二甲双胍通过激活 GPR40-PLC-IP3途径减轻 β- 细胞脂毒性后继炎症

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Metformin Reduces Lipotoxicity-Induced Meta-Inflammation in β-Cells through the Activation of GPR40-PLC-IP3 Pathway


Ximei Shen 深圳市西梅森科技有限公司,1,2 Beibei Fan Beibei,1 Xin Hu 新虎,1 Liufen Luo 罗,1 Yuanli Yan 袁丽燕,1 and 及Liyong Yang 杨1,2Show more展示更多Academic Editor: 学术编辑:Alexander Kokkinos 亚历山大 · 科基诺斯Received 收到15 May 2019 二零一九年五月十五日Revised 修订本01 Aug 2019 二零一九年八月一日Accepted 接受04 Sep 2019 二零一九年九月四日Published 出版19 Dec 2019 二零一九年十二月十九日

Abstract

摘要

Background and Purpose. Metformin, a widely used antidiabetic drug, has been shown to have anti-inflammatory properties; nevertheless, its influence on β-cell meta-inflammation remains unclear. The following study investigated the effects of metformin on meta-inflammatory in β-cells and whether the underlying mechanisms were associated with the G protein-coupled receptor 40-phospholipase C-inositol 1, 4, 5-trisphosphate (GPR40-PLC-IP3) pathway. Materials and Methods. Lipotoxicity-induced β-cells and the high-fat diet-induced obese rat model were used in the study. Results. Metformin-reduced lipotoxicity-induced β-cell meta-inflammatory injury was associated with the expression of GPR40. GPR40 was involved in metformin reversing metabolic inflammation key marker TLR4 activation-mediated β-cell injury. Furthermore, downstream signaling protein PLC-IP3 of GPR40 was involved in the protective effect of metformin on meta-inflammation, and the above process of metformin was partially regulated by AMPK activity. In addition, the anti-inflammatory effects of metformin were observed in obese rats. Conclusion. Metformin can reduce lipotoxicity-induced meta-inflammation in β-cells through the regulation of the GPR40-PLC-IP3 pathway and partially via the regulation of AMPK activity.

背景及目的。二甲双胍是一种广泛应用的抗糖尿病药物,已被证明具有抗炎作用,但其对 β- 细胞间充质炎的影响尚不清楚。接下来的研究探讨了二甲双胍对 β 细胞间接炎症的影响,以及其作用机制是否与 GPR40-PLC-IP3通路有关。物料及方法。本研究采用脂毒性诱导的 β 细胞和高脂饮食诱导的肥胖大鼠模型。结果。二甲双胍减轻脂毒性诱导的 β- 细胞间接炎症损伤与 GPR40的表达有关。GPR40参与二甲双胍逆转 TLR4激活介导的 β- 细胞损伤。此外,GPR40的下游信号蛋白 PLC-IP3参与了二甲双胍对间接炎症的保护作用,二甲双胍的上述过程部分受到 AMPK 活性的调节。此外,还观察了二甲双胍对肥胖大鼠的抗炎作用。总结。二甲双胍通过调节 GPR40-PLC-IP3途径减轻脂毒性诱导的 β 细胞间接炎症反应,部分通过调节 AMPK 活性。

1. Introduction

1. 引言

Metformin has been widely used for the treatment of type 2 diabetes. Metformin exerts its effects through AMPK activation. Increasing evidence indicates that metformin directly acts on pancreatic β-cells [1]; nevertheless, the effect of metformin on β-cells remains controversial. Studies have shown that metformin inhibits β-cell apoptosis induced by hyperglycemia or hyperlipidemia [23] and enhances the number of pancreatic progenitor cells in the mouse embryonic pancreas [4], while other studies have indicated that metformin has a dual role in MIN6 pancreatic β-cell function through the AMPK-dependent autophagy pathway [5]. Therefore, the exact regulation mechanisms of metformin on β-cells need to be further investigated.

二甲双胍已广泛用于治疗2型糖尿病。二甲双胍通过 AMPK 活化发挥作用。越来越多的证据表明,二甲双胍直接作用于胰岛 β 细胞[1] ,然而,二甲双胍对胰岛 β 细胞的作用仍然存在争议。研究表明二甲双胍能抑制高血糖或高脂血症诱导的小鼠胚胎胰腺 β 细胞凋亡[2,3] ,增加胰腺祖细胞的数量[4] ,而其他研究表明二甲双胍通过 ampk 依赖的自噬途径参与 MIN6胰腺 β 细胞功能[5]。因此,二甲双胍对 β 细胞的确切调控机制有待进一步研究。

Metabolic inflammatory injury is currently considered an important mechanism for lipotoxicity-induced β-cell injury. However, targeted interventions have not yet been identified. Toll-like receptor 4 (TLR4) is the key marker of meta-inflammation which has an important role in the pathogenesis of metabolic inflammation in type 2 diabetes [610]. Our previous studies have demonstrated that lipotoxicity directly activates the TLR4-JNK pathway cascade in islet β-cells and that it induces insulin secretion disorders and apoptosis of β-cells [11]. Whether the protective effect of metformin on β-cells is associated with TLR4 has not yet been reported. Studies have suggested that metformin has a clear inhibitory effect on TLR4 in the skeletal muscle of diabetic rats [12]. The aim of this study was to investigate whether metformin could inhibit the activation of TLR4 inflammatory signals in β-cells induced by lipotoxicity.

目前认为代谢性炎症损伤是脂毒性诱导 β- 细胞损伤的重要机制。然而,目标明确的干预措施尚未确定。Toll样受体受体4(TLR4)是变性炎症的关键标志物,在2型糖尿病代谢性炎症的发病机制中具有重要作用[6-10]。我们以前的研究表明,脂毒性直接激活胰岛 β 细胞 TLR4-JNK 通路级联反应,并诱导胰岛素分泌紊乱和 β 细胞凋亡[11]。二甲双胍对 β 细胞的保护作用是否与 TLR4有关尚未见报道。研究表明二甲双胍对糖尿病大鼠骨骼肌 TLR4有明显的抑制作用[12]。本研究旨在探讨二甲双胍能否抑制脂毒性诱导的 β 细胞 TLR4炎症信号的激活。

GPR40 is a long-chain FFA receptor that is specifically expressed on the membrane of pancreatic β-cells [13]. Although the early literatures reported that GPR40 has inconsistencies in beta cell dysfunction [1416], many studies supported the benefit of GPR40 on beta cells [1718]; and GPR40 agonist was already tested in several clinical trials [1921]. At present, the association between the protective effects of metformin and GPR40 has not yet been reported. However, preclinical studies have demonstrated that a combination therapy with GPR40 agonist TAK-875 and metformin could be a valuable strategy for glycemic control and β-cell preservation in type 2 diabetes [22]. Thus, this study is aimed at further exploring the relationship between GPR40 and the protective effects of metformin on lipotoxicity-induced metabolic inflammation.

GPR40是一种长链 FFA 受体,在胰岛 β 细胞膜上特异表达[13]。尽管早期文献报道 GPR40在 β 细胞功能障碍中存在不一致性[14-16] ,但许多研究支持 GPR40对 β 细胞的益处[17,18] ,而且 GPR40激动剂已经在多个临床试验中进行了测试[19-21]。目前,二甲双胍的保护作用与 GPR40之间的关系尚未见报道。然而,临床前研究表明,联合应用 GPR40激动剂 TAK-875和二甲双胍对于2型糖尿病患者的血糖控制和 β 细胞保存是一种有价值的策略[22]。因此,本研究旨在进一步探讨 GPR40与二甲双胍对脂毒性代谢性炎症的保护作用的关系。

In summary, β-cell lines and high-fat diet-induced obese rats were employed to investigate the protective effects of metformin on lipotoxicity in β-cells and the relationship between this protective effect and GPR40. Furthermore, it was confirmed that metformin inhibits lipotoxicity-induced TLR4 activation through the GPR40 pathway and AMPK activity, thereby preventing lipotoxicity-induced apoptosis of β-cells.

本研究以 β- 细胞系和高脂饮食诱导的肥胖大鼠为研究对象,探讨二甲双胍对 β- 细胞脂毒性的保护作用及其与 GPR40的关系。二甲双胍通过 GPR40通路和 AMPK 活性抑制脂毒性诱导的 TLR4活化,从而抑制脂毒性诱导的 β 细胞凋亡。

2. Materials and Methods

2. 材料和方法

2.1. Cell Culture
2.1. 细胞培养

Mouse islet cell line βTC6 was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were incubated with Dulbecco’s modified Eagle’s medium (DMEM, 25 mmol/L glucose), supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, and 10 mmol/L HEPES buffer solution in a humidified atmosphere containing 5% CO2/95% air at 37°C. All reagents were purchased from Gibco (Carlsbad, USA). Cells at passages 26 to 30 were used for all experiments.

