Stearic acid (S4751) was obtained from Sigma (St. Louis, MO, USA). We prepared its stock solution by dissolving stearic acid in ethanol and saponification with sodium hydroxide. After drying, the sodium salt was resuspended in saline, and then, heated at 80 °C until it was dissolved completely. Then, 20% (wt/vol) BSA was added. Then, the complex was sterilized and aliquoted. The final stock concentration was 3 mmol/L [29]. The working concentration of stearic acid was 400 µmol/L. Metformin (CAS No. 1115-70-4, Biotopped, Beijing, China) was dissolved in cell culture medium to prepare a stock solution of 100 mmol/L, which was diluted in cell culture medium.

Mouse $\beta$-TC6 cells were purchased from the Shanghai Academy of Chinese Sciences Cell Library and incubated in Dulbecco’s modified Eagle’s medium (12800017, Gibco/Life Technologies, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), 1.5 g/L NaHCO${}_3$, and 100 IU/mL penicillin–streptomycin mix [29]. The cell line has been authenticated by short tandem repeat, and mycoplasma testing has been done.

Cell viability was assessed using Cell Counting Kit 8 (C0038; Beyotime Biotechnology, Shanghai, China). For this purpose, $\beta$-TC6 cells were seeded in a 96-well plate and 10 µL of Cell Counting Kit 8 reagents were added to each well and incubated for 2 h at 37 °C. Absorbance was detected at 450 nm with a microplate reader (SpectraMax M2; Molecular Devices, San Jose, CA, USA), as described previously [29].

$\beta$-TC6 cells were transfected with miR-297b-5p mimics, anti-miR-297b-5p oligonucleotides (AMO-297b-5p), siRNA-Igf1r, or their negative controls using Lipofectamine 2000 (11668019; Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions and our previous study [29]. miR-297b-5p mimics, anti-miR-297b-5p oligonucleotides, and their negative controls were purchased from RiboBio Co. Ltd (Guangzhou, Guangdong, China). siRNA-Igf1r (sc-35638)and its negative control (sc-37007) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The sequences of these oligonucleotides are displayed in Table 1.

$\beta$-TC6 cells were preincubated in secretion buffer (129 NaCl, 4.8 KCl, 1.2 MgSO${}_4$, 1.2 KH${}_2$PO${}_4$, 2.5 CaCl${}_2$, 5.0 NaHCO${}_3$, 10 HEPES (all mmol/L) and 1 mg/mL bovine serum albumin, adjusted to pH 7.4) with 2.8 or 20 mmol/L glucose [5]. The supernatant was collected for insulin measurement and $\beta$-TC6 cells were, then, lysed to measure the total protein content using a bicinchoninic acid (BCA) protein assay reagent kit (Cat. No. P0010, Beyotime Biotechnology). Insulin levels were measured using a mouse/rat insulin ELISA kit (Cat. No. EZRMI-13K, Millipore, Burlington, MA, USA). The supernatants obtained after stimulation with 2.8 mmol/L and 20 mmol/L glucose were diluted at 1:10 and 1:30 for insulin measurement, respectively. Insulin levels were normalized to the milligrams of protein present in each well.

$\beta$-TC6 cells were seeded into a 24-well plate at 6 $\times$ 10${}^4$ cells/well and cultured at 37 °C in a 5% CO${}_2$ humidified incubator. The senescence status was analyzed using a Senescence $\beta$-Galactosidase Staining Kit (Cat. No. C0602, Beyotime Biotechnology). Cells were washed with phosphate-buffered saline (PBS) and fixed in the senescence-associated $\beta$-galactosidase fixative solution for 15 min at room temperature. After washing three times with PBS, the cells were incubated in senescence-associated $\beta$-galactosidase working solution overnight at 37 °C without CO${}_2$. To calculate the number of senescent cells, five images of each well were randomly selected and analyzed blindly. The percentage of senescence-associated $\beta$-galactosidase-positive cells (blue) was determined by dividing the number of positive cells by the total number of cells present in each image [31, 32], which was determined by Hoechst 33342 staining (C1022, Beyotime Biotechnology).

Immunocytofluorescence was performed as described [33]. Briefly, $\beta$-TC6 cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with PBS containing 0.1% Triton X-100 (P0096, Beyotime Biotechnology). After washing and blocking, the cells were incubated overnight at 4 °C with a primary antibody against the insulin-like growth factor-1 receptor (IGF1R) (AF6125, 1:250, Affinity Biosciences, OH, USA). Then, the cells were incubated in the dark for 1 h at room temperature in the presence of the secondary anti-rabbit IgG (#4413, 1:600, Cell Signaling Technology, Danvers, MA, USA) and subsequently counterstained with Hoechst 33342 (C1022, Beyotime Biotechnology) for counting. The slides were observed with a laser confocal microscope. Five random images of each slide were selected to quantify the fluorescence intensity.

