All animal experiments were approved by the Animal Care and Use Committee of Nanjing Drum Tower Hospital, Nanjing Medical University (approval no. 2019AE02013; Nanjing, China). The present study complies with all relevant ethical regulations of Nanjing Drum Tower Hospital Ethics Committee.

A total of 16 male Sprague-Dawley rats (weight, 150±10 g; age, 4 weeks) were purchased from the animal core facility of Nanjing Medical University (Jiangsu, China). The rats were housed in specific pathogen-free units of the Animal Center at Nanjing Drum Tower Hospital maintained at 60-70% relative humidity and 23±1˚C, with a 12-h light/dark cycle and ad libitum access to a 60% high-fat diet (HFD; cat. no. D12492; Research Diets, Inc.) and water. After 8 weeks on HFD, T2D was induced by intraperitoneal injection of 30 mg/kg streptozotocin (STZ; MilliporeSigma). Random blood glucose levels were measured 72 h following STZ injection with a hand-held glucose meter (OneTouch UltraVue; Johnson & Johnson). Rats with random blood glucose levels >16.0 mmol/l on three consecutive days were considered diabetic. Diabetic rats were randomly assigned to the sham or the SG group (n=8/group). Body weight, food intake and fasting glucose (FG) levels were measured pre-operatively and post-operatively at week 2, 4, 6 and 8. An oral glucose tolerance test (OGTT) was performed pre-operatively and at week 8 post-surgery. The body fat content and distribution were also measured at week 8 using dual-energy X-ray absorptiometry (DEXA). The ingWAT was then harvested for subsequent experiments.

Rats were fasted overnight and then anesthetized using isoflurane (3% for induction; 2% for maintenance). Under sterile conditions, a single 3-cm epigastric laparotomy was performed. As presented in Fig. 1A, the greater curvature of the stomach from the antrum to the fundus and glandular stomach was dissected, ~70% of the gastric volume was excised and the gastroesophageal junction and the pylorus were preserved. For the sham group, a similar length incision was made in the anterior gastric wall, then sutured using a 6-0 absorbable thread. Abdominal closure was performed using 3-0 silk suture with a continuous suture technique. The rats received ibuprofen (15 mg/kg of body weight daily) to minimize post-operative pain for 2 days. After allowing 24 h for the anastomoses to heal, the animals were kept on a liquid diet for 3 days before being returned to normal chow for an additional 8 weeks.

Primary pre-adipocytes were isolated from the ingWAT of 4-week-old SD rats. In brief, adipose tissue samples were collected from the inguinal region under sterile conditions and washed in PBS three times. The tissue mass was cut with scissors into ~1-mm sections and digested with 0.1% type-I collagenase at 37˚C for 1 h in a shaking water bath. High-glucose (4.5 g/l) DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was added to stop digestion. To remove undigested tissue fragments and large cell aggregates, the digested tissue suspension was filtered through 100-µm nylon mesh (Falcon). The cells were collected by centrifugation at 233 x g for 10 min at room temperature (RT). The cells were then resuspended in high-glucose DMEM supplemented with 10% FBS and 1% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO₂.

For white adipocyte in vitro differentiation, fully confluent pre-adipocytes were kept in DMEM + 10% FBS growth medium for 2 days, then treated with induction medium (DMEM + 10% FBS) supplemented with 10 µg/ml insulin, 1 µM dexamethasone and 0.5 mM isobutylmethylxanthine (MilliporeSigma). After 2 days, the cells were kept in medium supplemented with 10 µg/ml insulin for a further 2 days. The culture medium was then replaced with fresh DMEM containing 10% FBS every 2-3 days. The cells were fully differentiated into mature adipocytes on day 9.

Differentiated adipocytes were washed three times with phosphate-buffered saline and were subsequently fixed in 4% paraformaldehyde at RT for 30 min. Afterwards, adipocytes were exposed to Oil Red O for 1 h at RT. To remove the staining solution, cells were washed several times with distilled water. Representative images were taken using a light microscope (Zeiss AG).

The negative control (NC) and specific siRNA against Shp2 were purchased from HIPPOBIO, Inc. Pre-adipocytes were seeded in 6-well plates at a density of 60-70% confluence and transfected with 50 nM siRNA using Lipofectamine^® 3000 transfection reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. After 6 h of transfection, the medium was replaced with normal medium and the cells were cultured for another 18 h at 37˚C with 5% CO₂, and then harvested for transfection efficiency evaluation using western blot analysis. The transfected cells were collected to perform subsequent experiments at least 48 h after transfection and transfection was performed every 72 h during differentiation. The NC siRNA consisted of a scrambled sequence that would not cause the specific degradation of any known cellular mRNA. The siRNA sequences are presented in Table I.