小鼠胰岛细胞系 βtc6来源于美国弗吉尼亚州马纳萨斯的美国型培养基。在37 ° c、含5% CO2/95% 空气的湿化环境中,用含10% 胎牛血清(FBS)、2mmol/l l- 谷氨酰胺和10mmol/l HEPES 缓冲液的改良鹰氏培养基(DMEM,25mmol/l 葡萄糖)培养细胞。所有试剂都是从 Gibco (卡尔斯巴德)购买的。第26ー30代的细胞用于所有实验。

2.2. In Vitro Cell Experiments
2.2. 体外细胞实验
2.2.1. Palmitic Acid (PA) or Lipopolysaccharide (LPS) Treatment
2.2.1. 棕榈酸(PA)或脂多糖(LPS)治疗

βTC6 cells were incubated in DMEM (25 mmol/L glucose) for 24 h until the cells reached 50% confluence. Cell culture medium was then removed and washed twice with PBS then treated with 0.5 mmol/L PA (Sigma-Aldrich) or 1.0 mg/L LPS (Sigma-Aldrich) for 24 h in culture media. Cells treated with 0.5% BSA/ethanol in HDMEM without PA or LPS (uninduced) served as the control. PA and LPS solutions were prepared as described before [11].

βtc6细胞在25mmol/l 葡萄糖的 DMEM 培养液中孵育24h,直至细胞达到50% 汇合。将细胞培养液去除,用 PBS 洗涤两次,然后用0.5 mmol/L 的 Sigma-Aldrich (Sigma-Aldrich)或1.0 mg/L 的 LPS (Sigma-Aldrich)处理24h。用0.5% bsa/乙醇处理的 HDMEM 细胞作为对照,未经 PA 或 LPS 诱导。PA 和 LPS 溶液的制备方法与文献[11]相同。

2.2.2. Metformin (MF) Treatment
2.2.2. 二甲双胍治疗

βTC6 cells were treated with 0.5 mmol/L PA+DMEM (25 mmol/L glucose) or 1.0 mg/L LPS+DMEM (25 mmol/L glucose) for 24 h and then exposed to different concentrations (25, 50, and 100 μmol/L) of metformin (dissolved in 0.1% dimethylsulfoxide (DMSO) (); BN1504091105; Chia Tai Tianqing, China) [23] with complete medium for 72 h. PA-induced or LPS-induced cells without metformin and uninduced cells exposed to metformin served as controls.

用0.5 mmol/L pa + dmem (25mmol/l 葡萄糖)或1.0 mg/L lps + dmem (25mmol/l 葡萄糖)处理 βtc6细胞24h,然后分别接触不同浓度的二甲双胍(溶于0.1% 二甲基亚砜(DMSO)中)、 BN1504091105、中国正大天清[23] ,用完全培养液培养72 h paforming 或 LPS-induced 的细胞,无二甲双胍和无二甲双胍的细胞作为对照。

2.2.3. GPR40 Agonist and Inhibitor Treatment
2.2.3. GPR40激动剂和抑制剂治疗

βTC6 cells were pretreated with 1.0 mg/L LPS for 24 h. Afterwards, cells were washed twice with PBS and then treated with 10 μmol/L of TAK-875 (dissolved in 0.1% DMSO (); TRC-CANADA) [24] or 5 μmol/L of DC260126 (dissolved in 0.1% DMSO (); TRC-CANADA) [25] for 72 h. LPS-induced cells without TAK-875 or DC260126 and uninduced cells exposed to TAK-875 or DC260126 served as controls.

用1.0 mg/L LPS 预处理 βtc6细胞24小时后,用 PBS 洗涤2次,然后用10μmol/L TAK-875(溶于0.1% DMSO () ; TRC-CANADA)[24]或 DC260126(溶于0.1% DMSO () ; TRC-CANADA)[25]处理72小时 LPS 诱导的细胞,不用 ta875或 DC260126作为对照。

2.2.4. Protein Inhibitor Treatments
2.2.4. 蛋白质抑制剂处理

βTC6 cells were pretreated with 20 μmol/L PLC inhibitor (U73122, Sigma-Aldrich) [26] or 20 μmol/L IP3 inhibitor (2-APB, Sigma-Aldrich) [27], respectively, for 4 h before being exposed to 0.5 mmol/L PA+DMEM (25 mmol/L glucose)+100 μmol/L MF for 72 h. Cells exposed to U73122, 2-APB, or complete medium alone were used as controls.

用20μmol/L 的 PLC 抑制剂(U73122,Sigma-Aldrich)[26]和20μmol/L 的 IP3抑制剂(2-APB,Sigma-Aldrich)[27]分别预处理 βtc6细胞4h,然后用0.5 mmol/L 的 pa + dmem (25mmol/l 葡萄糖) + 100μmol/L MF 处理72h 作为对照。

2.2.5. AMPK Inhibitor and Agonist Treatment
2.2.5. AMPK 抑制剂和激动剂治疗

βTC6 cells were pretreated with 10 μmol/L AMPK inhibitor (compound C; Sigma-Aldrich, USA) [28] for 4 h before being exposed to 0.5 mmol/L PA+DMEM (25 mmol/L glucose)+100 μmol/L MF for 72 h. Cells exposed to compound C or complete medium alone were used as controls.

用10μmol/L AMPK 抑制剂(化合物 c; Sigma-Aldrich,USA)[28]预处理 βtc6细胞4h,然后用0.5 mmol/L pa + dmem (25mmol/l 葡萄糖) + 100μmol/L MF 暴露72h 作为对照。

βTC6 cells were pretreated with 0.5 mmol/L PA+DMEM (25 mmol/L glucose) for 24 h. Consequently, cells were treated with 1 mmol/L of AMPK agonist AICAR (dissolved in 0.1% DMSO (); Henan DaKen Chemical CO., LTD.) [29] for additional 72 h. PA-induced cells+DMEM (25 mmol/L glucose) without AICAR and uninduced cells exposed to AICAR served as controls.

用0.5 mmol/L pa + DMEM (25mmol/l 葡萄糖)预处理 βtc6细胞24h,然后用1mmol/l AMPK 激动剂 AICAR (溶于0.1% DMSO () ,河南大肯化工有限公司)[29]处理细胞72h 后,加入未经 AICAR 处理的 DMEM (25mmol/l 葡萄糖)作为对照。

2.3. GPR40 Small Interfering (si) RNA and GPR40 Cloning and Adenovirus Generation
2.3. GPR40小干扰 RNA 和 GPR40的克隆及腺病毒的制备

The specific experimental process was based on our previous methods [24].

具体的实验过程是基于我们以前的方法[24]。

2.4. Glucose-Stimulated Insulin Secretion (GSIS)
2.4. 葡萄糖刺激的胰岛素分泌

The detection steps were carried out according to our previous methods [11], and the total cellular protein concentration was used to correct the concentration of insulin.

检测步骤根据我们以前的方法[11]进行,并用细胞总蛋白质浓度来校正胰岛素浓度。

2.5. Animal Experiments
2.5. 动物实验

Five- to six-week-old, specific-pathogen-free grade, male Sprague-Dawley (SD) rats () were obtained from the laboratory animal facility at Fujian Medical University (animal certification number SYXK (min) 2016-0006). After a one-week acclimation period, the animals were randomly divided into two groups: normal control diet () and high-fat diet (HFD, ). Rats in the control group were additionally divided into two groups, half of which received metformin (50 mg/kg/d; fasting gavage, per day; ) [30], while others received no treatment (). All rats in the control group were fed a normal pellet diet for 16 weeks. Rats in the HFD group received a high-fat diet [11] for 16 weeks to induce obesity. The HFD group was then subdivided into two groups, half of which received metformin (50 mg/kg/d; fasting gavage, per day; ), while others received no treatment (). The body weight and length were recorded weekly.

5-6周大,无特异性病原体等级的雄性 SD 大鼠从福建医科大学的实验动物设施获得(动物认证号 SYXK (min)2016-0006)。经过一周的习服后,动物被随机分为两组: 正常对照组()和高脂饮食组()。对照组大鼠另外分为两组,其中一半给予二甲双胍(50mg/kg/d; 空腹灌胃,每天;)[30] ,另一半不给予治疗()。对照组大鼠以正常颗粒饲料喂养16周。HFD 组大鼠接受高脂饮食16周诱导肥胖。HFD 组随后再分为两组,其中一半给予二甲双胍(50mg/kg/d; 空腹灌胃,每天;) ,另一半不给予治疗()。每周记录体重和体长。

This study was approved by the Ethics Committee for Biomedical Research of the First Affiliated Hospital of Fujian Medical University.

这项研究得到了生物医学研究福建医科大学第一附属医院伦理委员会的批准。

2.6. ITT and IPGTT
2.6. ITT 和 IPGTT
2.6.1. Insulin Tolerance Test (ITT)
2.6.1. 胰岛素耐受性测试

All animals were fasted for 4 hours. Insulin was injected at 1 U/kg [31]. Blood glucose was measured before and 0, 30, 60, 90, and 120 min after the injection of insulin.

所有动物禁食4小时。胰岛素注射量为1 U/kg [31]。分别于注射胰岛素前及注射后0、30、60、90、120分钟测定血糖。

2.6.2. Intraperitoneal Glucose Tolerance Test (IPGTT)
2.6.2. 腹腔内糖耐力测试

Intraperitoneal glucose tolerance test was preformed according to a previously described protocol [32]. Briefly, rats were fasted for 8 hours. Then, all animals received an intraperitoneal injection of 50% glucose (2 g/kg). Blood glucose and insulin were measured before and 0, 30, 60, 90, and 120 min after the injection of glucose. After the experiment, the feed was supplemented.