Cells were harvested in PBS and lysed with intermediate RIPA lysis buffer (Cat. No. P0013C, Beyotime Biotechnology). Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay kit, Cat. No. P0012, Beyotime Biotechnology. Whole-cell lysates (50 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane [29]. Primary antibodies against insulin-like growth factor-1 receptor (IGF1R) (AF6125, 1:1000, Affinity Biosciences, OH) and $\beta$-actin (#4970S, 1:1000, Cell Signaling Technology, Massachusetts) were used. The secondary antibody was an anti-rabbit alkaline phosphatase-conjugated antibody (S373B, 1:7500, Promega, Wisconsin). Proteins were visualized using Stabilized Substrate for Alkaline Phosphatase (S3841, Promega) and the FluorChem R system (ProteinSimple, San Jose, CA, USA).

pmiR-RB-REPORT dual luciferase reporter vectors carrying the 3${}^{\prime }$-untranslated region (UTR) of insulin-like growth factor-1 receptor containing wildtype or mutated target sites for miR-297b-5p were constructed by RiboBio (Guangzhou, Guangdong, China). Plasmids (200 ng) were cotransfected into human embryonic kidney (HEK-293) cells (ATCC, Manassas, VA, USA) with miR-297b-5p mimics or the negative control. After transfection for 24 h, a dual luciferase reporter assay kit (E1910, Promega, Madison, WI, USA) was used to measure luciferase activity in a GloMax20/20 Luminometer (Promega) [29].

Overall, 7-week-old male C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Company (Beijing, China). After adaptation, they were randomly divided into two groups that were either fed a control diet or a diet high in stearic acid (n = 15 per group). The compositions of normal and high stearic acid diets were the same as described in our previous study [29]. After 12 weeks of feeding, the mice were sacrificed by CO${}_2$ asphyxiation followed by the collection of pancreatic tissue and blood samples. Mouse islets were isolated by Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, China).

After overnight fasting, the mice were administered glucose (0.75 g/kg) via the tail vein, as described previously [5]. Serum insulin and glucose concentrations were measured 0, 1, 5, 10, 20, 30, and 60 min after the administration of glucose.

Non-esterified fatty acid profile analysis of fasting serum was performed using a TRACE gas chromatograph with a Polaris Q mass spectrometer (Thermo Finnigan, Austin, TX, USA), as described previously [3]. Fasting glucose, total cholesterol (TC), triacylglycerol (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured by an automatic analyzer (Hitachi-7100, Hitachi, Tokyo, Japan). All kits were purchased from Biosino Biotechnology (Beijing, China). Serum insulin levels were measured using a mouse/rat insulin ELISA kit (Cat. No. EZRMI-13K, Millipore, Burlington, MA, USA) with a standard curve ranging from 0.2–10 ng/mL. Inter- and intra-assay variations of this kit were 6.0–17.9 and 0.9–8.4, respectively.

Pancreatic tissues were fixed and then embedded in paraffin. Insulin-positive $\beta$-cells were immunostained with anti-insulin antibody (BM1621, Boster), and glucagon-positive $\alpha$-cells were immunolabeled with anti-glucagon antibody (3014S, Cell Signaling Technology) [5].

Total RNA was extracted from $\beta$-TC6 cells using TRIzol reagent (15596026, Invitrogen, Carlsbad, CA, USA), as described in our previous study [29]. mirVana miRNA Isolation Kit (AM1561, Ambion, Austin, TX, USA) was used for miRNA isolation. Quantitative polymerase chain reaction was performed using SYBR Green PCR Master Mix (4367659, Applied Biosystems, Foster City, CA, USA). Levels were normalized to $\beta$-actin for mRNA and U6 for miRNA. All primers were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China), and their sequences are listed in Table 2.

All data were presented as mean $\pm$ standard deviation. SPSS version 21.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Differences between two groups were analyzed using a two-tailed Student t-test. One-way ANOVA followed by a Student-Newman–Keuls test was carried out to test differences among multiple groups. A two-sided p value 0.05 was considered statistically significant.