Lentivirus-containing expression cassettes were purchased from Syngentech Co. Ltd., and lentivirus preparation and packaging were performed by the company. Lentiviral transduction was performed following the manufacturer's instructions. Briefly, 5x10⁵ ingWAT pre-adipocytes were seeded in T25 flasks. The next day, these cells were infected with Shp2 lentiviral expression vector (pLV-hef1a-mNeongreen-P2A-Puro-WPRE-CMV-Ptpn11) or control lentiviral expression vector (pLV-hef1a-mNeongreen-P2A-Puro-WPRE-CMV-MCS) at MOI values of 50 at 37˚C with 5% CO₂. The culture media were replaced at 12 h post-transfection, and the cells were incubated for a further 48 h. At 72 h post-transfection, the transfected cells showing green fluorescence were observed under a fluorescence microscope and transduction efficiency was evaluated using western blot analysis. The transfected cells were collected to perform subsequent experiments 4 days after transfection. Stably transduced cells were selected using 2 µg/ml puromycin (Beyotime Institute of Biotechnology) for 3 days.

At week 8 post-surgery or if a humane endpoint, including listlessness, infection or eating cessation or drinking cessation could not be relieved, rats were euthanized by CO₂ inhalation. The flow rate of CO₂ was 20-30% of the chamber volume per minute. Death was confirmed based on the absence of a heartbeat, respiration and corneal reflex. After the rats were sacrificed, their ingWAT was fixed in 4% paraformaldehyde at RT for 24 h. The samples were then embedded in paraffin and cut into 5-µm coronal slides using a microtome (Thermo Fisher Scientific, Inc.). After at 60˚C for 30 min, the tissue slides from the sham and SG groups were deparaffinized in xylene, rehydrated in gradient ethanol (each for 5 min) and then washed under running water for 3 min. Tissue slides were stained using hematoxylin and eosin (Beyotime Institute of Biotechnology). The slides were immersed in hematoxylin at RT for 3 min and rinsed with running water, sequentially followed by differentiation for 30 sec in 1% acid-alcohol (hydrochloric acid and ethanol) at RT and then rinsed again with running water. The slides were then stained with eosin at RT for 30 sec and rinsed again with water. The slides were air-dried at RT. Subsequently, the slides were sequentially immersed in 75, 85, 95 and 100% ethanol, a solution of 50% ethanol and 50% xylene, and 100% xylene, each for 1 min. The slides were then observed under a light microscope (Zeiss AG). The diameter of the adipocytes was measured using ImageJ (version 1.8.0; National Institutes of Health).

After heating at 60˚C for 30 min, histological slides of ingWAT were deparaffinized in xylene, then rehydrated using decreasing concentrations of ethanol ranging from 100 to 50%. The slides were blocked with 5% bovine serum albumin (MilliporeSigma) for 1 h at RT, then incubated with a primary antibody against Shp2 (1:200 dilution; cat. no. sc-7384; Santa Cruz Biotechnology, Inc.) overnight at 4˚C. The slides were washed with PBS and incubated with tetramethylrhodamine-conjugated secondary antibody (1:200 dilution; cat. no. ab6786; Abcam) for 1 h at RT in the dark. The slides were counterstained with DAPI at RT in the dark, and fluorescence images of randomly selected fields (n=3) were obtained using a fluorescence microscope (Zeiss AG).

The triglyceride (cat. no. 1025; Applygen Technologies, Inc.) and leptin (cat. no. ml002969; Mlbio) levels in ingWAT samples were measured using ELISA kits according to the manufacturer's protocols. The absorbance was read at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

WAT and adipocytes were lysed with RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing 1 mM phenylmethanesulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.) and 1 mM phosphatase inhibitor cocktail (Bimake). Lysate protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Equal quantities of protein (10 µg/well) were separated using 10% (w/v) SDS-PAGE (Epizyme, Inc.) and were subsequently transferred to PVDF membranes (MilliporeSigma) according to standard procedures. After the membranes were blocked with 5% (w/v) milk (Biofroxx) for 1 h at RT, the membranes were incubated with primary antibodies (1:1,000 dilution) against Shp2 (cat. no. 3397), β-catenin (cat. no. 84441) (both Cell Signaling Technology, Inc.), peroxisome proliferator-activated receptor γ (PPARγ; cat. no. AP0686; Bioworld Technology, Inc.), CCAAT/enhancer-binding protein alpha (Cebpα; cat. no. ab40764; Abcam), adiponectin (cat. no. 2789; Cell Signaling Technology, Inc.), leptin (cat. no. DF8583; Affinity Biosciences) and β-actin (cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4˚C. A horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG (cat. no. BL003A/BL001A; 1:5,000; Biosharp Life Sciences) was used as a secondary antibody for 1 h at RT. All signals were detected using the ChemiDocXRS⁺ Imaging System (Tanon Science and Technology Co., Ltd.). Quantitative analysis of protein densitometry was performed using Image J (version 1.8.0; National Institutes of Health).