根据先前描述的治疗方案,腹腔内注射糖耐力测试。简单地说,老鼠禁食8小时。然后,所有动物腹腔注射50% 葡萄糖(2g/kg)。分别于注射葡萄糖前、注射葡萄糖后0、30、60、90、120分钟测定血糖和胰岛素。试验结束后,添加饲料。

2.7. Serum Insulin, FFA, and Biochemical Indicator Measurements
2.7. 血清胰岛素、游离脂肪酸和生化指标测量

Control and experimental rats were fasted overnight for 8 hours and then euthanized by intraperitoneal injection of 10% chloral hydrate (0.03 mL/kg). Blood samples were obtained from the abdominal aorta (serum tube, without anticoagulant); samples were centrifuged at 3500 rpm for 10 minutes at room temperature and then separated and stored at -80°C. Blood samples were used to measure fasting plasma glucose, fasting plasma insulin, free fatty acid (FFA), triglycerides (TG), total cholesterol (TC), HDL cholesterol (HDL-C), and LDL cholesterol (LDL-C), based on previously published methods [11].

对照组和实验组大鼠禁食8小时后腹腔注射10% 水合氯醛(0.03 mL/kg) ,实施安乐死。从腹主动脉(无抗凝剂的血管)采集血样,在室温下3500转/分钟离心10分钟,然后分离并保存在 -80 ° c。根据以前发表的方法[11] ,血液样本被用来测量空腹血糖、空腹胰岛素、游离脂肪酸(FFA)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-c)和低密度脂蛋白胆固醇(LDL-c)。

2.8. Enzyme-Linked Immunosorbent Assay
2.8. 酶联免疫吸附试验
2.8.1. Inflammatory Cytokine Measurements
2.8.1. 炎症因子测量

Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were detected by ELISA (Cusabio, China). Intra-assay coefficient of variation was <8% and interassay coefficient of variation was <10%.

应用 ELISA 法检测白细胞介素 -1(IL-1)、白细胞介素 -6(IL-6)和肿瘤坏死因子 -α (tnf-α)。分析内变异系数小于8% ,分析间变异系数小于10% 。

2.8.2. Cellular IP3 Measurements
2.8.2. 细胞 IP3测量

Cellular IP3 detection referenced the ELISA instructions (Cusabio, catalog number CSB-E13420m, http://www.cusabio.com,) and documentary reports by Li et al. [33]. Cells were thoroughly washed with precooled PBS (pH 7.2 to 7.4), then transferred to a suitable centrifuge tube, diluted with 1× PBS (pH 7.2 to 7.4) to a cell concentration of 100 million/mL, and stored at -20°C overnight. Repeat freeze-thaw cycles were used to disrupt the cell membrane. The lysed cells were then centrifuged at 1000 g for 2 min at 2-8°C, and the supernatant was used for analysis. Anti-IP3 detection antibody was added to the supernatant and incubated at 37°C for 1 hour and then incubated with substrate solution at 37°C for 15 min. The reaction was terminated following the addition of stop solution, and the OD value was measured at a wavelength of 450 nm using a microplate reader. Using the standard concentration as the ordinate (logarithmic coordinate) and the corresponding OD value as the abscissa (logarithmic coordinate), the standard curve was drawn using GraphPad Prism 5 software and the concentration for each sample was calculated.

细胞 IP3检测参考了酶联免疫吸附试验(ELISA)的说明(目录编号 CSB-E13420m, http://www.Cusabio.com )和 Li 等人的文献报告[33]。电池用预冷的 PBS (ph7.2ー7.4)彻底清洗,然后转移到合适的离心管中,用1 × PBS (ph7.2ー7.4)稀释至浓度为1亿 ml 的电池,在 -20 ° c 条件下储存过夜。重复冻融循环破坏细胞膜。1000g,2ー8 °c 离心2min,上清液进行分析。在培养上清中加入抗 ip3检测抗体,在37 ° c 温度下培养1h,与底物溶液在37 ° c 温度下培养15min。反应终止后,加入停止溶液,并在450纳米波长用微板阅读器测量 OD 值。以标准浓度为纵坐标(对数坐标) ,对应的 OD 值为横坐标(对数坐标) ,用 GraphPad Prism 5软件绘制标准曲线,计算各样品的浓度。

2.9. Apoptosis Analysis by TUNEL Assay
2.9. TUNEL 法检测细胞凋亡
2.9.1. Detection of βTC6 Cell Apoptosis
2.9.1. βtc6细胞凋亡的检测

The TUNEL was performed using the DeadEnd™ Fluorometric TUNEL System (DeadEnd™ Fluorometric TUNEL System, Promega Corporation, USA), according to the previously published method [11]. Mechanism: the DeadEnd™ Fluorometric TUNEL System measures the fragmented DNA of apoptotic cells by catalytically incorporating fluorescein-12-dUTP at 3-OH DNA ends using the Terminal Deoxynucleotidyl Transferase, Recombinant, enzyme (rTdT), which forms a polymeric tail using the principle of the TUNEL (TdT-mediated dUTP Nick-End Labeling) assay. The fluorescein-12-dUTP-labeled DNA then can be visualized by fluorescence microscopy or quantified by flow cytometry. In addition, the nucleus is stained with red fluorescence by PI (https://www.promega.com.cn).

TUNEL 是使用 DeadEndTM 荧光 TUNEL 系统(DeadEndTM 荧光 TUNEL System,Promega Corporation,USA) ,根据以前发表的方法[11]进行的。机制: DeadEndTM 荧光 TUNEL 系统测量凋亡细胞的 DNA 片段,催化结合荧光素 -12-dUTP 在3-OH DNA 末端使用末端脱氧核苷酸转移酶,重组,酶(rTdT) ,形成一个聚合物的尾巴使用 TUNEL 原理(tdt 介导的 dUTP 缺口末端标记)。荧光素 -12-dutp 标记的 DNA 可以用荧光显微镜观察到,也可以用流式细胞仪定量。此外,细胞核被 PI ( https://www.promega.com.cn )染上红色荧光。

2.9.2. Apoptosis Detection in Pancreatic Tissue Samples
2.9.2. 胰腺组织标本中细胞凋亡的检测

Paraffin sections of pancreatic tissue were dewaxed and hydrated and then washed twice with xylene for 5 min. Tissue samples were then treated with different concentrations of ethanol (100, 95, 90, 80, and 70%) for 3 minutes and then treated with 1% Triton-100 and 3% H2O2-methanol solution for 15 min. Consequently, samples were treated with proteinase K at 37°C for 30 min. Streptavidin-FITC-labeled working solution was added to each section, and the reaction was incubated in a humidified chamber at 37°C for 1 h in the dark. Afterwards, each slice was dripped in prepared DAPI dye solution for 5 min at room temperature in the dark. Apoptotic cells were observed by fluorescence microscopy and imaged.

胰腺组织石蜡切片脱蜡水化后,用二甲苯洗涤2次,每次5min。然后用不同浓度的乙醇(100,95,90,80,70%)处理组织标本3min,再用1% Triton-100和3% h2o2-甲醇溶液处理15min。用蛋白酶 k 在37 ° c 处理样品30min。每段加入链霉亲和素 -fitc 标记的工作液,反应在37 ° c 的湿化室中在黑暗中孵育1h。然后,将每片切片滴入准备好的 DAPI 染料溶液中,在室温黑暗条件下浸泡5分钟。荧光显微镜下观察细胞凋亡情况。

2.10. Protein Extraction and Western Blotting
2.10. 蛋白质提取和西方墨点法
2.10.1. Protein Extraction
2.10.1. 蛋白质提取

Whole cell and nuclear extracts were prepared for Western blot analysis. Control and experimental rats were fasted overnight for 8 h and then euthanized by intraperitoneal injection of 10% chloral hydrate (0.03 mL/kg). Islets were isolated from each SD rat pancreas according to previously described approach [11]. Samples were then washed twice with PBS and lysed in radioimmune precipitation assay buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS with protease inhibitors on ice for 30 min.

制备全细胞和细胞核提取物进行免疫印迹分析。对照组和实验组大鼠禁食8小时,腹腔注射10% 水合氯醛(0.03 mL/kg)。根据以前描述的方法[11]从每个 SD 大鼠的胰腺分离胰岛。样品用 PBS 洗涤两次,在含有50mm Tris (ph7.4)、150mm NaCl、1% Triton X-100、1% 脱氧胆酸钠和0.1% SDS 的冰蛋白酶抑制剂的放射免疫沉淀试验缓冲液中溶解30min。

2.10.2. Western Blotting
2.10.2. 西方墨点法

Protein concentration from cells and islet tissue was determined by bicinchoninic acid protein assays. Protein samples were separated by SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Sigma). Membranes were blocked with 5% fat-free milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 and incubated overnight at 4°C with anti-TLR4 (1 : 1000, Abcam, UK, ab13867), anti-NF-κB p65 (1 : 1000, Abcam, UK, ab16502), anti-GRP40 (1 : 200, Santa Cruz, sc-32905), anti-PLC (1 : 500, Abcam, UK, ab243181), anti-pAMPKα1 (1 : 1000, Abcam, UK, ab23875), anti-AMPK (1 : 500, Abcam, UK, ab3759), or anti-β-actin antibody (1 : 200, Abcam, UK, ab8226). Membranes were then incubated with horseradish peroxidase-labeled secondary antibody (1 : 500) for 1 hour at room temperature. Protein signals were visualized using the enhanced chemiluminescence detection system.