We assessed the effect of metformin on the survival rate of $\beta$-TC6 cells and we observed a significant destructive effect only at concentrations $\ge$400 µmol/L (Fig. 1A, Supplementary Fig. 1). Incubation of $\beta$-TC6 cells, for various times (6, 12, 24, and 48 h), with 50 µmol/L metformin, a concentration previously used by others [23, 34], showed that cell viability was significantly impacted only after 48 h incubation (Fig. 1B). Treatment with 50 µmol/L metformin for 24 h significantly reversed stearic acid-reduced $\beta$-cell viability (Fig. 1C). Moreover, stearic acid-impaired glucose-stimulated insulin secretion was remarkably recovered upon metformin treatment (Fig. 1D). Additionally, metformin reversed the upregulation of aging (Igf1r and Bambi) and senescence markers (Cdkn2a and Trp53bp1), senescence-associated secretory phenotype factors (Ccl2, Il6, Tnfa, and Cd99), and forbidden genes (Cat and Ldha), and the downregulation of $\beta$-cell identity genes (Ins1 and Mafa) (Fig. 1E) induced by stearic acid, along with the increase in senescence-associated $\beta$-galactosidase activity (Fig. 1F). Immunofluorescence analysis also demonstrated the ability of metformin to attenuate the rise in fluorescence intensity of insulin-like growth factor-1 receptor observed in the presence of stearic acid (Fig. 1G).

Fig. 1.

Metformin protects against stearic acid-induced senescence in mouse $\beta$-TC6 cells. (A) Cytotoxic effect of metformin on the survival rate of $\beta$-TC6 cells at various concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, 1600, 3200, 6400, and 12800 µmol/L). ${}^{*}$p 0.05, ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. 0 mmol/L group. (B) Cytotoxic effect of 50 µmol/L metformin on $\beta$-cell viability at various times (0, 6, 12, 24, and 48 h). ${}^{*}$p 0.05 vs. 0 h group. (C,D) Protective effect of metformin on the cell survival rate and glucose-stimulated insulin secretion in stearic acid-treated $\beta$-TC6 cells. (E) Changes in expression of senescence-related genes after treatment of $\beta$-TC6 cells with metformin in the presence or absence of stearic acid. (F) Effect of metformin on $\beta$-galactosidase activity with or without stearic acid treatment. (G) Immunofluorescence analysis shows the expression of insulin-like growth factor-1 receptor (IGF1R) in $\beta$-TC6 cells treated with metformin in the presence or absence of stearic acid. Hoechst, blue; IGF1R, red. For C–G, ${}^{*}$p 0.05, ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. Ctrl group; ${}^{\mathrm{^}}$p 0.05, ${}^{\mathrm{^}\mathrm{^}}$p 0.01, ${}^{\mathrm{^}\mathrm{^}\mathrm{^}}$p 0.001 vs. SA group. Ctrl, control group; SA, stearic acid; Met, metformin. For F and G, scale bar: 100 µm. Each independent experiment was repeated three times.

The level of miR-297b-5p was significantly decreased in stearic acid-treated $\beta$-TC6 cells (Fig. 2). The effect observed in $\beta$-TC6 cells was reversed by metformin. However, no change in miR-297b-5p expression was observed after metformin treatment in the absence of stearic acid (Fig. 2).

Fig. 2.

Alteration of miR-297b-5p expression in stearic acid-treated $\beta$-TC6 cells. miR-297b-5p expression in stearic acid-treated $\beta$-TC6 cells in the absence or presence of metformin. ${}^{*}$p 0.05 vs. Ctrl group, ${}^{\mathrm{^}}$p 0.05 vs. SA group. n = 3. Ctrl, control group; SA, stearic acid; Met, metformin.

Transfection of miR-297b-5p mimics into pancreatic $\beta$-TC6 cells resulted in the upregulation of miR-297b-5p expression in the absence or presence of stearic acid (Fig. 3A). Overexpression of miR-297b-5p improved the survival rate of $\beta$-TC6 cells (Fig. 3B) and glucose-stimulated insulin secretion (Fig. 3C) in the presence of stearic acid. Additionally, stearic acid induced the upregulation of senescence-related genes (Cat, Ldha, Igf1r, Bambi, Cdkn2a, Trp53bp1, Cd99, Ccl2, Il6, and Tnfa), while the downregulation of Ins1 and Mafa largely returned to normal after the transfection of miR-297b-5p mimics (Fig. 3D). Moreover, the increase in the percentage of senescence-associated $\beta$-galactosidase-positive cells and the fluorescence intensity of the insulin-like growth factor-1 receptor caused by stearic acid was reversed after overexpressing miR-297b-5p (Fig. 3E,F). Conversely, transfection of the miR-297b-5p inhibitor decreased the level of this miRNA (Fig. 4A). In the absence of stearic acid, the inhibition of miR-297b-5p had no significant effect on cell viability (Fig. 4B); however, it led to impaired insulin secretion (Fig. 4C), abnormal changes in senescence-related genes expression (Fig. 4D), increased senescence-associated $\beta$-galactosidase activity (Fig. 4E), and increased the fluorescence intensity of the insulin-like growth factor-1 receptor (Fig. 4F), which were reversed by miR-297b-5p overexpression.

