LNK deficiency decreases obesity-induced insulin resistance by regulating GLUT4 through the PI3K-Akt-AS160 pathway in adipose tissue

Expression of LNK in adipose tissue and correlations with serum glucose and insulin in obese people

Seventy-two patients were included from January 1, 2017 to June 30, 2018. Adipose tissue samples were obtained from 31 patients who received laparoscopic surgery for fallopian tube or pelvic disorders in the Gynecologic department of Sun Yat-sen Memorial Hospital of Sun Yat-sen University, and 41 patients received abdominal surgery for gastrointestinal tumors or extreme obesity in the First Affiliated Hospital of Guangzhou Medical University. Ages of patients ranged from 21 to 65 years old. Body mass index (BMI) of patients ranged from 16.40 to 36.36 kg/m².

The relationships between the expression of LNK in human omental adipose tissue and metabolic features are shown in Figure 1 and Table 1. LNK showed a decreasing trend with the elevation of BMI and then an increasing trend after a BMI level of 24-28 kg/m² (Figure 1A). The mRNA expression of GLUT4 in human omental adipose tissue showed a downward trend with an elevated BMI (Figure 1B). Expression of LNK was positively correlated to fasting serum glucose (r=0.745, P<0.05), 2-h serum glucose (r=0.924, P<0.05) and fasting serum insulin (r=0.940, P<0.05) in the group of patients with BMI ≥28 kg/m² (Table 1). The expression of LNK in mice visceral adipose tissue decreased after 4 weeks of high-fat diet and subsequently increased after 8 weeks of high-fat diet (Figure 1C). Consistent with this change, LNK relieved impaired glucose tolerance in HFD feeding mice for 4 weeks (Supplementary Figure 2) Immunohistochemistry showed that LNK was mainly expressed in the cytoplasm and a small part of the protein was localized in the plasma membrane of mice adipocytes (Figure 1D).

Figure 1. Expression of LNK in adipose tissue. The mRNA expression of LNK (A) and GLUT4 (B) in human omental adipose tissue (n=72). The representative image (C) of LNK protein expression in adipose tissue of mice with HFD feeding for 0, 4, 8 and 16 weeks were shown (n=6 mice per group). Localization of LNK in adipose tissue of wild type mice with both normal chow diet and high-fat diet (D). Data were shown as mean ± SD.

LNK influences glucose and lipid metabolism in the in vivo model of diet-induced obesity (DIO) mice

An in vivo DIO mice model was successfully built by detecting the body weight, blood glucose level in GTT as well as ITT, and MRI of the abdominal fat tissue volume. There were significant differences in food intake (Figure 2B) and body weight (Figure 2C) between LNK^-/- and WT mice in HFD group (P<0.05). The abdominal anatomy of the mice under high-fat diet and normal chow diet (Figure 2F) were shown. VAT volume of HFD-fed LNK^-/- mice was significantly higher than that of WT mice (Figure 2G).

Figure 2. LNK resulted in worse diet-induced obesity, impaired glucose tolerance and increased insulin resistance under HFD feeding for 16 weeks. Body weights (A) were measured in 6 to 8-week-old LNK^-/- (n=13) or WT mice (n=13). (B–E) Food intake and body weight after HFD or NCD feeding for 16 weeks were shown. Abdominal pathologic specimen (F) and MRI analyses (G) were shown to compare abdominal fat volume. GTTs (H and J) and ITTs (I and K) in WT (filled symbols) or LNK^-/- mice (open symbols) were measured. Blood glucose concentrations were measured at the indicated times. Fasting insulin concentration (L) were measured in mice after NCD or HFD feeding for 16 weeks. HFD: LNK^-/- n=7, WT n=6; NCD: LNK^-/- n=6, WT n=7. Data were shown as mean ± SD. Statistical analysis was performed by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Given that LNK-deletion promoted an insulin-resistant phenotype, we then wanted to examine whether glucose and lipid metabolism were altered in these mice. Compared with LNK^-/- mice, blood glucose levels at 15, 30, 60 min of GTTs and 0, 15 min of ITTs were higher in WT mice after 16-week HFD (P<0.05, Figure 2H and 2I). The fasting insulin levels (32.58±1.64 μIU/mL vs. 41.76±3.24 μIU/mL, P<0.05, Figure 2L) were significantly increased after 16-week HFD in WT mice. There were no significant differences in food intake, body weight, VAT volume, GTTs, ITTs and fasting insulin level in WT and LNK^-/- mice with standard chow diet (P>0.05, Figure 2D, 2E, 2J, 2K and 2L).