采用二辛可尼酸蛋白测定法测定细胞和胰岛组织蛋白质含量。蛋白质样品用 sds- 聚丙烯酰胺凝胶分离,并转移到聚偏二氟乙烯(PVDF)膜上。用含0.1% Tween-20的5% 脱脂牛奶(TBS)缓冲生理盐水(Tris-buffered saline,TBS)阻断细胞膜,并在4 ° c 条件下与抗 tlr4(1:1000,Abcam,UK,ab13867)孵育一夜,抗 nf-κb p65(1:1000,Abcam,UK,ab16502) ,抗 grp40(1:200,Santa Cruz,sc-32905) ,抗 -plc (1:500,Abcam,UK,ab243181) ,抗 -pampkα1(1:1000,Abcam,UK,ab23875) ,抗 -ampk (1:500,Abcam,UK,ab3759) ,或抗 -β-actin(1:200,Abcam,UK,ab8226).然后与辣根过氧化物酶标记的二级抗体(1:500)在室温下孵育1小时。蛋白质信号可视化使用增强的化学发光检测系统。

2.11. Immunofluorescence
2.11. 免疫荧光

Pancreatic paraffin sections from each group were dewaxed, hydrated, and pretreated using the heat-induced antigen retrieval technique. Each section was enzyme inactivated using 3% H2O2 in methanol for 10 min and blocked with ready-to-use goat serum for 20 min. The sections were then incubated with 1 : 50 diluted primary antibody (anti-insulin and anti-glucagon) incubated in a wet box for 2 hours at 37°C and 1 : 50 diluted second antibody FITC/TRITC incubated in the dark for 1 hour at 37°C. Each slice was then incubated in prepared DAPI dye solution for 5 min at room temperature in the dark and then sealed with antiextraction sealant. Cellular protein expression was observed under a fluorescence microscope, and three random regions were imaged.

每组的胰腺石蜡切片均用热诱导抗原修复技术脱蜡、水化和预处理。每组酶用3% H2O2在甲醇中灭活10min,用即食山羊血清封闭20min。这些切片随后用1:50稀释的初级抗体(抗胰岛素和抗胰高血糖素)在37 ° c 的湿箱中孵育2小时,1:50稀释的第二抗体 FITC/TRITC 在37 ° c 的黑暗中孵育1小时。每片切片在准备好的 DAPI 染料溶液中室温孵育5分钟,在黑暗中,然后用抗萃取密封剂密封。在荧光显微镜下观察细胞蛋白的表达,并对3个随机区域进行成像。

2.12. Statistical Analysis
2.12. 统计分析

Results were expressed as  Comparisons between groups were performed using ANOVAs. The LSD test was performed to compare the two groups.  was considered to be statistically significant.

结果表明,组之间的比较进行了使用 ANOVAs。采用 LSD 试验对两组进行比较。被认为具有统计学意义。

3. Results

3. 结果

3.1. The Protective Effect of Metformin on Lipotoxicity-Induced Meta-Inflammation in β-Cells Is Regulated by the GPR40 Expression
3.1. 二甲双胍对脂毒性诱导的 β 细胞间接炎的保护作用受 GPR40表达的调节

To observe the effect of metformin on β-cell lipotoxicity injury, lipotoxicity-injured β-cells were incubated with different concentrations of metformin, and TLR4 expression was assessed as a marker of meta-inflammation. We observed that metformin decreased apoptosis of β-cells (Figure 1(a)) in a concentration-dependent manner and increased insulin secretion, BIS, and GSIS (Figure 1(b)). Simultaneously, metformin concentration-dependently increased expression of GPR40 and decreased TLR4 and NF-κB subunit P65 expression (Figure 1(c)).

为了观察二甲双胍对 β- 细胞脂毒性损伤的影响,用不同浓度的二甲双胍对脂毒性损伤的 β- 细胞进行孵育,并以 TLR4的表达作为间接炎症的标志物。我们观察到二甲双胍以浓度依赖的方式降低 β 细胞凋亡(图1(a)) ,增加胰岛素分泌、 BIS 和 GSIS (图1(b))。同时,二甲双胍浓度依赖性增加了 GPR40的表达,降低了 TLR4和 NF-κB P65的表达(图1(c))。(a)
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(h)Figure 1 图1Adjustment GPR40 expression altered metformin’s protective function on lipotoxicity in 调节 GPR40表达改变二甲双胍对脂毒性的保护作用β-cells. All data is presented as – 细胞。所有数据表示为 of three independent experiments. (a) Metformin reduces PA-induced apoptosis in (a)二甲双胍减少 pa 诱导的细胞凋亡β-cells. (A1) Representative images from fluorescent microscopy in each group. The white arrow indicates apoptotic cells. (A2) Collective analyses of all three independent experiments. (b) Detection of basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS) by ELISA. (c) Expression of TLR4, NF- – 细胞。(A1)每组荧光显微镜的代表性影像。白色箭头显示凋亡细胞。(A2)三个独立实验的集体分析。(b) ELISA 检测基础胰岛素分泌(BIS)和葡萄糖刺激的胰岛素分泌(GSIS)。(c) TLR4,NF-的表达κB subunit P65, and GPR40 detected by Western blotting. (C1) Representative Western blot images for each group. (C2) The ratio of target protein to 西方墨点法检测到 b 亚基 P65和 GPR40。(C1)每组有代表性的免疫印迹图像。(C2)目标蛋白与β-actin. (d, e) GPR40 protein expression levels detected by Western blotting in GPR40-overexpressing transfected cells and siRNA transfected cells (siRNA). Regulation of GPR40 expression and the protective effects of metformin on PA -induced – 肌动蛋白。(d,e) GPR40转染细胞和 siRNA 转染细胞(siRNA)中 GPR40蛋白表达水平的检测,结果表明: 转染细胞和 siRNA 转染细胞中 GPR40蛋白表达水平的差异有统计学意义(p & lt。GPR40的表达调控及二甲双胍对 PA 诱导的保护作用β-cell apoptosis (f), insulin secretion disorder (g), and TLR4 and NF- – 细胞凋亡(f) ,胰岛素分泌紊乱(g) ,TLR4和 NF-κB subunit P65 protein expression (h). (a–c) B 亚基 P65蛋白表达(h) . (a-c)A vs. NC group (without PA and MF), 与 NC 组(不包括 PA 和 MF) ,B vs. 0.5 mmol/L PA group, 与0.5 mmol/L PA 组比较,Cvs. 0.5 mmol/L PA+25  0.5 mmol/L PA + 25μmol/L MF group, and 分子量为10mol/l 的 MF 基团,以及D vs. 0.5 mmol/L PA+50  0.5 mmol/L PA + 50μmol/L MF group. (d, e) (d,e)A vs. NC group, 对北卡罗来纳州的小组,B vs. NC+vector group. (f–h) 与 NC + 矢量组。(f-h)A vs. NC group (without PA and MF), 与 NC 组(不包括 PA 和 MF) ,B vs. 0.5 mmol/L PA group, and 与0.5 mmol/L PA 组比较,差异有统计学意义C vs. 0.5 mmol/L PA+100  0.5 mmol/L PA + 100μmol/L MF group. mol/L MF 基团

We further validated whether GPR40 was involved in the protective effects of metformin using lentivirus-mediated overexpression (Figure 1(d)) or silencing of GPR40 in β-cells (Figure 1(e)). Our results showed that the upregulation of GPR40 improved the effect of metformin on lipotoxicity-induced apoptosis (Figure 1(f)), increased BIS and GSIS (Figure 1(g)), and reduced inflammation-related protein expression (Figure 1(h)). Conversely, the downregulation of GPR40 decreased the protective effects of metformin on β-cells (Figures 1(f)1(h)).

我们进一步验证了 GPR40是否参与了二甲双胍的保护作用,使用慢病毒介导的过度表达(图1(d))或沉默 GPR40在 β 细胞(图1(e))。我们的结果表明,GPR40的上调改善了二甲双胍对脂毒性诱导的细胞凋亡的影响(图1(f)) ,增加了 BIS 和 GSIS (图1(g)) ,并降低了炎症相关蛋白的表达(图1(h))。相反,GPR40的下调降低了二甲双胍对 β 细胞的保护作用(图1(f)-1(h))。

3.2. GPR40 Was Involved in Metformin Reversing Metabolic Inflammation Key Marker (TLR4) Activation-Mediated β-Cell Injury
3.2. GPR40参与二甲双胍逆转 TLR4激活介导的 β- 细胞损伤

Next, we investigated the specificity of the relationship between protective effects of metformin and metabolic inflammation key marker TLR4. We used LPS, a specific agonist of TLR4, to induce β-cell inflammatory injury and then treated cells with metformin. We found that metformin improved LPS-induced inflammatory apoptosis in β-cells (Figure 2(a)), increased insulin secretion (Figure 2(b)) and GPR40 expression, and inhibited the expression of TLR4 and NF-κB subunit P65 (Figure 2(c)) in a concentration-dependent manner.

