA-548ag mimic adenovirus vector and inhibitor used in this study were constructed by Shanghai Genepharma. The adenovirus was amplified in 293A cells. In brief, 293A cells cultured in 10% foetal bovine serum about 90% confluence were added with adenovirus seed. 48 h post-infection, visible regions of cytopathic effect (CPE) are observed. Harvest adenovirus stock until approximately 90% CPE is observed (typically 72 h post-infection). The cell suspension was freeze-thawed repeatedly 3 times in a methanol dry ice mixing bath and 37 °C water bath, and the supernatant was collected by centrifugation, which can be frozen at −80 °C for long-term preservation. For specific purification procedures, we referred to the instructions of ViraTrap “M Adenovirus Purification Maxiprep KitViraTrap” adenovirus mass purification kit, and the reagent dosage was adjusted according to the proportion of virus suspension quantity.

Intraperitoneal injection of adenovirus vector in mice

Sixteen 4-week-old C57BL/6 male mice were fed with normal diet (D12450J, 10% energy from fat) until the 16th week. They were divided into two groups: intraperitoneal injection of empty adenovirus group (n = 8) and intraperitoneal injection of adenovirus particles encoding miR-548ag -mimic group (n = 8, 1 × 1011 VP/ mice, once a week) for 6 weeks. Sixteen 4-week-old C57BL/6 male mice were fed with high-fat diet (D12494, 60% energy from fat) until the 16th week. They were divided into two groups: intraperitoneal injection of empty adenovirus group (n = 8) and intraperitoneal injection of adenovirus particles with miR-548ag-inhibitor group (n = 8, 1 × 1011 VP/ mice, once a week) for 6 weeks. Twelve 4-week-old male db/db mice were reared, acclimatised and fed for 1 week and then divided into an intraperitoneally injected empty adenovirus group (n = 6) and an intraperitoneally injected miR-548ag inhibitor adenoviral vector group (n = 6, 1 × 1011 VP/pupil, 1 injection/week) for 6 consecutive weeks.

Cell transfection

Lipofectamine 2000 (Catalogue#: 11668–019; Invitrogen, USA) was used to transfect miR-548ag-mimic, miR-548ag-inhibitor and TLR7/8 interference fragment. HepG2 cells were transfected with miR-548ag-mimic, miR-548ag-inhibitor, TLR7/8 interference fragment at concentrations of 50 nM, 75 nM and 80 nM respectively. L02 cells were transfected with miR-548ag-mimic, miR-548ag-inhibitor, TLR7/8 interference fragment were at concentrations of 50 nM, 75 nM and 80 nM respectively. After 4–6 h of transfection, the transfection reagent was replaced with DMEM containing 10% foetal bovine serum. 24 h later, RNA or protein was extracted,or other treatments were performed.

Cellular glucose consumption and insulin sensitivity assay

Cellular glucose consumption assay: After 12 h of serum-free starvation, cells were transfected with mimic/inhibitor for 24 h, and the culture medium was collected for measuring glucose concentration using the glucose oxidase method.

Cellular insulin sensitivity assay: After 12 h of serum-free starvation, cells were transfected with mimic/inhibitor for 24 h, and Cells were continuously stimulated with 100 nmol/L insulin + low-glucose DMEM (1 g/L) + 10% FBS mixed medium. Culture medium was collected at 0 min, 15 min, 30 min, 45 min, 60 min, 90 min, and 120 min, and glucose concentration was measured.

Western blotting and antibodies

Total proteins from the liver and HepG2, L02 cells were extracted in RIPA Lysis Buffer (Cat#R0010, Solarbio) containing 1% PMSF (Cat#P8340, Solarbio). Lysates were then quantitated and equal amounts of protein were subjected to SDS-PAGE and immunoblotted with antibodies against GAPDH, p65, p-p65, IκB, p-IκB and DPP4. Antibodies against GAPDH (Cat#4967s) were from ZSGB-BIO, antibodies against DPP4 (ab23841) were from Abcam. And antibodies against p65(4764), p-p65 (3033), IκB (4812) and p-IκB (2859) were from Cell Signalling.

Real-time PCR

The miRcute Plus microRNA first-strand cDNA kit and miRcute plus microRNA qPCR Kit used in the experiment were purchased from Beijing TIANGEN, the total mRNA reverse transcription kit was purchased from American Thermo Fisher Scientific, and the real-time quantitative PCR kit was purchased from German QIAGEN. The operation was performed according to the instructions, the primers are shown in Supplementary Table 1.

Plasmid amplification and extraction

The bacteria were incubated in LB medium (peptone: 10 g/L, yeast powder: 5 g/L, NaCl: 10 g/L). and after adding antibiotics, the bacteria were enriched in a constant temperature shaker for 12 hours. The plasmid was extracted according to the instructions of the plasmid extraction kit (TIANGEN).

Intraperitoneal glucose-tolerance and insulin sensitivity tests in mice

Glucose-tolerance tests were performed in mice after 12-h overnight fasting. After determination of FBG levels, an intraperitoneal bolus of 2 g glucose per kg body weight was administered to each mouse. Blood glucose levels were detected after 15, 30, 45, 60, and 120 min.

Insulin-sensitivity tests were performed in mice after 6-h fasting. After determination of FBG levels, an intraperitoneal bolus of 0.5 UI insulin per kg body weight was administered to each mouse. Blood glucose levels were detected after 15, 30, 45, 60, and 120 min.

Detection of DPP4 in human serum

DPP4 in the serum of normal subjects(n = 20) and subjects with obesity (n = 20) was detected. (According to China’s BMI classification: Low weight: BMI < 18.5 kg/m2; Normal weight: 18.5 kg/m2 ≤ BMI < 24 kg/m2; Overweight: 24 kg/m2 ≤ BMI < 28 kg/m2; Obese: BMI ≥ 28 kg/m2). Human DPP4/CD26 ELISA kit was purchased from proteintech. Add 100 µL of sample diluent to the zero well and 100 µL of gradient diluted standard or sample to be tested to the remaining wells, incubate at 37 °C for 2 h, then wash the plate. After shaking off the liquid and tapping the slats, add 100 µL of HRP-labelled detection antibody (1×) to each well and incubate for 40 min at 37 °C. Add 100 µL of TMB Developing Solution to each well and develop the colour for 15–20 min at 37 °C, protected from light, and add 100 µL of Final Solution to each well to change the colour from blue to grey. Measure the optical density (OD) of each well at 450 nm with an enzyme counter using 630 nm as the calibration wavelength.

DPP4 inhibitor and miR-548ag inhibitor administration

Eighteen 4-week-old male C57BL/6 mice, fed a high-fat diet until week 8, were divided into a blank control group (n = 6), a tail-vein injection of miR-548ag inhibitor adeno-associated virus group (n = 6, 1 × 1012vg/ml) and a gavage of DPP4 inhibitor group (n = 6, 30 mg/kg for 4 weeks, 3 times per week gavages). The consent form and ethical approval were provided by the Medical Ethics Committee at the First Affiliated Hospital, Shihezi University School of Medicine (reference number 2019–029–01).

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 analyses

The SPSS statistical package (version 17.0, SPSS Inc, Chicago, IL, USA) was used for statistical data analyses. For data that fitted a normal distribution, statistical differences between groups were determined using an unpaired Student’s t test. For data not fitting a normal distribution, a rank-sum test was performed. Chi square test was used for comparison of counting data. P < 0.05 was considered statistically significant.