Overexpression of miR-297b-5p in Mouse Insulin-Secreting Cells Promotes Metformin-Mediated Protection Against Stearic Acid-Induced Senescence by Targeting Igf1r

Background : A long-term consumption of saturated fat significantly increases the concentration of saturated fatty acids in serum, which accelerates the appearance of senescence markers in β -cells and leads to their dysfunction. An understanding of the mechanisms underlying β -cell senescence induced by stearic acid and the exploration of effective agents preventing it remains largely unclear. Here, we aimed to investigate the protective effect of metformin against stearic acid-treated β -cell senescence and to assess the involvement of miR-297b-5p in this process. Methods : To identify senescence, we measured senescence-associated β -galactosidase activity and the expression of senescence-related genes. Gain and loss of function approaches were applied to explore the role of miR-297b-5p in stearic acid-induced β -cell senescence. Bioinformatics analysis and a luciferase activity assay were used to predict the downstream targets of miR-297b-5p. Results : Stearic acid markedly induced senescence and suppressed miR-297b-5p expression in mouse β -TC6 cells, which were significantly alleviated by metformin. After transfection of miR-297b-5p mimics, stearic acid-evoked β -cell senescence was remarkably prevented. Insulin-like growth factor-1 receptor was identified as a direct target of miR-297b-5p. Inhibition of the insulin-like growth factor-1 receptor prevented stearic acid-induced β -cell senescence and dysfunction. Moreover, metformin alleviates the impairment of the miR-297b-5p inhibitor in β -TC6 cells. Additionally, long-term consumption of a high-stearic-acid diet significantly increased senescence and reduced miR-297b-5p expression in mouse islets. Conclusions : These findings imply that metformin alleviates β -cell senescence by stearic acid through upregulating miR-297b-5p to suppress insulin-like growth factor-1 receptor expression, thereby providing a potential target to not only prevent high fat-diet-induced β -cell dysfunction but also for metformin therapy in type 2 diabetes.


Introduction
Type 2 diabetes (T2D) is one of the fastest-growing non-communicable chronic diseases and a serious threat to human health with approximately 415 million people currently affected worldwide [1].A high-fat diet has been closely associated with the development of type 2 diabetes.Strong evidence indicates that long-term excessive consumption of foods rich in animal fat significantly increases the serum concentration of saturated fatty acids (SFAs), which leads to β-cell dysfunction and eventually accelerates the development of type 2 diabetes [2].Stearic and palmitic acids are two major saturated fatty acids.Although the proportion of stearic acid is lower than palmitic acid in fatty foods and human serum, growing evidence shows that the increase in circulating stearic acid in the profile of free fatty acids is significantly higher, while its detrimental ef-fect on β-cells is stronger than that of palmitic acid in patients with hyperlipidemia and mice fed high-fat diets [3][4][5].However, the understanding of the role of stearic acid in β-cell impairment remains incomplete.
The proposed mechanisms responsible for saturated fatty acid-induced β-cell failure mainly include endoplasmic reticulum stress, apoptosis, inflammation, dedifferentiation, aging, and senescence [6][7][8][9][10].Among them, the contribution of β-cell senescence to this process has attracted more attention in recent years.Cellular senescence is a stress response that can occur at any time and is sensitive to various stimuli, such as DNA damage, endoplasmic reticulum stress, reactive oxygen species, and oncogene activation [11][12][13][14][15].It is characterized by a decline in cell proliferation [16] and increases in senescence-associated β-galactosidase activity and secre-tion of senescence-associated secretory phenotype (SASP) factors [17,18].Senescent β-cells accumulate and increase in islets with age and under certain conditions, including peripheral insulin resistance, a high body mass index, and type 2 diabetes [19,20].Similarly, a high-fat diet significantly increases the accumulation of senescent β-cells, whereby decreasing the number of senescent cells obviously improves β-cell function [20].These findings imply that senescence is a promising target in saturated fatty acidinduced β-cell dysfunction during type 2 diabetes development.However, the development of an effective drug for the management of β-cell senescence remains challenging.
Metformin is a first-line drug in the management of type 2 diabetes, mostly via the inhibition of hepatic gluconeogenesis and promotion of glucose uptake in skeletal muscles [21].Researchers are also currently focusing on metformin use in other fields because this drug has been shown to have pleiotropic effects, such as weight loss, cancer prevention, and anti-aging and senescence [22].Although metformin is an interesting candidate as an antiaging treatment, clinical evidence of this effect is still lacking and the precise mechanisms have not been completely elucidated.In particular, evidence demonstrating the potential role of metformin in the protection against β-cell senescence is at present quite limited [23][24][25].There is no doubt that the identification of novel potential targets of metformin that prevent β-cell aging is important for slowing type 2 diabetes development.
High-throughput sequencing technologies and bioinformatics analysis have significantly expanded our knowledge about the important role of non-coding RNAs in gene regulation at multiple levels and have provided a large number of novel targets for the treatment of human diseases.MicroRNAs (miRNAs)-a class of endogenous ~20 nucleotide RNAs-have been strongly suggested to participate in the regulation of β-cell function, such as miR-375, miR-7, and miR-184 [26][27][28].In our previous studies, we found that miR-297b-5p was significantly downregulated in stearic acid-treated β-TC6 cells and in the islets of mice fed a high-fat diet.Overexpression of miR-297b-5p effectively alleviates stearic acid-induced β-cell dysfunction through its anti-apoptotic and anti-inflammatory effects [29,30].However, whether miR-297b-5p is also involved in the anti-senescence effect of metformin in β-cells exposed to stearic acid remains unknown.
In this study, we aimed to investigate the protective effect of metformin on stearic acid-evoked β-cell senescence in β-TC6 cells and to examine the involvement of miR-297b-5p in this process.We found that the upregulation of miR-297b-5p promotes the anti-senescence effect of metformin on stearic acid-treated β-TC6 cells by decreasing the level of the insulin-like growth factor-1 receptor (Igf1r).These results provide a potential mechanism to not only prevent the induction of β-cell dysfunction by a high-fat diet but also for the therapeutic use of metformin to prevent or delay the onset of type 2 diabetes.