其次,我们研究了二甲双胍保护作用与代谢性炎症关键标志物 TLR4之间关系的特异性。我们用 TLR4特异性激动剂 LPS 诱导 β 细胞炎症损伤,然后用二甲双胍处理细胞。我们发现二甲双胍改善 lps 诱导的 β- 细胞炎性凋亡(图2(a)) ,增加胰岛素分泌(图2(b))和 GPR40的表达,并以浓度依赖方式抑制 TLR4和 NF-κB P65的表达(图2(c))。(a)
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(l)Figure 2 图2GPR40 and its involvement in metformin improves LPS-induced inflammatory injury. All data is presented as gPR40及其参与二甲双胍改善 lps 诱导的炎症损伤 of three independent experiments. Metformin improves LPS-induced inflammatory apoptosis (a), insulin secretion disorder (b), and TLR4 and NF- 二甲双胍改善 lps 诱导的炎症细胞凋亡(a)、胰岛素分泌紊乱(b)、 TLR4和 NF-κB subunit P65 expression (c). Regulation of GPR40 expression and the protective effects of metformin on LPS-induced B 亚基 P65的表达(c) . GPR40的表达调控及二甲双胍对 lps 诱导的保护作用β-cell apoptosis (d), insulin secretion disorder (e), and TLR4 and NF- – 细胞凋亡(d)、胰岛素分泌紊乱(e)、 TLR4和 NF-κB subunit P65 expression (f). Activation of GPR40 by TAK-875 protects LPS-induced TAK-875对脂多糖诱导的 GPR40表达的保护作用β-cell apoptosis (g), insulin secretion disorder (h), and TLR4 and NF- – 细胞凋亡(g)、胰岛素分泌紊乱(h)、 TLR4和 NF-κB subunit P65 expression (i). Inhibition of GPR40 by DC260126 aggravated LPS-induced apoptosis (j), insulin secretion disorder (k), and TLR4 and NF- B 亚基 P65的表达(i)。DC260126抑制 GPR40加重 lps 诱导的细胞凋亡(j)、胰岛素分泌紊乱(k)、 TLR4和 NF-κB subunit P65 expression (l). (a–c) B 亚基 P65表达(l)。(a-c)A vs. NC group (without LPS and MF), 对比 NC 组(无 LPS 和 MF) ,B vs. 1.0 mg/L LPS group, 1.0 mg/L LPS 组,C vs. 1.0 mg/L LPS+25  1.0 mg/L LPS + 25μmol/L MF group, and 分子量为10mol/l 的 MF 基团,以及D vs. 1.0 mg/L LPS+50  1.0 mg/L LPS + 50μmol/L MF group. (d–f) 摩尔/l MF 组(d-f)A vs. NC group (without LPS and MF), 对比 NC 组(无 LPS 和 MF) ,B vs. 1.0 mg/L LPS group, and 1.0 mg/L LPS 组C vs. 1.0 mg/L LPS+100  1.0 mg/L LPS + 100μmol/L MF group. (g–i)mol/L MF 基团(g-i)A vs. NC group (without LPS and TAK-875), 正常对照组(无 LPS 和 TAK-875) ,B vs. TAK-875 group, and 对 TAK-875组,和C vs. LPS group. (j–l) 与 LPS 组的比较A vs. NC group (without LPS and DC260126), 与 NC 组(无 LPS 和 DC260126) ,B vs. DC260126 group, and 与 DC260126组,和C vs. LPS group. 与 LPS 组的比较

We then used lentivirus-mediated silencing or overexpression of GPR40 to verify the effect of GPR40 expression on metformin’s protective role in LPS-injured β-cells. Our results demonstrated that the upregulation of GPR40 improved the effect of metformin on LPS-induced apoptosis (Figure 2(d)), increased BIS and GSIS (Figure 2(e)), and reduced inflammation-related protein expression (Figure 2(f)). Conversely, the downregulation of GPR40 attenuated the protective effect of metformin on β-cells (Figures 2(d)2(f)).

然后利用慢病毒介导的 GPR40基因沉默或过度表达来验证 GPR40基因表达对二甲双胍在 lps 损伤的 β- 细胞中保护作用的影响。我们的结果表明,GPR40的上调改善了二甲双胍对 lps 诱导的细胞凋亡的影响(图2(d)) ,增加了 BIS 和 GSIS (图2(e)) ,并降低了炎症相关蛋白的表达(图2(f))。相反,GPR40的下调减弱了二甲双胍对 β 细胞的保护作用(图2(d)-2(f))。

To further confirm the effect of GPR40 on β-cell inflammatory injury, we treated cells with GPR40 agonists or inhibitors after LPS-induced β-cell inflammatory injury. Activation of GPR40 improved lipotoxic-induced apoptosis (Figure 2(g)), increased BIS and GSIS (Figure 2(h)), and decreased expression of TLR4 and NF-κB subunit P65 (Figure 2(i)); and inhibition of GPR40 activity significantly aggravated the inflammatory injury of LPS on β-cells (Figures 2(j)2(l)).

为了进一步证实 GPR40对 β 细胞炎症损伤的影响,我们在 lps 诱导的 β 细胞炎症损伤后,用 GPR40激动剂或抑制剂处理细胞。GPR40的激活促进脂毒性诱导的凋亡(图2(g)) ,增加 BIS 和 GSIS (图2(h)) ,降低 TLR4和 NF-κB P65的表达(图2(i)) ,抑制 GPR40活性显著加重 LPS 对 β 细胞的炎性损伤(图2(j)-2(l))。

3.3. Downstream Target of GPR40 (PLC-IP3) Is Involved in the Protective Effect of Metformin on Lipotoxicity Inflammatory β-Cells
3.3. GPR40(PLC-IP3)下游靶点参与二甲双胍对脂毒性炎症 β 细胞的保护作用

We then investigated the possible association of GPR40 downstream signaling (PLC-IP3) with metformin treatment. We first observed the effect of metformin on PLC-IP3 and then used PLC-IP3 specific inhibitors to interfere with the effects of metformin on lipotoxicity in β-cells. Our results showed that metformin concentration-dependently increased the level of IP3 in β-cells (Figure 3(a)) and PLC (Figure 3(b)) expression. Inhibition of PLC or IP3 reduced the protective effect of metformin on lipotoxicity inflammatory β-cells, which was characterized by increased apoptosis of lipotoxicity inflammatory β-cells (Figures 3(c) and 3(f)), decreased insulin secretion (Figures 3(d) and 3(g)), reduced expression of GPR40, and increased expression of TLR4 and NF-κB subunit P65 (Figures 3(e) and 3(h)).

然后我们研究了 GPR40下游信号通路(PLC-IP3)与二甲双胍治疗的可能联系。我们首先观察了二甲双胍对 PLC-IP3的影响,然后用 PLC-IP3特异性抑制剂干预二甲双胍对 β- 细胞脂毒性的影响。我们的结果显示二甲双胍浓度依赖性地增加了 β 细胞(图3(a))和 PLC (图3(b)表达的 IP3水平。抑制 PLC 或 IP3可降低二甲双胍对脂毒性炎症 β 细胞的保护作用,即增加脂毒性炎症 β 细胞的凋亡(图3(c)和图3(f)) ,减少胰岛素分泌(图3(d)和图3(g)) ,降低 GPR40的表达,增加 TLR4和 NF-κB P65的表达(图3(e)和图3(h))。(a)
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(h)Figure 3 图3The relationship between PLC-IP3 and the protective effects of metformin on lipotoxicity inflammatory injury. Results shown are representative of at least 3 experiments. Metformin concentration-dependently increased the expression of PLC (a) and IP3 (b) in PLC-IP3与二甲双胍抗脂毒性炎症损伤的关系。所显示的结果至少代表了3个实验。二甲双胍浓度依赖性地增加了肝癌细胞 PLC (a)和 IP3(b)的表达β-cells. (c–h) Inhibition of PLC or IP3 expression attenuated the protective effects of metformin on lipotoxicity inflammatory injury. (c, f) The rate of apoptosis in – 细胞。(c-h)抑制 PLC 或 IP3表达可减轻二甲双胍对脂毒性炎症损伤的保护作用。(c,f)细胞凋亡率β-cells. (d, g) Levels of BIS and GSIS. (e, h) Expression levels of TLR4, NF- – 细胞。(d,g) BIS 和 GSIS 水平。(e,h) TLR4,NF-表达水平κB subunit P65, and GPR40. (a, b) B 亚基 P65和 GPR40。(a,b)A vs. NC group (without PA and MF), 与 NC 组(不包括 PA 和 MF) ,B vs. 0.5 mmol/L PA group, 与0.5 mmol/L PA 组比较,C vs. 0.5 mmol/L PA+25  0.5 mmol/L PA + 25μmol/L MF group, and 分子量为10mol/l 的 MF 基团,以及D vs. 0.5 mmol/L PA+50  0.5 mmol/L PA + 50μmol/L MF group. (c–h) 摩尔/l MF 组(c-h)A vs. NC group (without PA and MF), 与 NC 组(不包括 PA 和 MF) ,B vs. 0.5 mmol/L PA+100  0.5 mmol/L PA + 100μmol/L MF group. mol/L MF 基团

3.4. The Protective Effect of Metformin on Lipotoxicity Was Partially Regulated by AMPK Activity
3.4. AMPK 活性部分调节二甲双胍对脂毒性的保护作用

First, we observed the effect of metformin on AMPK activity in lipotoxicity β-cells and found that metformin can increase the activation of AMPK in a concentration-dependent manner (Figure 4(a)). Consequently, we investigated whether the AMPK inhibitor compound C may interfere with the effect of metformin on lipotoxicity. Our data suggested that the inhibition of AMPK activity partially reduced the protective effect of metformin (Figures 4(b)4(e)). Furthermore, we examined the effect of AMPK specific activator AICAR on lipotoxicity β-cells. We found that AICAR inhibits β-cell apoptosis (Figure 4(f)), improves insulin secretion (Figure 4(g)), increases GPR40-IP3-PLC expression, and inhibits TLR4 and NF-κB subunit P65 expression (Figures 4(h) and 4(i)).