Fig. 3.

Role of miR-297b-5p in cellular senescence and impaired glucose-stimulated insulin secretion induced by stearic acid in $\beta$-TC6 cells. (A) Changes in miR-297b-5p expression after transfection of miR-297b-5p mimics in the presence of stearic acid. (B,C) Effect of miR-297b-5p mimics on the stearic acid-decreased cell survival rate and glucose-stimulated insulin secretion. (D–F) miR-297b-5p overexpression reverses senescence-related genes expression, $\beta$-galactosidase activity, and the expression of insulin-like growth factor-1 receptor (IGF1R) (red) in stearic acid-treated $\beta$-TC6 cells. ${}^{*}$p 0.05, ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. Ctrl+MIC-NC group; ${}^{\mathrm{^}}$p 0.05, ${}^{\mathrm{^}\mathrm{^}}$p 0.01, ${}^{\mathrm{^}\mathrm{^}\mathrm{^}}$p 0.001 vs. SA+MIC-NC group. Ctrl, control group; MIC-NC, miR-297b-5p mimics negative control; SA, stearic acid; 297MIC, miR-297b-5p mimic; Glu, glucose. For (E) and (F), scale bar: 100 µm. Each independent experiment was repeated three times.

Fig. 4.

Role of miR-297b-5p in $\beta$-TC6 cell senescence and glucose-stimulated insulin secretion in the absence of stearic acid. (A) Changes in miR-297b-5p expression upon transfection of anti-miR-297b-5p oligonucleotides alone. (B,C) Effect of anti-miR-297b-5p oligonucleotides on cell viability and glucose-stimulated insulin secretion in the absence of stearic acid. (D) Alteration of senescence-related gene expression after inhibition of miR-297b-5p. (E) X-galactosidase staining showing an increase in $\beta$-galactosidase activity after transfection of anti-miR-297b-5p oligonucleotides. (F) Immunofluorescence of insulin-like growth factor-1 receptor was enhanced after inhibition of miR-297b-5p. Hoechst, blue; IGF1R, red. ${}^{*}$p 0.05, ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. Ctrl+AMO-NC group; ${}^{\mathrm{^}}$p 0.05, ${}^{\mathrm{^}\mathrm{^}}$p 0.01, ${}^{\mathrm{^}\mathrm{^}\mathrm{^}}$p 0.001 vs. 297AMO group. Ctrl, control group; AMO-NC, anti-miR-297b-5p oligonucleotide negative control; 297AMO, anti-miR-297b-5p oligonucleotides; 297MIC, miR-297b-5p mimics; Glu, glucose. For (E) and (F), scale bar: 100 µm. Each independent experiment was repeated three times.

Prediction of the binding site of miR-297b-5p in the 3${}^{\prime }$-untranslated region of the insulin-like growth factor-1 receptor is shown in Fig. 5A. Insulin-like growth factor-1 receptor expression at both the mRNA and protein levels was significantly increased in the $\beta$-TC6 cells by stearic acid, an effect that was markedly reversed by miR-297b-5p mimics. Furthermore, overexpression of miR-297b-5p alone inhibited insulin-like growth factor-1 receptor expression (Fig. 5B,C). Conversely, inhibition of miR-297b-5p increased the level of insulin-like growth factor-1 receptor (Fig. 5D,E). Moreover, miR-297b-5p overexpression significantly decreased luciferase activity in human embryonic kidney (HEK293) cells transfected with a plasmid carrying the wildtype 3${}^{\prime }$-untranslated region of insulin-like growth factor-1 receptor (Fig. 5F). Additionally, metformin prevented the rise of the insulin-like growth factor-1 receptor induced by stearic acid (Fig. 5G).

Fig. 5.