Total RNA was extracted from WAT and adipocytes using the RNA-quick Purification Kit (ES Science) according to the manufacturer's instructions. cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Vazyme Biotech Co., Ltd.) in accordance with the manufacturer's instructions. qPCR was performed using a SYBR Green QPCR kit (Vazyme Biotech Co., Ltd.) on a LightCycler 480 PCR System (Roche Diagnostics). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 2 min, followed by 40 cycles of 95˚C for 20 sec, 60˚C for 20 sec and 72˚C for 20 sec, and then a final step at 78˚C for 5 min. The fold changes in the expression levels of each gene were calculated using the 2^-ΔΔCq method (27). The mRNA expression levels for each target gene were normalized to those of β-actin. The primer sequences are presented in Table II.

The viability of pre-adipocytes was assessed using CCK-8 (Dojindo Laboratories, Inc.) assays according to the manufacturer's instructions following treatment with recombinant leptin rat (0, 5, 25, 50, 100 and 200 ng/ml) (Bioworld Technology, Inc.), SHP099 (0, 1, 5, 10, 50, 100 and 200 µM) or PNU-74654 (0, 0.5, 1, 10, 25, 50 and 100 µM) (MedChemExpress) at 37˚C with 5% CO₂ for 24 h. The absorbance at 450-nm wavelength was detected using a microplate reader (Thermo Fisher Scientific, Inc.).

SPSS (version 26.0; IBM, Corp.) and GraphPad Prism software (version 8.4; GraphPad Software, Inc.) were used for statistical analysis. Quantitative data are representative of at least three independent experiments. Unpaired two-tailed Student's t-tests were used to compare the means of two groups. Differences between multiple groups were detected using one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test as appropriate. The data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

The body shapes of rats 8 weeks after surgery are presented in Fig. 1B. There were no significant differences in body weight or food intake between the sham and the SG group pre-operatively. Throughout the post-operative period, the SG group had a significantly lower body weight and food intake compared with those of the sham group (P<0.01; Fig. 1C and D). During the post-operative period, the FG levels declined significantly in the SG group compared with the sham group (P<0.05; Fig. 1E). Compared with week 0, the FG levels in the SG group were significantly reduced (70.8±10.1% reduction; P<0.001) at week 8 post-surgery. Compared with the pre-operative results (Fig. 1F), the SG group demonstrated marked improvements in glucose control following surgery (Fig. 1G). Moreover, the SG group revealed significantly improved glucose control compared with the sham group post-surgery (P<0.001; Fig. 1H). The pre- and post-operative results of the sham group did not differ significantly.

The SG group had significantly lower body fat compared with the sham group (P<0.01; Fig. 1I and J). As presented in Fig. 1K, the volume of adipose tissue of the SG group was smaller than those of the sham group. Histological analysis of adipose tissue sections revealed smaller adipocyte sizes in rats from the SG group compared with the sham (P<0.001; Fig. 1L and M). At week 8 post-surgery, the SG group exhibited significantly lower triglyceride (P<0.001) and leptin (P<0.001) levels in adipose tissue than the sham group (Fig. 1N and O). Furthermore, the expression of the adipogenic markers, PPARγ and Cebpα, were significantly downregulated in ingWAT from the SG group compared with the sham group (P<0.05; Fig. 1P and Q).

To investigate the effect of leptin on the expression of Shp2, ingWAT pre-adipocytes were treated with different concentrations of leptin (0, 5, 25, 50, 100 and 200 ng/ml) during differentiation. The results indicated that Shp2 and β-catenin protein levels were markedly reduced by leptin in a dose-dependent manner (Fig. 2A). Marked changes in Shp2 and β-catenin protein expression were observed after 100 and 200 ng/ml leptin treatment. CCK-8 assay was performed to evaluate the cell viability, the results of which suggested that there was no effect on cell viability at 100 ng/ml leptin (Fig. 2B). Subsequently, 100 ng/ml leptin was used to stimulate ingWAT pre-adipocytes during differentiation. As presented in Fig. 2C, leptin treatment markedly increased the lipid accumulation of adipocytes. In addition, the mRNA expression level of Shp2 and fatty acid-binding protein 4 (Fabp4), and the mRNA or protein expression levels of the other adipogenic markers, including PPARγ, Cebpα, adiponectin and leptin significantly increased (all P<0.05; Fig. 2D-F).

The mRNA and protein expression levels of Shp2 and β-catenin were significantly upregulated following SG (P<0.01 and P<0.001; Fig. 3A-C). Moreover, the fluorescence intensity of Shp2 in ingWAT was significantly increased in the SG group compared with the sham group (P<0.05; Fig. 3D and E).