Chemicals
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.

Cell Culture
Mouse β-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 Assay
Cell viability was assessed using Cell Counting Kit 8 (C0038; Beyotime Biotechnology, Shanghai, China).For this purpose, β-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].
glucose [5].The supernatant was collected for insulin measurement and β-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.

Senescence-Associated β-Galactosidase (SA-β-gal) Staining
β-TC6 cells were seeded into a 24-well plate at 6 × 10 4 cells/well and cultured at 37 °C in a 5% CO 2 humidified incubator.The senescence status was analyzed using a Senescence β-Galactosidase Staining Kit (Cat.No. C0602, Beyotime Biotechnology).Cells were washed with phosphate-buffered saline (PBS) and fixed in the senescence-associated β-galactosidase fixative solution for 15 min at room temperature.After washing three times with PBS, the cells were incubated in senescenceassociated β-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 β-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).
Briefly, β-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.

Animal Experiments
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).

Intravenous Glucose Tolerance Testing
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.

Serum Fatty Acid Profile Analysis and Lipid Measurements
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 lowdensity 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 intraassay variations of this kit were 6.0-17.9 and 0.9-8.4,respectively.

Real-Time Quantitative Polymerase Chain Reaction
Total RNA was extracted from β-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 β-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.

Statistical Analysis
All data were presented as mean ± 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.

Results
Accumulated evidence indicates that β-cell senescence is a promising target to prevent β-cell dysfunction elicited by a long-term high-fat diet during type 2 diabetes development.However, the mechanism underlying saturated fatty acid-induced β-cell senescence is not yet understood and there are currently no effective agents to prevent this effect.In this study, we aimed to investigate the protective effect of metformin on stearic acid-promoted β-cell senescence and to explore the potential role of miR-297b-5p in this process.We found that metformin dramatically ameliorates stearic acid-evoked β-cell senescence through the upregulation of miR-297b-5p, which effectively reverses the increase in insulin-like growth factor-1 receptor expression triggered by stearic acid.These results provide a potential target to not only prevent high saturated fat diet-induced β-cell dysfunction but also for the therapeutic use of metformin to prevent or delay the onset of type 2 diabetes.

Metformin Reverses the Decrease in β-Cell miR-297b-5p Expression Caused by Stearic Acid
The level of miR-297b-5p was significantly decreased in stearic acid-treated β-TC6 cells (Fig. 2).The effect observed in β-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).

Validation of Insulin-Like Growth Factor-1 Receptor as the Direct Target of miR-297b-5p
Prediction of the binding site of miR-297b-5p in the 3 ′ -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 β-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 ′ -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).

Inhibition of Insulin-Like Growth Factor-1 Receptor Ameliorates Stearic Acid-Stimulated the Impairment in Glucose-Stimulated Insulin Secretion and Prevents Senescence of β-TC6 Cells
In β-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 insulinlike growth factor-1 receptor reversed the abnormal expression of senescence-related genes (Fig. 6D), the increase in senescence-associated β-galactosidase-positive cells (Fig. 6E), and the rise in fluorescence intensity of insulin-like growth factor-1 receptor (Fig. 6F), triggered by stearic acid.