首先,我们观察了二甲双胍对脂毒性 β 细胞 AMPK 活性的影响,发现二甲双胍能以浓度依赖的方式增加 AMPK 的活性(图4(a))。因此,我们研究了 AMPK 抑制剂化合物 c 是否可能干扰二甲双胍对脂毒性的影响。我们的数据表明,抑制 AMPK 活性部分降低了二甲双胍的保护作用(图4(b)-4(e))。此外,我们还观察了 AMPK 特异性激活剂 AICAR 对脂毒性 β 细胞的影响。我们发现 AICAR 抑制 β 细胞凋亡(图4(f)) ,改善胰岛素分泌(图4(g)) ,增加 GPR40-IP3-PLC 的表达,抑制 TLR4和 NF-κB P65亚单位的表达(图4(h)和4(i))。(a)
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(i)Figure 4 图4The protective effect of metformin was partially independent of AMPK activity. (a) Metformin concentration-dependently increased the expression of AMPK. (A1) Representative Western blot images for each group. (A2) The ratio of target protein to 二甲双胍的保护作用与 AMPK 活性无关。(a)二甲双胍浓度依赖性增加腺苷酸活化蛋白激酶的表达。(A1)每组有代表性的免疫印迹图像。(A2)目的蛋白质与蛋白质的比例β-actin. (b–e) The relationship between metformin-regulated lipotoxicity and AMPK inhibitor. (b) The rate of apoptosis in – actin。(b-e)二甲双胍调节脂毒性与 AMPK 抑制剂的关系。(b)细胞凋亡率β-cells. (c) Levels of BIS and GSIS. (d) Expression of IP3 in- 细胞。(c) BIS 和 GSIS 的水平。(d) IP3的表达β-cells. (e) Expression levels of TLR4, NF- – 细胞。(e) TLR4,NF-的表达水平κB subunit P65, and GPR40. (f–i) Effect of AICAR on PA-induced 乙亚基 P65,GPR40。(f-i) AICAR 在 pa 诱导中的作用β-cell injury. (f) The rate of apoptosis in 细胞损伤。(f)细胞凋亡率β-cells. (g) Levels of BIS and GSIS. (h) Expression of IP3 in – 细胞。(g) BIS 和 GSIS 的水平。(h) IP3的表达β-cells. (i) Expression levels of TLR4, NF- – 细胞。(i) TLR4,NF-的表达水平κB subunit P65, and GPR40. (a) B 亚基 P65和 GPR40A vs. NC group (without PA and MF), 与 NC 组(不包括 PA 和 MF) ,B vs. 0.5 mmol/L PA group, 与0.5 mmol/L PA 组比较,C vs. 0.5 mmol/L PA+25  0.5 mmol/L PA + 25μmol/L MF group, and 分子量为10mol/l 的 MF 基团,以及D vs. 0.5 mmol/L PA+50  0.5 mmol/L PA + 50μmol/L MF group. (b–e) 摩尔/l MF 组(b-e)A vs. NC group, 对北卡罗来纳州的小组,B vs. 0.5 mmol/L PA+100  0.5 mmol/L PA + 100μmol/L MF group, and 分子量为10mol/l 的 MF 基团,以及C vs. 10  对10μmol/L compound C group. (f–i) 摩尔/l 化合物 c 基团(f-i)A vs. NC group, 对北卡罗来纳州的小组,B vs. 1.0 mmol/L AICAR group, and 1.0 mmol/L AICAR 组C vs. 0.5 mmol/L PA+1.0 mmol/L AICAR group. 与0.5 mmol/L PA + 1.0 mmol/L AICAR 组比较

3.5. Effect of Metformin on High-Fat Diet-Induced Inflammatory Injury in Obese Rats
3.5. 二甲双胍对高脂饮食诱导的肥胖大鼠炎症损伤的影响

Diet-induced obese SD rats were used to verify our findings in vitro. The results revealed that metformin reduces body weight (Figure 5(a)), Lee’s index (Figure 5(b)), HOMA-IR (Figure 5(c)), FFA (Figure 5(d)), TNF-α, IL-1, and IL-6 (Figure 5(e)) levels, and the AUC of glucose IPGTT (Figure 5(g)) and ITT (Figure 5(i)) and increases the HOMA-B (Figure 5(j)) and the AUC for plasma insulin concentration during IPGTT (Figure 5(l)) in rats fed with HFD. However, there was no significant difference in fasting blood glucose (Figure 5(m)), total cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein levels (Figure 5(n)) between the obese group and the metformin intervention obese group. In addition, metformin decreased the pancreatic cell apoptosis induced by HFD (Figure 5(o)), increased the levels of IP3 (Figure 5(p)) and the expression of GPR40, PLC, and pAMPKα1, inhibited the expression of TLR4 and NF-κB subunit P65 (Figure 5(q)), and improved the distribution and quantity of α-cells and β-cells in the pancreas (Figure 5(r)).

用饮食诱导肥胖的 SD 大鼠进行体外实验验证我们的发现。结果显示,二甲双胍降低体重(图5(a))、 Lee’s 指数(图5(b))、 HOMA-IR (图5(c))、 FFA (图5(d))、 tnf-α、 IL-1和 IL-6(图5(e))水平,以及葡萄糖 IPGTT (图5(g))和 ITT (图5(i)的 AUC,并增加胰岛素喂养期间 HOMA-B (图5(j))和 AUC 的血浆浓度(图5(l))。然而,空腹血糖(图5(m))、总胆固醇、甘油三酯、高密度脂蛋白和低密度脂蛋白水平(图5(n))在肥胖组和二甲双胍干预肥胖组之间没有显著差异。此外,二甲双胍降低了 HFD 诱导的胰腺细胞凋亡(图5(o)) ,增加了 IP3(图5(p))和 GPR40、 PLC 和 pAMPKα1的表达,抑制了 TLR4和 NF-κB P65的表达(图5(q)) ,改善了胰腺中 α 细胞和 β 细胞的分布和数量(图5(r))。(a)
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(r)Figure 5 图5Protective effects of metformin on high-fat diet-induced inflammatory injury in obese rats. (a) The average weight of each treatment group. (b) Lee’s index. (c) HOMA-IR. (d) Free fatty acid A (FFA). (e) Metformin decreases the levels of IL-1, IL-6, and TNF- 二甲双胍对高脂饮食诱导的肥胖大鼠炎症损伤的保护作用。(a)每个治疗组的平均体重。(b)李索引。(c) HOMA-IR.(d)游离脂肪酸 a。(e)二甲双胍能降低 IL-1、 IL-6和 TNF-的水平α. (f) IPGTT. (g) Glucose AUC of IPGTT. (h) ITT. (i) Glucose AUC of ITT for each group. (j) HOMA- . (f) IPGTT. (g) IPGTT 的葡萄糖 AUC. (h) ITT. (i)各组 ITT 的葡萄糖 AUC. (j) HOMA-β. (k) Plasma insulin levels during IPGTT. (l) The AUC for plasma insulin concentration during IPGTT. (m) Fasting blood glucose levels. (n) Blood lipid levels for each group: total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL). (o) Metformin reduces pancreatic cell apoptosis induced by high-fat diet. (O1) Representative images from fluorescent microscopy for each group. The white arrows indicate apoptotic cells. (O2) Collective analyses of all three independent experiments. (p) Metformin increases the levels of IP3. (q) Expression of TLR4, NF- .(k) IPGTT 时血浆胰岛素水平。(1) IPGTT 期间血浆胰岛素浓度的 AUC。(m)空腹血糖水平。(n)各组血脂水平: 总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL)和低密度脂蛋白(LDL)。(o)二甲双胍减少高脂饮食诱导的胰腺细胞凋亡。(O1)每组的荧光显微镜代表图像。白色箭头显示凋亡细胞。(O2)所有三个独立实验的集体分析。(p)二甲双胍增加 IP3水平。(q) TLR4,NF-的表达κB subunit P65, GPR40-PLC, AMPK, and pAMPK- B 亚基 P65、 GPR40-PLC、 AMPK 和 pAMPK-α1 detected by Western blot. (Q1) Representative Western blot images for each group. (Q2) The ratio of target protein to (Q1)每组均有代表性的免疫印迹图像。(Q2)靶蛋白与蛋白的比值β-actin. (r) Representative images of immunofluorescence from pancreatic tissue for each group. 肌动蛋白(r)每组胰腺组织中免疫荧光的代表性图像A vs. NC group, 对北卡罗来纳州的小组,B vs. HFD group. 与 HFD 组比较

4. Discussion

4. 讨论

In this study, we found that metformin could reduce the lipotoxicity-induced insulin secretion deficiency in β-cells, decrease cell apoptosis, and inhibit the activation of metabolic inflammation key marker (TLR4-NF-κB). In addition, we discovered that the GPR40 expression alters metformin’s protective function on lipotoxicity β-cells. We also found that GPR40 affected metformin’s role in inhibiting activated TLR4-induced β-cell injury. Furthermore, downstream signaling protein PLC-IP3 of GPR40 was involved in the protective effect of metformin on meta-inflammation, and the above process of metformin was partially regulated by AMPK activity. We also observed that metformin could reduce body weight and inflammatory-related factors, improve β-cell function, increase GPR40-PLC-IP3 expression, and inhibit activation of the TLR4-NF-κB pathway in an obese rat model.