Serum lipid and liver enzyme levels were examined in both the HFD and NCD groups. The levels of fasting triglyceride (0.99 ± 0.16 mmol/L vs. 0.75 ± 0.15 mmol/L, P<0.05, Figure 3B), free fatty acids (1373.67 ± 229.25 μmol/L vs. 1051.71 ± 148.00 μmol/L, P<0.05, Figure 3F) and high sensitivity C reactive protein (0.14±0.02 mg/L vs. 0.10±0.02 mg/L, P<0.01, Figure 3G) were elevated in WT mice relative to LNK^-/- mice in the HFD group. Total cholesterol (Figure 3A), HDL (Figure 3C), LDL (Figure 3D), and ApoE (Figure 3E) levels were not significantly different between LNK^-/- and WT mice after 16-week HFD. However, these serum biochemical indexes were not different between LNK^-/- and WT mice which were fed a standard chow diet. These data implied that the presence of LNK may impair glucose and lipid metabolism under a circumstance of obesity-induced insulin resistance.

Figure 3. LNK deficiency influences adipose metabolism and systemic inflammation. Fasting total cholesterol (A), triglyceride (B), HDL (C), LDL (D), apolipoprotein (E), free fatty acid (F), high-sensitivity c-reactive protein (G) levels in mice serum at week 16 on the HFD or NCD feeding. HFD: LNK^-/- n=7, WT n=6; NCD: LNK^-/- n=6, WT n=7. Bars indicate mean ± SD. Statistical analysis was performed by One-way analysis of variance (ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001.

LNK knockout mice show ameliorative hepatic steatosis

HFD-induced obesity can lead to hepatosteatosis, and we found that the deletion of LNK alleviated the hepatic lipid profile in HFD mice (Figure 4). The anatomy and out-of-phase MRI of the liver showed increased lipid deposition in WT mice (Figure 4A–4C). Histological analysis of the liver showed that the severity of hepatic steatosis in the liver was increased with HFD; and WT mice developed more severe steatosis than LNK^-/- mice, with increased hepatic triglycerides (Figure 4D–4F). Markers of adipose differentiation (Figure 4G), lipolysis (Figure 4H) and gluconeogenesis (Figure 4I) were not different between genotypes. CD206 and binding immunoglobulin protein (BIP) were decreased in the livers of LNK^-/- HFD mice (Figure 4J, 4K), which indicated a reduction in inflammation and endoplasmic reticulum stress.

Figure 4. LNK deficiency relieves hepatic steatosis. Representative images of pathologic specimen (A) of liver tissue were shown. Liver weight (B) was compared in WT or LNK^-/- mice after HFD or NCD feeding for 16 weeks. Out-of-phase MRI for diagnosis of fatty liver of mice with HFD (C) and representative images of H&E staining of the liver (D, HFD: LNK^-/- n=7, WT n=6; NCD: LNK^-/- n=6, WT n=7. 10 images were quantified per mouse) were shown. Scale bars: 500 μm, 100 μm and 50 μm. Percentage of fat area in liver pathological tissue slice (E) and liver triglycerides (F) were shown. The mRNA expression of adipose differentiation-related molecule (G), lipolysis-related molecule (H), gluconeogenesis-related molecule (I), inflammation-related molecule (J) and endoplasmic reticulum stress-related molecule (K) in liver tissue of mice fed with HFD after 16 weeks were compared. HFD: LNK^-/- n=7, WT n=6; NCD: LNK^-/- n=6, WT n=7. Data were shown as mean ± SD. Statistical analysis was performed by ANOVA (B, C, E and F) and Student’s t-test (G to K). *p < 0.05, **p < 0.01, ***p < 0.001.

