∆nFGF1 Protects β-Cells against High Glucose-Induced Apoptosis via the AMPK/SIRT1/PGC-1α Axis

Long-term exposure to high glucose leads to β-cell dysfunction and death. Fibroblast growth factor 1 (FGF1) has emerged as a promising diabetes treatment, but its pharmaceutical role and mechanism against glucolipotoxicity-induced β-cell dysfunction remain uncharacterized. Wild-type FGF1 (FGF1WT) may exhibit in vivo mitogenicity, but deletion of N-terminal residues 1-27 gives a nonmitogenic variant, ∆nFGF1, that does not promote cell proliferation and still retains the metabolic activity of FGF1WT. To investigate the roles of ∆nFGF1 on glucose regulation and potential islet β-cell dysfunction, db/db mice were used as a model of type 2 diabetes. The results showed that insulin secretion and apoptosis of islet β-cells were dramatically improved in ∆nFGF1-treated db/db mice. To further test the effects of ∆nFGF1 treatment, pancreatic β-cell (MIN6) cells were exposed to a mixture of palmitic acid (PA) and high glucose (HG) to mimic glucolipotoxic conditions in vitro. Treatment with ∆nFGF1 significantly inhibited glucolipotoxicity-induced apoptosis. Mechanistically, ∆nFGF1 exerts a protective effect on β-cells via activation of the AMPK/SIRT1/PGC-1α signaling pathway. These findings demonstrate that ∆nFGF1 protects pancreatic β-cells against glucolipotoxicity-induced dysfunction and apoptosis.


Introduction
Type 2 diabetes mellitus (T2DM) is characterized by persistent hyperglycemia in the context of insulin resistance [1,2]. Insulin resistance and pancreatic β-cell dysfunction are considered the central characteristics of T2DM [3,4]. Longterm high blood glucose can induce the loss of β-cells, considered a key step in the development of diabetes [5]. Therefore, an effective strategy to prevent diabetes mellitus may be the reduction of pathological β-cell apoptosis induced by high blood glucose levels.
The fibroblast growth factor (FGF) family consists of 22 members that play important roles in regulating the function of endocrine-relevant tissues and various metabolic processes [6][7][8][9]. The biological activities of FGFs are regulated by FGF receptors, and multiple FGFs may share the same FGF receptors to modulate cellular activity [10,11]. FGF21 increases insulin secretion in diabetic islets and protects βcells against apoptosis through extracellular signalregulated kinase 1/2 (Erk1/2) and AKT pathways [12]. As a recently found metabolic modulator, FGF1 has been reported to improve insulin resistance and β-cell insulin secretion in diabetic mouse models [13,14]. However, the underlying mechanisms of these effects to counter glucolipotoxicity-induced β-cell dysfunction remain unclear. Wild-type FGF1 (FGF1 WT ) has mitogenic activity, limiting its therapeutic potential, but a nonmitogenic FGF1 (ΔnFGF1) lacking N-terminal residues 1-27 of FGF1 WT exhibits no apparent proliferative activity [15]. The goals of this study were to investigate the roles of ΔnFGF1 on glucose control and its protective effect against islet β-cell apoptosis after chronic administration in a diabetes model and determine the underlying mechanisms of these effects.
Adenosine 5′-monophosphate-activated protein kinase (AMPK) is highly expressed in metabolically active tissues and plays a crucial role in whole-body energy homeostasis [16][17][18]. AMPK dysregulation has been implicated in metabolic disorders like insulin resistance and T2DM [19][20][21]. AMPK activation may mediate the effect of FGF1 to protect against nonalcoholic fatty liver disease (NAFLD) in T2DM mice [22], and FGF4 improves blood glucose through activation of the AMPK pathway [23], implicating the AMPK pathway in the protective effects of FGF treatment on pancreatic islet β-cells.
In this study, we explored the glucose-lowering effects of ΔnFGF1 in insulin resistance mouse models. Our results show that ΔnFGF1 significantly inhibits β-cell apoptosis and improves β-cell function through the AMPK/SIRT1/ PGC-1α pathway. Overall, these results indicate the therapeutic potential of ΔnFGF1 to prevent high glucoseinduced β-cell apoptosis for T2DM treatment.