Long-Term Exposure to Stearic Acid Results in Impaired Insulin Secretion and β-cell Senescence in Mice
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 α-cell to β-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 β-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).

Discussion
Prolonged exposure of β-cells to elevated concentrations of saturated fatty acids results in the accumulation of senescent cells, which leads to a progressive decline in in- sulin secretion.Exploring potential targets and effective drugs capable of preventing β-cell senescence represents a promising strategy to overcome the deleterious effects of saturated fatty acids.In our study, we found that metformin showed a remarkable protective effect against the senescence of β-cells caused by stearic acid.Our findings highlight the involvement of miR-297b-5p in stearic acidincreased β-cell senescence.
Although miR-297b-5p was initially characterized in cancers [35], our recent studies proposed a novel role of miR-297b-5p in stearic acid-induced β-cell dysfunction via inhibiting the expression of both proapoptotic and proinflammatory factors.In this study, we found an alternative mechanism to explain the anti-senescence effect by miR-297b-5p.Indeed, overexpression of miR-297b-5p resulted in the considerable recovery of stearic acid- 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.increased senescence-related genes in β-TC6 cells, including disallowed genes, aging and senescence markers, and senescence-associated secretory phenotype factors.These genes were selected based on previous studies [19,36] and our initial comparison analysis (Supplementary Fig. 2).
There is strong evidence indicating that miRNAs conduct their regulatory activity through multiple targets [37].Here, we confirmed by computational analysis and using a luciferase reporter assay that insulin-like growth factor-1 receptor-an aging marker in β-cells that is associated with type 2 diabetes [36]-is a downstream target of miR-297b-5p.We found that miR-297b-5p exerts a negative effect on insulin-like growth factor-1 receptor expression.Additionally, silencing this receptor effectively reversed βcell senescence induced by stearic acid and the impairment in insulin secretion.These findings suggest that stearic acid causes cellular senescence and dysfunction through the miR-297b-5p/Igf1r axis in β-TC6 cells.Future studies will need to determine whether miR-297b-5p exerts a similar role in human β-cells.
Early lifestyle intervention and pharmacological treatment to restore β-cell function is a well-accepted strategy to prevent the onset and progression of type 2 diabetes [38].Metformin is a well-tolerated and safe drug that delays type 2 diabetes [39].However, its pleiotropic effects in various tissues increase the difficulty of establishing specific targets, especially in β-cells.In this study, we observed a significant protective effect of metformin on βcell function through the clearance of senescent cells.In this process, metformin significantly restored stearic aciddecreased miR-297b-5p expression and inhibited the upregulation of insulin-like growth factor-1 receptor expression caused by the fatty acid.Moreover, the reduction in cell viability observed in the presence of stearic acid was partially reversed after metformin treatment.These results indicate that miR-297b-5p likely mediates the protective effect of metformin and that this drug may be useful in improving and restoring β-cell function in subjects who have developed type 2 diabetes as a consequence of a long-term highfat diet.
This study has several limitations.Firstly, further studies are needed to confirm whether this conclusion remains in primary mouse and human β-cells.Secondly, it will be essential to confirm the protective effect alongside the potential dose of metformin required to prevent β-cell senescence in mice fed a high stearic acid diet and to perform RNA-sequencing analysis on mouse islets.Thirdly, the causal relationship between senescence and inflamma- tion in stearic acid-induced β-cell dysfunction needs to be determined because the release of senescence-associated secretory phenotype proteins worsens surrounding cells leading to senescence [40] and inflammation [41].Additionally, whether metformin directly interacts with stearic acid and how it increases miR-297b-5p expression are interesting points to be addressed and can promote the use of metformin to prevent the induction of type 2 diabetes by high-fat diets.

Conclusions
We found that metformin protects against stearic acidstimulated β-cell senescence through the upregulation of miR-297b-5p expression and the reduction of insulin-like growth factor-1 receptor expression (Fig. 9).After these metabolic stresses ceased, β-cell function largely returned to normal, indicating that lowering the consumption of stearic acids and reversing the hallmarks of cellular aging is a potential strategy for type 2 diabetes therapies.Our findings also suggest that metformin is probably repurposed to preserve β-cell function in type 2 diabetes induced by a high-fat diet, through its anti-senescence effect.

Fig. 8 .
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.

Table 3 . The profile of fasting serum NEFAs in normal and HSD mice at 12 weeks.
p < 0.05, * * p < 0.01, compared to the value in normal mice.