LNK influenced glucose translocation by regulating insulin signaling

Since insulin regulates glucose transporter GLUT4 in adipose and muscle tissues, it seemed probable that adipose GLUT4 levels were elevated in LNK^-/- mice. To test this hypothesis, we measured adipose GLUT4 by WB and IHC, and found that high-fat diet-fed LNK^-/- mice showed marked improvement in glucose transport, however on the chow diet, LNK deficiency did not significantly alter glucose transport (Figure 5A–5C). Furthermore, we found that the insulin signaling pathway was involved. Levels of IRS1 serine phosphorylation, PI3K tyrosine phosphorylation, Akt serine phosphorylation and AS160 threonine phosphorylation adipose tissue decreased in wild-type mice compared with LNK^-/- mice after 16-weeks of HFD. There were no significant differences in the phosphorylation levels of insulin signaling molecules between WT and LNK^-/- mice with standard chow diet (Figure 5E, 5F). Insulin-stimulated glucose uptake in primary adipocytes was significantly improved with LNK knockout. Ex vivo administration of 50 μM LY294002, an inhibitor of phosphatidylinositol 3-kinase, attenuated the insulin-induced glucose uptake in adipocytes derived from VAT of LNK^-/- mice (Figure 5G).

Figure 5. LNK deficiency altered insulin signaling and the expression of GLUT-4 protein in adipose tissues in obesity-induced insulin resistance model. (A, B) Representative images of immunohistochemical stain of GLUT4 in adipose tissue were shown (HFD: LNK^-/- n=7, WT n=6; NCD: LNK^-/- n=6, WT n=7. Five images were quantified per mouse). (C, D) LNK impairs IRS1 serine phosphorylation, PI3K tyrosine phosphorylation, Akt serine phosphorylation and AS160 threonine phosphorylation as shown by WB in HFD-fed mice visceral adipose tissue (LNK^-/- n=7, WT n=6 with three replicates. Representative images were shown). (E, F) GLUT4 expression and IRS1 serine phosphorylation, PI3K tyrosine phosphorylation, Akt serine phosphorylation and AS160 threonine phosphorylation as shown by WB in NCD-fed mice adipose tissue (LNK^-/- n=6, WT n=7 with three replicates. Representative images were shown). (G) LNK deficiency improves glucose uptake in primary adipocyte from mice after HFD feeding for 16 weeks (n=6 per group). The primary adipocytes were differentiated into mature adipocytes and were then stimulated by LY294002 (50 μM) and insulin (10 μM) for 30 min or insulin (10 μM) for 30 min alone. Scale bars: 100 μm. Data were shown as mean ± SD. Statistical analysis was performed by ANOVA (B and G) and Student’s t-test (D and F). *p < 0.05, **p < 0.01, ***p < 0.001.

Human samples

The study protocol was approved by the Research Ethics Board of Sun Yat-sen memorial hospital of Sun Yat-sen University and the first affiliated hospital of Guangzhou Medical University. All participants signed a consent form before the surgery. Seventy-two patients were included from January 1, 2017 to June 30, 2018. Adipose tissue samples were obtained from 31 patients who received laparoscopic surgery for fallopian tube or pelvic disorders in the Gynecologic department of Sun Yat-sen Memorial Hospital of Sun Yat-sen University, and 41 patients received abdominal surgery for gastrointestinal tumors or extreme obesity in the First Affiliated Hospital of Guangzhou Medical University. Individuals with a history of cardiovascular or cerebrovascular disease, severe hepatic disease, severe renal disease, autoimmune diseases, immunodeficiency, or individuals receiving immunosuppressive treatment were excluded from this study. Omental adipose tissue (4–6 g) from the peripheral portion of the omentum majus was collected from the patients during surgery. Samples were immediately put in liquid nitrogen for further experiments.

Animals

All of the experimental procedures were approved by the Committee for Animal Research of Sun Yat-sen University and were in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Male C57BL/6 wild-type mice were purchased from the animal research center of Sun Yat-sen University. Whole-body LNK-deficient on C57BL/6 genetic background mice were created by CRISPR/Cas-mediated genome engineering by Cyagen Biosciences Inc. The mouse Sh2b3 gene (GenBank accession number: NM_001306127.1; Ensembl: ENSMUSG00000042594) is located on mouse chromosome 5. Exon 1 to exon 3 were selected as target sites. Cas9 mRNA and gRNA generated by in vitro transcription were then injected into fertilized eggs for knock-out mouse production. The founders were genotyped by PCR followed by DNA sequencing analysis. The positive founders were bred to the next generation (F1) and subsequently genotyped by PCR and DNA sequencing analysis. (Supplementary Figure 1).