Cell Culture and Treatments.
Mouse pancreatic β-cell line MIN6 (Keygen Biotech, Nanjing, China) cells were cultured in RPMI1640 medium (Gibco) accompanied with 10% fetal bovine serum (FBS, Gibco) and 1% penicillinstreptomycin. Cells were passaged every three days. Culture media was changed every 24 h.
For intracellular signaling studies, MIN6 cells were starved for 12 h followed by incubation with or without ΔnFGF1 (50 ng/mL) for 1 h. Cells were then exposed to media containing normal glucose (NG, 11.1 mM) as a control or high glucose (HG, 33 mM)+palmitic acid (PA, 0.5 mM) for 24 h and then lysed to detect protein expression by Western blot. For inhibitor experiments, MIN6 cells were also starved for 12 h and treated with AMPK inhibitor Compound C (10 μM, Selleck Chemicals, S7306) or SIRT1 inhibitor EX-527 (10 μM, MedChemExpress, HY-15452) or PGC-1α inhibitor SR-18292 (10 μM, MedChemExpress, HY-101491) for 1 h and then incubated in NG or HG+PA or HG+PA+ΔnFGF1 for 24 h and lysed to detect protein expression by Western blot. For siRNA knockdown experiments, MIN6 cells were seeded and grown in six-well plates for 24 h to achieve 70% confluence. Cell transfection was performed with the transfection reagent Lipofectamine 3000 in accordance with the manufacturer's instructions. A 24 h transfection of AMPK siRNA (Santa Cruz Biotechnology, sc45313) was followed by starvation and treatment as described above.

Cell
Counting Kit-8 Assay. MIN6 cells were seeded into a 96-well plate (3000 cells/well) and serum starved in RPMI1640 medium for 24 h. The MIN6 cells were then exposed to 50 ng/mL ΔnFGF1 or FGF1 WT for 24 h or exposed to 33 mM HG and 0.5 mM PA in the presence or absence of 50 ng/mL ΔnFGF1 for 24 h. Cell Counting Kit-8 (CCK-8, Jiancheng Bioengineering Institute, China) was used to assess cell viability according to the manufacturer's instructions. In brief, cells treated as described above were incubated with 10 μL of CCK-8 solution at 37°C for 1 h. The optical density (OD) value was detected by a microplate reader (Thermo, USA).
2.3. TUNEL Labeling. MIN6 cells were seeded at a total concentration of 3 × 10 5 on a glass coverslip in six-well plates for 24 h, followed by treatment with 33 mM HG and 0.5 mM PA in the presence or absence of 50 ng/mL ΔnFGF1 for 24 h. Then, cells were washed with sterile PBS twice, fixed using 4% paraformaldehyde (PFA) for 15 min, and then permeabilized with 0.2% Triton X-100 for 5 min. Subsequently, each slide was incubated with a terminal deoxynucleotidyl transferase-(TdT-) labeled reaction mix at 37°C for 60 min in the dark. Cell nuclei were counterstained with DAPI for 10 min. Finally, fluorescent images were captured by Leica SP8 confocal microscopy using the FITC channel (Leica, Wetzlar, Germany).

Flow Cytometry
Analysis. MIN6 cells were seeded at a total concentration of 3 × 10 5 and cultured in six-well plates. Apoptosis of MIN6 cells was detected using an Annexin V-FITC Apoptosis Detection Kit (A211, Vazyme, Shanghai, China). In brief, treated and untreated cells were washed with cold PBS and resuspended in 500 μL binding buffer and then incubated with 5 μL of Propidium Iodide (PI) and 5 μL of Annexin V-FITC in the dark for 10 min. The fluorescence intensity was measured using a flow cytometer (Beckman, USA) within 1 h and monitoring 1 × 10 5 cells per sample. Apoptosis was analyzed using FlowJo VX10.