本研究发现二甲双胍能减轻脂毒性诱导的胰岛素分泌缺陷,减少细胞凋亡,抑制代谢性炎症关键标志物 TLR4-NF-κB 的活化。此外,我们还发现 GPR40的表达改变了二甲双胍对脂毒性 β 细胞的保护作用。我们还发现 GPR40影响二甲双胍抑制 tlr4活化诱导的 β- 细胞损伤的作用。此外,GPR40的下游信号蛋白 PLC-IP3参与了二甲双胍对间接炎症的保护作用,二甲双胍的上述过程部分受到 AMPK 活性的调节。我们还观察到二甲双胍能降低肥胖大鼠的体重和炎症相关因子,改善 β 细胞功能,增加 GPR40-PLC-IP3的表达,抑制 TLR4-NF-κB 通路的活化。

The present study showed that metformin has a direct protective effect on lipotoxicity β-cells. Yet, the administration of metformin alone did not cause a decrease in cells or an increase in insulin secretion. This phenomenon is consistent with the clinic findings, which indicated that metformin alone does not increase the risk of hypoglycemia in diabetic patients [34]. Hence, metformin could be administered to nondiabetic patients, such as patients with fatty liver [35] and polycystic ovary syndrome (PCOS) [36], without causing hypoglycemia. Moreover, clinical studies have suggested that metformin has a protective effect on β-cells, independent of its hypoglycemic effect [37]. These data suggest that metformin has an additional protective effect on β-cells; however, the specific mechanisms need to be investigated [3839].

研究表明二甲双胍对脂毒性 β 细胞有直接的保护作用。然而,单独使用二甲双胍并没有引起细胞减少或胰岛素分泌增加。这一现象与临床结果一致,表明单用二甲双胍并不增加糖尿病患者发生低血糖的风险[34]。因此,二甲双胍可以用于非糖尿病患者,如脂肪肝和多囊卵巢综合症,而不会引起低血糖。此外,临床研究表明二甲双胍对 β 细胞有保护作用,与其降血糖作用无关[37]。这些数据表明二甲双胍对 β 细胞有额外的保护作用,然而,具体的机制需要研究[38,39]。

Furthermore, we examined whether GPR40 was involved in metformin’s lipotoxicity protection. Our results demonstrated that high expression of GPR40 was directly associated with the protective effect induced by metformin, while its inhibition reduced the effects of metformin. So far, a number of studies, including our previous work, have confirmed the benign intervention of GPR40 on β-cells [1140] and the protective effect of GPR40 on islet β-cells [41]. In addition, decreased GPR40 expression was found in diabetic patients [42]. The above evidence indicates that the upregulation and activation of GPR40 have a protective role on β-cells, which is consistent with our findings. Moreover, studies have suggested that PA-induced TLR4-NF-κB activation in β-cells leads to inflammatory apoptosis and insulin secretion deficiency [2443]. However, whether the positive-mediated effect of GPR40 on metformin is related to the inhibition of TLR4-NF-κB activity still remains unclear and needs to be further investigated.

此外,我们还检测了 GPR40是否参与了二甲双胍的脂毒性保护作用。结果表明,GPR40的高表达与二甲双胍的保护作用有直接关系,而其抑制作用减少了二甲双胍的保护作用。到目前为止,许多研究,包括我们以前的工作,都证实了 GPR40对胰岛 β 细胞的良性干预[11,40]和 GPR40对胰岛 β 细胞的保护作用[41]。此外,在糖尿病患者中发现 GPR40表达减少[42]。上述证据表明 GPR40的上调和激活对 β 细胞具有保护作用,这与我们的研究结果一致。此外,研究表明 pa 诱导的 β 细胞 TLR4-NF-κB 激活导致炎症性凋亡和胰岛素分泌缺乏[24,43]。然而,GPR40对二甲双胍的正向介导作用是否与抑制 TLR4-NF-κB 活性有关仍不清楚,有待进一步研究。

In order to confirm whether GPR40 mediates the effect of metformin through the inhibition TLR4-NF-κB activity, we used LPS as a specific agonist for TLR4 to induce β-cell damage. We found that GPR40 was involved in metformin reversing LPS-induced β-cell inflammatory injury. From the above experiments, we confirmed that GPR40 could inhibit LPS-induced or lipotoxicity-induced TLR4-NF-κB activation by metformin. It has been demonstrated that GPR40 has anti-inflammatory actions [44], thus supporting our conclusion.

为了确定 GPR40是否通过抑制 TLR4-NF-κB 活性介导二甲双胍的作用,我们以 LPS 作为 TLR4特异性激动剂诱导 β 细胞损伤。我们发现 GPR40参与二甲双胍逆转 lps 诱导的 β- 细胞炎症损伤。通过以上实验,我们证实 GPR40能够抑制脂多糖诱导的或脂毒性诱导的二甲双胍对 TLR4-NF-κB 的激活。已证明 GPR40具有抗炎作用[44] ,从而支持我们的结论。

The exact mechanisms through which GPR40 mediates metformin-regulated β-cell lipotoxicity inflammatory injury still remain unclear. PLC-IP3 is a downstream signaling pathway of GPR40, which regulates β-cell insulin secretion [45]. Our previous study has shown that PLC is involved in GPR40-mediated pioglitazone antagonism of lipotoxic β-cell apoptosis and oxidative stress [24]. Several other studies have also shown that PLC-IP3 is involved in GPR40-mediated DHA-induced neuronal differentiation and neurite outgrowth in adult rat stem cells [46]. In this study, we further verified that PLC-IP3 is involved in the mediating effects of GPR40 on metformin. There are several publications supporting our findings; existing studies have suggested that palmitate-induced ER Ca (2+) depletion by PLC-IP3R signaling upregulates ER stress proteins and that mitochondrial dysfunction leads to beta cell dysfunction. In addition, studies have reported that metformin has protective effects on palmitic acid-induced ER-mitochondrial dysfunction [47]. Another study performed in breast cancer patients found that metformin inhibited the activity of PLC [48], which is inconsistent with our findings. We hypothesized that the possible reason was the cell-specific or bidirectional effects of metformin on cells; metformin may inhibit tumor cell growth by inhibiting the activity of PLC. In our study, metformin alleviated β-cell apoptosis induced by lipotoxicity via PLC.

GPR40介导二甲双胍调节 β- 细胞脂毒性炎症损伤的确切机制尚不清楚。PLC-IP3是 GPR40的下游信号通路,调节 β 细胞的胰岛素分泌[45]。我们以前的研究表明,PLC 参与了 gpr40介导的吡格列酮拮抗脂毒性 β 细胞凋亡和氧化应激的作用[24]。其他一些研究也表明 PLC-IP3参与了 gpr40介导的 dha 诱导的神经元分化和成年大鼠干细胞的神经突起生长[46]。在本研究中,我们进一步证实了 PLC-IP3参与了 GPR40对二甲双胍的调节作用。已有的研究表明,palmitate 诱导的 ER Ca (2 +)缺失通过 PLC-IP3R 信号传导上调 ER 应激蛋白,线粒体功能障碍导致 β 细胞功能障碍。此外,研究报道二甲双胍对棕榈酸诱导的雌激素受体线粒体功能障碍有保护作用[47]。另一项在乳腺癌患者中进行的研究发现二甲双胍抑制了 PLC 的活性,这与我们的发现不一致。我们推测可能的原因是二甲双胍对细胞的特异性或双向作用,二甲双胍可能通过抑制肿瘤细胞的活性来抑制肿瘤细胞的生长。二甲双胍通过可编程序控制器减轻脂毒性诱导的 β 细胞凋亡。

Next, we investigated whether the GPR40-IP3-PLC pathway was mediated or affected by the AMPK activation. Our results indicated that metformin could activate AMPK phosphorylation in lipotoxicity β-cells. We further found that the activation of AMPK can antagonize the damage to lipotoxicity, while its inhibition can partially decrease the protective effect of metformin. The above results suggest that the effect of metformin on lipotoxicity is partially dependent on AMPK activity. In addition, recent studies have confirmed that metformin could suppress the formation of fat by regulating the activity of Runx2 and PPARγ [49]. This effect appears to be independent of the activity of AMPK.