To confirm that the observed changes in Shp2 were associated with inhibition of pre-adipocyte differentiation at day-2 post-confluence, growth-arrested ingWAT pre-adipocytes were induced into mature adipocytes. The differentiation status of ingWAT pre-adipocytes was monitored by Oil Red O staining, which is indicative of terminal differentiation (Fig. 4A). The results suggested that the mRNA and protein levels of Shp2 were significantly reduced following treatment with inducers (P<0.001), and the mRNA expression levels of adipogenic markers significantly increased (P<0.01 and P<0.001; Fig. 4B and C).

To examine the role of Shp2 during ingWAT pre-adipocyte differentiation, siRNA was used to knock-down the expression of Shp2. The results demonstrated that Shp2 knockdown resulted in increased lipid accumulation throughout differentiation (Fig. 5A). Additionally, Shp2 siRNA significantly inhibited both the mRNA and protein expression of Shp2 at day 9 post-induction (P<0.05 and P<0.001; Fig. 5B-D). Triglyceride levels were also significantly increased (P<0.001; Fig. 5E) and there were significant increases in the mRNA levels of adipogenic markers (P<0.01; Fig. 5F). Analysis of protein expression confirmed the adipogenic effects of Shp2 knockdown. PPARγ, Cebpα and adiponectin protein levels were significantly increased at differentiation day 9 in the Shp2 knockdown group compared with the NC group (P<0.05; Fig. 5G and H). Although the protein expression levels of leptin increased following Shp2 knockdown, this was not statistically significant. Furthermore, the expression of β-catenin, a protein involved in the Wnt signaling pathway, was significantly reduced in the Shp2 knockdown group compared with the NC group (P<0.05; Fig. 5G and H).

Furthermore, similar experiments were carried out in ingWAT pre-adipocytes in the presence of the Shp2 inhibitor, SHP099, in the induction medium at different time points. IngWAT pre-adipocytes were treated with different concentrations of SHP099, and cell viability was measured using CCK-8 assays, with the results showing that cell viability decreased with increasing SHP099 concentration (Fig. 6A). The optimal protective effect was conferred by 10 µM, so this concentration was selected for subsequent experiments. As presented in Fig. 6B-F, SHP099 had a significantly positive effect on cellular lipid accumulation (P<0.001; Fig. 6B and C) and significantly increased the mRNA and protein levels of adipogenic markers compared with the NC group (P<0.05; Fig. 6D-F). Moreover, the protein expression of β-catenin was decreased significantly compared with that in the NC group (P<0.05; Fig. 6E and F). These results suggested that knockdown or inhibition of Shp2 promoted ingWAT adipocyte differentiation, and these adipogenic effects may be mediated by the Wnt/β-catenin signaling.

To determine whether the Wnt/β-catenin signaling pathway was involved in the effects of Shp2 on pre-adipocyte differentiation, a small-molecule inhibitor of the Wnt/β-catenin pathway, PNU-74654 (28,29), was used to test this hypothesis. The pre-adipocytes were treated with different concentrations for 24 h, cell viability was measured using CCK-8 assays. When the concentration reached 10 µM, cell viability was not significantly affected, and an optimal concentration of 10 µM was selected for subsequent experiments (Fig. 7A). Shp2 was overexpressed in ingWAT pre-adipocytes using lentiviral transduction. At day 9 of differentiation, lipid accumulation markedly decreased in the Shp2-overexpressing cells (Fig. 7B). Moreover, the triglyceride levels of Shp2-overexpressing adipocytes were significantly reduced compared with the vector group (P<0.01; Fig. 7C). Furthermore, the overexpression of Shp2 decreased the mRNA and protein levels of adipogenic markers (P<0.05; Fig. 7D-F), while the protein expression level of β-catenin was significantly upregulated (P<0.05; Fig. 7E and F). These results suggested that Shp2 may suppress adipocyte differentiation through the Wnt/β-catenin pathway. Next, PNU-74654 was added to the inducing medium at different time points, and the protein expression of β-catenin was significantly decreased compared with the Shp2 overexpression group (P<0.05; Fig. 7E and F). As presented in Fig. 7B, Shp2-overexpressing adipocytes treated with PNU-74654 displayed markedly increased lipid accumulation. In addition, triglyceride levels were also significantly increased compared with the Shp2 overexpression group (P<0.01; Fig. 7C). Furthermore, the mRNA and protein expression of adipogenic markers were significantly increased compared with the Shp2 overexpression group following treatment with PNU-74654 (P<0.05; Fig. 7D-F). These results suggested that Shp2 suppressed ingWAT adipocyte differentiation by activating the Wnt/β-catenin signaling pathway.