Animal Models.
Eight-week-old male db/db (C57BLKS/J Leprdb/db) mice and normal db/m mice were purchased from GemPharmatech Co. Ltd. (Nanjing, China) and allowed to adapt to the new environment for no less than 7 days. All animals were fed with basal rodent chow and tap water ad libitum and housed at 22-24°C. The animal study was approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University.
10-week-old db/db mice received every other day intraperitoneal injection of ΔnFGF1 protein (0.5 mg/kg) for eight weeks, whereas nondiabetic db/m and db/db control mice were injected with the equal amount of 0.9% saline. Body weights were recorded before injection every two days, and glucose tests were performed weekly. Random nonfasted blood glucose was measured in mouse tail venous blood using an automatic glucose monitor (Roche, Germany). Finally, mice in each group were sacrificed and the pancreatic tissues were collected for further analysis.
2.6. Hematoxylin and Eosin (H&E) Staining. After dehydration and hydration, tissue sections were stained with H&E (Beyotime Biotech, Nantong, China) and captured by light microscopy (Nikon, Tokyo, Japan) to evaluate histological 2 Oxidative Medicine and Cellular Longevity changes. The pancreatic islet areas were measured by ImageJ (National Institutes of Health, USA).
2.9. Statistical Analysis. Statistical analysis was performed using GraphPad Prism version 8.0. All data are presented as mean values ± SEM. For the comparison of two groups, unpaired Student's t-test (two-tailed) was performed. Oneway ANOVA was used to compare more than two groups. A value of p < 0:05 was considered statistically significant.

ΔnFGF1
Ameliorates Diabetes in db/db Mice. To evaluate the protective effects of long-term treatment with ΔnFGF1 on type 2 diabetes, db/db mice received every other day intraperitoneal injection of ΔnFGF1 (0.5 mg/kg) for eight weeks (Figure 1(a)). Compared with saline-treated db/m mice, the blood glucose level was markedly increased in db/db mice from day 6 but restored to near normal level after ΔnFGF1 treatment (Figures 1(b) and 1(c)). The db/db mice treated with ΔnFGF1 exhibited effectively reduced food intake and water intake, with maintenance of a continuous upward trend in the saline-treated mice (Figures 1(d) and 1(e)), suggesting that ΔnFGF1 treatment significantly improved polydipsia and polyphagia caused by diabetes. As a consequence, the db/db mice treated with ΔnFGF1 showed notably reduced body weight compared with db/db mice treated with saline (Figures 1(f) and 1(g)). Thus, these data suggest that ΔnFGF1 suppressed body weight gain and blood glucose levels of db/db mice.

ΔnFGF1
Relieves β-Cell Apoptosis in db/db Mice. Given that β-cell apoptosis is the major risk factor for diabetes, we next investigated whether ΔnFGF1 protected against pancreatic β-cell apoptosis. We found markedly decreased areas of pancreatic islets in db/db mice injected with saline for 8 weeks compared to that in db/m mice, with the areas significantly increased nearly to normal level in ΔnFGF1treated db/db mice (Figure 2(a)). Caspases are crucial mediators of apoptotic process, and cleaved-(C-) caspase 3 is frequently activated cell death protease that catalyzes the specific cleavage of numerous crucial cellular proteins [24,25]. To further investigate whether ΔnFGF1 countered βcell apoptosis in db/db mice, we performed coimmunostaining of insulin and C-caspase 3 in pancreatic islets. Compared with the saline-treated db/m mice, the numbers of insulin/Ccaspase 3 double-positive β-cells were significantly increased in the saline-treated db/db mice. Conversely, ΔnFGF1 treatment notably decreased the numbers of double-positive pancreatic β-cells (Figure 2(b)). Additionally, there was no significant increase in immunostaining for proliferating cell nuclear antigen (PCNA) in pancreatic islets of db/db mice treated with ΔnFGF1 compared with that of db/db mice treated with saline ( Figure 2(c)). Collectively, these data suggest that ΔnFGF1 protects against diabetes by inhibiting βcell apoptosis without affecting cell proliferation in db/db mice.

ΔnFGF1
Protects MIN6 Cells against Glucolipotoxicity-Induced Apoptosis. Long-term hyperglycemia and high levels of circulating free fatty acids (FFAs) are related to pancreatic β-cell dysfunction [26]. To further evaluate the effect of ΔnFGF1, we next explored the damage effects of hyperglycemia and/or hyperlipidemia on MIN6 cells. Exposure to both 33 mM high glucose (HG) and 0.5 mM palmitic acid (PA) resulted in a notable decrease in antiapoptotic protein Bcl-2 expression compared with the untreated group (Figure 3(a)). Based on the established cell apoptosis model, we asked if ΔnFGF1 could inhibit cell apoptosis. Western blot analysis showed that the expression levels of proapoptotic protein C-caspase 3 and Bax significantly decreased in a dose-depended manner in ΔnFGF1-treated MIN6 cells, with an optimal concentration of ΔnFGF1 at 50 mg/mL (Figure 3(b)).