Mice were housed in numbers of four to six per cage with standard laboratory conditions (12 h light:12 h darkness cycle) and free access to rodent feed and water. Mice (4-5 weeks old) were fed with a high-fat diet (HFD, 60% calories from fat, D12492; Research Diets Inc.) or normal chow diet (NCD, 10% calories from fat, D12450; Research Diets Inc.) for 0, 2, 4, 8 or 16 weeks (n=6 or 7 per group). At the end of the diet intervention, mice were anesthetized with sodium pentobarbital (Sigma-Aldrich) at 50 mg/kg (intraperitoneal, i.p.). Blood, liver, epididymal fat pads, perirenal fat and other visceral adipose tissue were collected.

Measurements of metabolic parameters

Food intake and body weight were measured once a week. Briefly, for the glucose tolerance test (GTT), mice which were fasted overnight were injected (i.p.) with D-glucose (1 g/kg body weight; Sigma-Aldrich) after 12 h fasting. For the insulin tolerance test (ITT), mice were injected (i.p.) with insulin (0.75 units/kg body weight; Novolin R, Novo Nordisk, Copenhagen, Denmark) after 6 h fasting. Glucose concentrations were measured in tail venous blood using an automated glucometer (Roche Diagnostics) up to 90 min or 120 min after injection.

Fasting insulin was measured by the insulin ELISA kit (Cusabio, Wuhan, China). Liver concentrations of triglyceride (TG) was measured by the triglyceride assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Serum concentrations of total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL), apolipoprotein E (ApoE), FFA and hsCRP were measured by enzymatic method using an automatic biochemical analyzer (Beckman AU5800, USA).

Magnetic resonance imaging (MRI) of lower abdominal visceral adipose tissue (VAT)

The volume of mouse abdominal VAT was measured in vivo via MRI (Philips Ingenia 3.0T, Netherlands) equipped with a mouse 8-channel coil. After 16 weeks of HFD feeding, mice were anesthetized with pentobarbital (50 mg/kg) and fixed in an entirely stretched supine position to minimize posing error. MRI T1WI-TSE-TRA, T1WI-TSE-COR and T1WI-HR-mDIXON-COR sequences were conducted to image the abdominal region. The scan was constructed for the whole abdominal region, which begins from the diaphragm to the pelvic floor. The volume of total abdominal adipose tissue was calculated using the regions of interest (ROI) by Image-Pro Plus 6.0 (Media Cybernetics, Inc., Singapore).

Histological and immunohistochemical staining

The mouse epididymal fat and liver tissue were fixed in 4% paraformaldehyde (Sigma-Aldrich), embedded in paraffin and sectioned at 10 μm. Paraffin-embedded tissue sections were stained with H&E. Immunohistochemistry was then performed to detect the location of LNK and the expression of GLUT4 in adipose tissue. Briefly, five randomly selected sections in each sample were exposed to primary antibodies (LNK, 1:50, Abcam; GLUT4, 1:200, Servicebio) or goat serum as the negative control at 4°C overnight, followed by incubation with corresponding secondary antibodies. Histological images were captured using a Nikon Instruments Eclipse Ni-U microscope (Japan) with plan×4, ×10, ×20, or ×40 objective lenses.