Insulin-like growth factor-1 receptor is the direct target of miR-297b-5p. (A) Prediction of the binding site between 3${}^{\prime }$-untranslated region (UTR) of insulin-like growth factor-1 receptor and miR-297b-5p. (B,C) Effect of miR-297b-5p on Igf1r expression at both mRNA and protein levels in $\beta$-TC6 cells treated with or without stearic acid. ${}^{*}$p 0.05, ${}^{***}$p 0.001 vs. Ctrl+MIC-NC group, ${}^{\mathrm{^}}$p 0.05, ${}^{\mathrm{^}\mathrm{^}}$p 0.01, ${}^{\mathrm{^}\mathrm{^}\mathrm{^}}$p 0.001 vs. SA+MIC-NC group. For (B), n = 4; For C, n = 3. MIC-NC, miR-297b-5p mimics negative control; SA, stearic acid; 297MIC, miR-297b-5p mimic. (D,E) Changes in insulin-like growth factor-1 receptor expression at mRNA and protein levels after inhibition or overexpression of miR-297b-5p. ${}^{*}$p 0.05, ${}^{**}$p 0.01 vs. the Ctrl+AMO-NC group, ${}^{\mathrm{^}}$p 0.05, ${}^{\mathrm{^}\mathrm{^}}$p 0.01, ${}^{\mathrm{^}\mathrm{^}\mathrm{^}}$p 0.001 vs. 297AMO group. For (D), n = 3; For (E), n = 5. AMO-NC, anti-miR-297b-5p oligonucleotide negative control; 297AMO, anti-miR-297b-5p oligonucleotides; 297MIC, miR-297b-5p mimics. (F) Luciferase reporter assay verifying the inhibitory effect of miR-297b-5p mimics on luciferase activity of a plasmid carrying 3${}^{\prime }$-untranslated region (UTR) of insulin-like growth factor-1 receptor (Igf1r) (wildtype). ${}^{***}$p 0.001 vs. m-Igf1r+MIC-NC group. n = 5. m-Igf1r+MIC-NC, plasmid carrying the wildtype 3${}^{\prime }$-untranslated region (UTR) of the Igf1r gene + miR-297b-5p mimics negative control; m-Igf1r-Mut+MIC-NC, plasmid carrying a mutant 3${}^{\prime }$-untranslated region (UTR) of the Igf1r gene + miR-297b-5p mimics negative control. (G) Effect of metformin on insulin-like growth factor-1 receptor (IGF1R) protein expression in the presence of stearic acid. ${}^{**}$p 0.01 vs. Ctrl group. ${}^{\mathrm{^}}$p 0.05, ^^p 0.01 vs. SA group. n = 4. Met, metformin; SA, stearic acid.

In $\beta$-TC6 cells, transfection of siRNA-Igf1r efficiently decreased insulin-like growth factor-1 receptor expression with or without stearic acid treatment (Fig. 6A). Silencing insulin-like growth factor-1 receptor significantly blocked the reduction in insulin secretion caused by stearic acid (Fig. 6B), yet not the decrease in cell viability (Fig. 6C). Furthermore, knockdown of insulin-like growth factor-1 receptor reversed the abnormal expression of senescence-related genes (Fig. 6D), the increase in senescence-associated $\beta$-galactosidase-positive cells (Fig. 6E), and the rise in fluorescence intensity of insulin-like growth factor-1 receptor (Fig. 6F), triggered by stearic acid.

Fig. 6.

Inhibition of insulin-like growth factor-1 receptor ameliorates stearic acid-stimulated glucose-stimulated insulin secretion impairment and senescence of $\beta$-TC6 cells. (A) Changes in insulin-like growth factor-1 receptor expression in stearic acid-treated $\beta$-TC6 cells. (B,C) Effect of siRNA-Igf1r on insulin secretion and cell viability in the presence of stearic acid. (D) Alteration of senescence-related gene expression after silencing insulin-like growth factor-1 receptor in $\beta$-TC6 cells treated with stearic acid. (E,F) Knockdown of insulin-like growth factor-1 receptor alleviated stearic acid-increased percentage of $\beta$-galactosidase-positive cells and immunofluorescence intensity of IGF1R (red). ${}^{*}$p 0.05, ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. Ctrl+si-NC group; ${}^{\mathrm{^}}$p 0.05, ${}^{\mathrm{^}\mathrm{^}}$p 0.01, ${}^{\mathrm{^}\mathrm{^}\mathrm{^}}$p 0.001 vs. SA+si-NC group. Ctrl, control group; si-NC, siRNA-Igf1r negative control; SA, stearic acid; si-Igf1r, siRNA-Igf1r; Glu, glucose. For (E) and (F), scale bar: 100 µm. Each independent experiment was repeated three times.