Oxidative Medicine and Cellular Longevity
To mimic in vivo hyperglycemia and hyperlipidemia conditions, MIN6 cells were treated with 0.5 mM PA and 33 mM HG for 24 h and cell viability was detected by CCK-8 assays. As shown in Figure 3(c), HG and PA combined treatment significantly decreased cell viability and ΔnFGF1 treatment notably increased cell survival. Importantly, compared with the increased cell proliferation in the wild-type FGF1-(FGF1 WT -) treated group, ΔnFGF1 treatment showed no effect on cell proliferation (Figure 3(d)).
To confirm this effect of ΔnFGF1 was due to cell apoptosis, we next performed TUNEL assay to directly examine cell apoptosis. We found that ΔnFGF1 alleviated HG+PAinduced apoptosis of MIN6 cells as evidenced by decreased numbers of TUNEL-positive cells (Figures 3(e) and 3(g)).
In parallel, immunofluorescence staining showed that ΔnFGF1 treatment markedly reduced C-caspase 3 expression induced by glucolipotoxicity in MIN6 cells (Figures 3(f) and 3(g)). Consistent with the immunostaining results, Western blot analysis revealed that treatment of ΔnFGF1 notably reversed the HG+PA-induced increased Bax and C-caspase 3 expression and decreased Bcl-2 ( Figure 3(h)). Taken together, these results demonstrate that ΔnFGF1 can effectively inhibit glucolipotoxicity-induced apoptosis in MIN6 cells.

ΔnFGF1
Induces the Activation of the AMPK/SIRT1/ PGC-1α Signaling Pathway. Numerous studies have demonstrated that FGF1 can activate the AMPK signaling pathway, which plays important pleiotropic roles in cellular responses to metabolic stress [27]. Activated AMPK further stimulates SIRT1 and subsequently increases the PGC-1α expression, thus playing a central regulatory role in energy metabolism [28]. We next evaluated the AMPK/SIRT1/PGC-1α signaling pathway in MIN6 cells after ΔnFGF1 treatment. We  Figure 1: The glucose-lowering effect of ΔnFGF1 in db/db mice. (a) The 10-week-old db/db mice received every other day intraperitoneal injection of ΔnFGF1 (0.5 mg/kg) or saline for eight weeks, with age-matched nondiabetic db/m littermates served as the normal control group. (b, c) Random nonfasted blood glucose and changes, (d, e) average food intake and water intake per mouse, and (f, g) body weight alteration. All data are presented as mean values ± SEM. n = 6 mice per group. * p < 0:05, * * p < 0:01, and * * * p < 0:001. n.s.: no significance. 4 Oxidative Medicine and Cellular Longevity

5
Oxidative Medicine and Cellular Longevity found that ΔnFGF1 significantly increased AMPK phosphorylation in HG+PA-induced MIN6 cells (Figure 4(a)). The ΔnFGF1 treatment group also exhibited increased expression of SIRT1 and PGC-1α (Figure 4(b)). Further, we examined the expression of SIRT1 and PGC-1α in pancreatic tissues and found higher protein levels of SIRT1 and PGC-1α in islets of ΔnFGF1-treated db/db mice than of db/db control mice (Figures 4(c)-4(e)). Therefore, we speculate that ΔnFGF1 modulates the AMPK/SIRT1/PGC-1α axis. Representative immunofluorescence images of MIN6 cells stained with (e) TUNEL (green) and (f) C-caspase 3 (red). DAPI was used to stain nuclei. (g) The numbers of TUNEL-positive cells (upper panel) and the fluorescence intensity of C-caspase 3 were quantified using ImageJ (lower panel). (h) Bax, Bcl-2, and C-caspase 3 expression was analyzed by Western blot (left panel) and quantitated by ImageJ (right panels). β-Actin was used as a control. All data are presented as mean values ± SEM. NG: normal glucose; HG: high glucose; PA: palmitic acid. * p < 0:05, * * p < 0:01, and * * * p < 0:001. n.s.: no significance. 6 Oxidative Medicine and Cellular Longevity apoptosis resulted from ΔnFGF1-induced activated AMPK signaling, we used the AMPK inhibitor Compound C (CC) to inhibit AMPK activity of MIN6 cells. Western blot result revealed that ΔnFGF1 increased the expression of p-AMPK, SIRT1, and PGC-1α, while CC abolished the ΔnFGF1induced expression increase of these proteins ( Figure 5(a)). Next, we investigated the effect of AMPK inhibition on apoptosis of islet β-cells and found that ΔnFGF1 treatment significantly increased the antiapoptotic protein expression of Bcl-2 and decreased the proapoptotic protein expression of Bax and C-caspase 3. Conversely, the antiapoptotic effects induced by ΔnFGF1 were impeded by CC treatment (Figure 5(b)). To further confirm that ΔnFGF1 inhibits cell apoptosis through AMPK signaling, we used siRNA to knockdown AMPK expression in MIN6 cells. We found that AMPK siRNA treatment notably inhibited the activation of SIRT1 and PGC-1α signaling by ΔnFGF1 (Figure 5(c)). In parallel, the antiapoptotic signaling induced by ΔnFGF1 was inhibited by AMPK siRNA (Figure 5(d)). Moreover, flow cytometry assay showed that ΔnFGF1 significantly alleviated glucolipotoxicity-induced apoptosis of cells, whereas cotreatment with AMPK inhibitor CC counteracted this effect of ΔnFGF1 ( Figure 5(e)), suggesting the importance of AMPK in the regulation of apoptosis in MIN6 cell.