Cell culture

The primary preadipocytes were firstly isolated from the mouse epididymal fat tissue. The adipose tissue was minced in PBS with 0.5% BSA, and then incubated with digestion buffer [DMEM, 12.5 mM HEPES (pH 7.4), 2% BSA, and 10 mg collagenase type I (Sigma)] in a 37°C shaking water bath (115 r/min) for 40 min. The digested tissue was filtered through a 250-μM nylon sieve and centrifuged at 500×g for 5 min. The pellet containing preadipocytes was incubated with an erythrocyte-lysing buffer, washed and spun at 300×g for 5 min to obtain the preadipocytes. Primary preadipocytes were cultured in DMEM medium (GIBCO) containing 10% fetal bovine serum (FBS, GIBCO) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO₂. Differentiation of preadipocytes to adipocytes was induced by an adipogenic cocktail. Briefly, after primary preadipocytes reached confluency, the cells were incubated in differentiation medium (DM) containing 1 μM DEX (Sigma), 0.5 mM IBMX (Sigma), and 10 μg/mL insulin (BI) in DMEM with 10% FBS for 6 days. The cell culture medium was changed to post-DM containing 10 μg/mL insulin in DMEM with 10% FBS and post-DM was freshly replaced every 48 h. Differentiated cells were used when at least 95% of the cells showed an adipocyte phenotype by accumulation of lipid droplets. Differentiated cells were stimulated by LY294002, a PI3K inhibitor (Absin, Shanghai, China, 50 μM) for 30 min or insulin (Biological Industries, Israel, 10 μM) for 30 min.

Insulin stimulation studies and glucose uptake assays

For insulin stimulation studies, cells were washed twice with warm PBS and serum starved in serum-free DMEM containing 0.5% bovine serum albumin 16 h before insulin stimulation. On the day of the assay, the medium was replaced with 100 μL of serum-free DMEM with or without 50 μM LY294002 and incubate for 30 min at 37°C in 5% CO₂. 10 μL of serum-free DMEM with or without 10 μM insulin was added and incubated for 30 min. Glucose uptake was assessed on Day-10 of differentiation in 96-well plates using the Glucose Uptake-Glo™ Assay (Promega, WI, USA). The medium was removed and washed with 100 μL of PBS if glucose was present. 50 μL of the prepared 1 mM 2DG was added per well, shaken briefly, and incubated for 10 min at room temperature. 25 μL of Stop Buffer was added and shaken briefly. 25 μL of Neutralization Buffer was then added and shaken briefly. Subsequently, 100 μL of 2DG6P Detection Reagent was added and shaken briefly. Incubation was performed for 30 min at room temperature and the luminescence was recorded with 0.3-1 s integration on a luminometer.

Quantitative real-time PCR

Total RNA was extracted from tissues or purified cells using RNAiso Plus reagent (9109, TAKARA, Japan) according to the manufacturer’s protocols. RNA was reverse-transcribed using the Primescriptor RT Master Mix (RR036A, TAKARA, Japan). Quantitative RT-PCR was performed using the TB Green Premix Ex Taq II (RR820A, TAKARA, Japan). Primer sequences are provided in Supplementary Table 1. As for the calculation, we selected one of the patients as a reference, and the 2^-ΔCT value of all other patients was divided by the 2^-ΔCT value of this patient. "1.0" represent for the mRNA value of the selected patient.

Western blotting

Tissue or cells were lysed using RIPA lysis buffer (Beyotime, Nanjing, China) containing protease inhibitors and phosphatase inhibitors (Complete; Roche). Equal amounts of protein (quantified by BCA protein assays, Thermo Scientific) in each sample were analyzed by SDS-PAGE and immunoblotting. Protein samples were stored at -80°C before the experiments. Samples were separated on 10% SDS polyacrylamide gels and then transferred to polyvinylidene difluoride membrane (Bio-Rad). The membrane was incubated with corresponding primary antibodies overnight. On the next day, the membrane was further incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h. The bands were detected by enhanced chemiluminescence (ECL) detection system (Affinity Biosciences, OH, USA). Invitrogen iBright CL1000 Imaging System was used to expose the bands. The primary antibodies are provided in Supplementary Table 2. Densitometric analysis was performed with ImageJ software (NIH, Bethesda, MD).

Statistical analysis

All data were presented as mean ± SEM. Statistical analyses were performed using Student’s t-test or one-way ANOVA followed by Bonferroni post-hoc test. Pearson correlation analysis was used to determine the relationship of LNK mRNA expression in adipose tissue and metabolic indexes in patients. A P-value <0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS 22.0 (SPSS, Inc., Chicago, IL, USA). Figures were created with GraphPad Prism 8.0 (San Diego, CA, USA).