Metformin markedly reversed the suppressive effect of anti-miR-297b-5p oligonucleotides on miR-297b-5p expression (Fig. 7A) and glucose-stimulated insulin secretion (Fig. 7B) in $\beta$-TC6 cells. However, metformin had no significant effect on cell viability after transfection of anti-miR-297b-5p oligonucleotides (Fig. 7C). Moreover, metformin effectively improved $\beta$-cell senescence observed upon silencing of miR-297b-5p and reversed the level of dysregulated senescence-related genes (Fig. 7D), the percentage of senescence-associated $\beta$-galactosidase-positive cells (Fig. 7E), and the enhanced staining for insulin-like growth factor-1 receptor (Fig. 7F). Additionally, metformin treatment was able to block the elevation in insulin-like growth factor-1 receptor protein expression induced by the miR-297b-5p inhibitor (Fig. 7G).

Fig. 7.

Metformin significantly alleviates anti-miR-297b-5p oligonucleotides-induced senescence and glucose-stimulated insulin secretion impairment of $\beta$-TC6 cells. (A) Alteration of miR-297b-5p expression after transfection of anti-miR-297b-5p oligonucleotides in the presence of metformin. (B,C) Effect of metformin on the cell survival rate and glucose-stimulated insulin secretion after inhibition of miR-297b-5p. (D–F) Reversal effect of metformin on the expression of senescence-related genes, $\beta$-galactosidase activity, and immunofluorescence of insulin-like growth factor-1 receptor (IGF1R) (red) after transfection of anti-miR-297b-5p oligonucleotides. (G) Effect of metformin on insulin-like growth factor-1 receptor (IGF1R) protein expression in the presence of anti-miR-297b-5p oligonucleotides in $\beta$-TC6 cells. ${}^{*}$p 0.05, ${}^{**}$p 0.01,${}^{***}$p 0.001 vs. Ctrl+AMO-NC group; ${}^{\mathrm{^}}$p 0.05, ${}^{\mathrm{^}\mathrm{^}}$p 0.01 vs. 297AMO group. Ctrl, control group; AMO-NC, anti-miR-297b-5p oligonucleotide negative control; 297AMO, anti-miR-297b-5p oligonucleotides; Met, metformin; Glu, glucose. For (E) and (F), scale bar: 100 µm. Each independent experiment was repeated three times.

As evidenced by the profile of serum fatty acids (Table 3), mice fed with a high stearic acid diet displayed high circulating levels of stearic acid. Table 4 summarizes the characteristics of the mice. Long-term feeding of a high stearic acid diet led to a significant impairment of glucose tolerance (Fig. 8A) and enhanced the second phase of insulin secretion in response to glucose (Fig. 8B). Meanwhile, the $\alpha$-cell to $\beta$-cell ratio was significantly higher in the islets of mice fed with a high stearic acid diet than in mice fed with a normal diet (Fig. 8C). Moreover, a high stearic acid diet dramatically upregulated the expression of aging (Igf1r and Bambi) and senescence markers (Cdkn2a and Trp53bp1), senescence-associated secretory phenotype factors (Ccl2, Il6, Tnfa, and Cd99), and forbidden genes (Cat and Ldha), while downregulating the level of $\beta$-cell identity genes (Ins1 and Mafa) in mouse islets (Fig. 8D). Additionally, the expression of miR-297b-5p was significantly reduced in the islets of mice fed a high stearic acid diet (Fig. 8E).

Fig. 8.

Long-term high stearic acid diet leads to senescence in mouse islets. (A,B) Detection of impaired glucose tolerance and insulin secretion by intravenous glucose tolerance testing. (C) Double immunohistochemical staining for insulin and glucagon in islets of mice receiving a normal diet or high stearic acid diet. Scale bar: 200 µm. (D) Alterations of the expression of senescence-related genes in mouse islets after high stearic acid diet feeding. ${}^{*}$p 0.05, ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. Ctrl group. For (A) and (B), n = 5 mice per group. For (C) and (D), n = 3 mice per group. Ctrl, normal diet; HSD, high stearic acid diet. (E) Downregulation of miR-297b-5p in high stearic acid diet-fed mouse islets. ${}^{***}$p 0.001 vs. Ctrl group. n = 4. Ctrl, control group; HSD, mice were fed a high stearic acid diet.