Discussion
Stable β-cell numbers are critical in insulin resistance and secretion, with significant reductions in β-cell functions in T2DM [29][30][31]. In this study, we determined that ΔnFGF1, a nonmitogenic truncation of FGF1, effectively reduces blood glucose in T2DM mice. More importantly, ΔnFGF1 can protect β-cells from apoptosis induced by high glucose levels through AMPK/SIRT1/PGC-1α signaling in vivo and in vitro.
Administration of ΔnFGF1 to db/db mice resulted in a notable decrease of blood glucose. Additionally, insulin secretion and apoptosis of islet β-cells were dramatically improved in ΔnFGF1-treated db/db mice. Immunofluorescence staining revealed significant more insulin-positive pancreatic β-cells in the ΔnFGF1-treated db/db mice compared with that in the db/db control mice. Interestingly, ΔnFGF1 treatment of db/db mice for 8 weeks did not increase the number of PCNA-positive islet cells. As a crucial cellular energy sensor, AMPK regulates energy metabolism and promotes glucose uptake through the modulation    Oxidative Medicine and Cellular Longevity of metabolic cell signaling molecules [32][33][34]. Previous studies identified the activation of the AMPK/SIRT1 pathway as a potential target to inhibit apoptosis in diabetic mouse models [35,36]. We tested the effects of ΔnFGF1 treatment on this pathway using MIN6, a pancreatic β-cell line, and found that treatment with ΔnFGF1 significantly inhibited glucolipotoxicity-induced apoptosis, and consistent with previous reports, we observed a reduction in phosphorylated-(p-) AMPK and Sirtuin-1 (SIRT1) expression. We demonstrated that ΔnFGF1 can attenuate glucolipotoxicity-induced apoptosis of MIN6 cells via activation of the AMPK/SIRT1 signaling pathway, indicating that ΔnFGF1 promotes SIRT1 signaling via AMPK activation. AMPK can promote mitochondrial synthesis via the direct phosphorylation of PGC-1α [37]. SIRT1 also acts upstream of PGC-1α [38]. Our results showed that ΔnFGF1 acts to phosphorylate AMPK and increase SIRT1 and PGC-1α expression. We also found that ΔnFGF1 can directly inhibit proapoptotic protein C-caspase 3 and Bax as well as increase antiapoptotic protein Bcl-2 in HG+PA-treated MIN6 cells. Further, the use of an AMPK inhibitor, Compound C, abolished the protective effects of ΔnFGF1. Overall, our results revealed that ΔnFGF1 induces AMPK phosphorylation to increase SIRT1 and PGC-1α expression and inhibit apoptosis in pancreatic β-cells ( Figure 5(f)).
In summary, this study uncovered a crucial role of ΔnFGF1 to inhibit β-cell apoptosis and promote cell survival in diabetes. Long-term application of ΔnFGF1 significantly reduced circulating blood glucose and increased the number of β-cells in db/db mice, by promoting insulin biosynthesis and increasing β-cell survival. Further investigation of the mechanism revealed that ΔnFGF1 inhibits β-cell apoptosis via activating AMPK/SIRT1/PGC-1α signaling to improve β-cell function. Our results illustrate the effectiveness of ΔnFGF1 as a potential treatment for high glucose-induced β-cell apoptosis and T2DM.

Data Availability
The data used to support the findings of this study are included within the article.