接下来,我们研究是否 GPR40-IP3-PLC 通路是介导或影响 AMPK 的活化。结果表明,二甲双胍能够激活脂毒性 β 细胞的 AMPK 磷酸化。我们进一步发现 AMPK 的激活可以拮抗脂毒性损伤,而其抑制作用可以部分降低二甲双胍的保护作用。上述结果表明,二甲双胍对脂毒性的影响部分依赖于 AMPK 活性。此外,最近的研究已经证实二甲双胍可以通过调节 Runx2和 pparγ 的活性来抑制脂肪的形成。这种效应似乎与 AMPK 的活性无关。

Moreover, we further confirmed the protective effect of metformin in vivo.Metformin reduced body weight, decreased the levels of inflammatory factors, improved islet cell function, increased the expression of GPR40-PLC-IP3 and pAMPKα1, and inhibited the activation of TLR4-NF-κB in HFD-induced obese SD rats. In order to further explain the effect of metformin on islet beta-cell function, we used statistics to control the effects of weight and insulin resistance, and we analyzed the effect of metformin on islet β-cells. The results showed that the protective effect of metformin on islet cell function was still present (, see supplementary materials 1). Our results suggested that metformin could improve β-cell function. Other studies have also reported the direct improvement of metformin in animal and human β-cells [5051], which further supported our experimental conclusions. However, metformin administration in normal SD rats did not affect the animal body weight or insulin secretion, which is consistent with clinical studies suggesting that metformin has self-limiting effects to reduce body weight [52]. Previous experiments have also demonstrated that metformin improves the metabolism of obese rats [53]. Our results showed that the high-fat diet caused a decrease in fasting insulin concentration in obese rats, which may be related to longer high-fat diet intervention time. Studies have suggested that the intervention time of high-fat diet is 4-8 weeks [5455]. And in the experiment, the high-fat diet lasted 16 weeks. Thus, we speculated that the metabolic stress induced by prolonged high-fat diet gradually leads to a decrease in islet function from decompensation to decompensation.

进一步证实了二甲双胍的体内保护作用。二甲双胍降低肥胖大鼠体重,降低炎症因子水平,改善胰岛细胞功能,增加 GPR40-PLC-IP3和 pAMPKα1的表达,抑制 TLR4-NF-κB 的活化。为了进一步解释二甲双胍对胰岛 β 细胞功能的影响,我们采用统计学方法控制体重和胰岛素抵抗的影响,并分析二甲双胍对胰岛 β 细胞的影响。结果显示二甲双胍对胰岛细胞功能的保护作用仍然存在(见补充材料1)。结果提示二甲双胍能改善 β 细胞功能。其他研究也报道了二甲双胍在动物和人 β 细胞中的直接改善作用[50,51] ,这进一步支持了我们的实验结论。然而,正常 SD 大鼠给予二甲双胍并不影响动物体重或胰岛素分泌,这与临床研究表明二甲双胍具有减轻体重的自限作用相一致[52]。以前的实验也表明二甲双胍能改善肥胖大鼠的代谢[53]。结果表明,高脂饮食引起肥胖大鼠空腹胰岛素浓度下降,这可能与高脂饮食干预时间延长有关。研究表明,高脂饮食的干预时间为4-8周[54,55]。在实验中,高脂肪饮食持续了16周。因此,我们推测长期高脂饮食引起的代谢应激逐渐导致胰岛功能从失代偿到失代偿的下降。

To sum up, we found that metformin could inhibit lipotoxicity-induced meta-inflammation damage in β-cells by regulating the GPR40-PLC-IP3 signaling pathway. Nevertheless, this study has certain limitations. Firstly, we did not investigate the mechanism of metformin acting on GPR40. Secondly, we used mouse islet cell line, which is different from islet β-cells found in humans. Third, our research lacked some negative control group, so our conclusions needed to be verified by subsequent experiments.

综上所述,我们发现二甲双胍通过调节 GPR40-PLC-IP3信号通路,抑制脂毒性诱导的 β 细胞间接炎症损伤。然而,这项研究有一定的局限性。首先,我们没有研究二甲双胍对 GPR40的作用机制。其次,我们使用小鼠胰岛细胞系,它不同于人类胰岛 β 细胞。第三,我们的研究缺乏一些负对照组,所以我们的结论需要通过后续的实验来验证。

Abbreviations

缩写

AECs: 美国电脑学会:Alveolar epithelial cells 肺泡上皮细胞
AICAR:5-Aminoimidazole-4-carboxamide1- 5- 氨基咪唑 -4- 羧酸 -1-β-D-ribofuranoside – d- 呋喃核苷
AMPK:AMP-activated protein kinase AMP活化蛋白激酶
ANOVAs: 阿诺瓦斯:Analyses of variance 方差分析
BIS: 国际清算银行:Basal insulin secretion 基础胰岛素分泌
DAPI:4,6-Diamidino-2-phenylindole 4,6-二氨基 -2- 苯基吲哚
DHA: 二十二碳六烯酸:Docosahexaenoic acid 二十二碳六烯酸
DMEM:Dulbecco’s modified Eagle’s medium Dulbecco 的改良鹰牌中号
DMSO: 二甲基亚砜:Dimethylsulfoxide 二甲基亚砜
FBS: 福布斯新闻:Fetal bovine serum 胎牛血清
FFA: 返回文章页面游离脂肪酸:Free fatty acid A 游离脂肪酸 a
GPR40:G protein-coupled receptor 40 G蛋白连接接受器40
GSIS: 国际战略研究中心:Glucose-stimulated insulin secretion 葡萄糖刺激的胰岛素分泌
HbA1c: 返回文章页面 HbA1c:Glycosylated hemoglobin 糖化血红蛋白
HDL-C: 高密度脂蛋白 c:High-density lipoprotein cholesterol 高密度脂蛋白胆固醇
HEPES: 赫普斯:Hydroxyethyl piperazine ethanesulfonic acid 羟乙基哌嗪脂肪酸甲脂磺酸钠
HSFD: 健康基金:High-sugar, high-fat diet 高糖高脂肪的饮食
HOMA-B: 返回文章页面 HOMA-B:Homeostasis model assessment of beta cell function β 细胞功能稳态模型评价
HOMA-IR: 返回文章页面霍马ー ir:Homeostasis model assessment to quantify insulin resistance 稳态模型在胰岛素抵抗量化中的应用
HOMA-S: 霍马斯:Homeostasis model assessment of insulin sensitivity 稳态模型评价胰岛素敏感性
IL-1: 白介素 -1:Interleukin-1 白细胞介素 -1
IL-6: 白介素 -6:Interleukin-6 白介素 -6
IP3:Inositol 1, 4, 5-trisphosphate 1,4,5-三磷酸肌醇
IPGTT:Intraperitoneal glucose tolerance test 腹腔内糖耐力测试
ITT:Insulin tolerance test 胰岛素耐受性试验
JNK:c-Jun N-terminal protein kinase c-Jun 氨基末端蛋白激酶
LDL-C:Low-density lipoprotein cholesterol 低密度脂蛋白胆固醇
LPS:Lipopolysaccharide 脂多糖
MF: 译者:Metformin 二甲双胍
MyD88:Myeloid differentiation factor 88 骨髓分化因子88
NC group: NC 组:Normal control group 正常对照组
NF-κB: 乙:Nuclear factor-kappa B 核因子 -kappa b
NLR: 美国国家航空航天中心:Neutrophil to lymphocyte ratio 中性粒细胞与淋巴细胞比率
OD value: OD 值:Optical density value 光密度值
PA: 英国广播公司:Palmitic acid 棕榈酸
PBS: 美国公共电视网:Phosphate buffer saline 磷酸盐缓冲盐水
PCOS: 多囊卵巢综合征:Polycystic ovary syndrome 多囊卵巢综合症
PLC: 法国法典委员会:Phospholipase C 磷脂酶 c
PVDF: 聚偏氟乙烯:Polyvinylidene fluoride 聚偏二氟乙烯
siRNA: 小干扰 rna:Small interfering RNA 小干扰RNA
TBS:Tris-buffered saline 三缓冲盐水
TC:Total cholesterol 总胆固醇
TG: 格洛克:Triglycerides 甘油三酯
TLR4: 4:Toll-like receptor 4 Toll样受体4
TNF- 肿瘤坏死因子 –α:Tumor necrosis factor- 肿瘤坏死因子-αα
TUNEL: 原子发射器:Terminal-deoxynucleoitidyl transferase-mediated nick end labeling. 末端脱氧核苷酸转移酶介导的缺口末端标记

Data Availability

数据可用性

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

支持这项研究结果的数据包括在本文中。

Conflicts of Interest

利益冲突

The authors have no conflicts to disclose.

两位作者之间没有任何冲突需要披露。

Authors’ Contributions

作者贡献

Ximei Shen wrote the manuscript; Liyong Yang conducted the design of the study and reviewed/edited the drafts and is the guarantor; Beibei Fan researched the data and edited the drafts; Liufen Luo and Yuanli Yan contributed to the discussion; Xin Hu cowrote the final draft. Liyong Yang is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Ximei Shen and Beibei Fan are co-first authors.

手稿是沈写的,杨负责研究的设计、审查/编辑草稿并担任担保人,Beibei 范研究数据并编辑草稿,罗和阎参与讨论,胡共同撰写草稿定稿。杨是这项工作的保证人,因此,他能够充分获取研究中的所有数据,并负责数据的完整性和数据分析的准确性。沈和 Beibei 范是第一作者之一。

Acknowledgments

鸣谢

This study was supported by the Science and Technology Innovation Joint Fund Project Fujian Province (grant number 2016Y9102) and Grants from the National Natural Science Foundation of China (grant numbers 81500632 and 81870572), Natural Science Foundation of Fujian Province (grant numbers 2019J01455 and 2015J01453), Fujian Province Higher Education Institute New Century Research Project (grant number 2018B049), and Medical Innovation Fund Project of Fujian Province (grant number 2018-CX-23).

本研究得到了福建省科技创新联合基金项目(资助号: 2016Y9102)和国家自然科学基金项目(资助号: 81500632、81870572)、福建省自然科学基金项目(资助号: 2019J01455、2015J01453)、福建省高等教育学院新世纪研究项目(资助号: 2018B049)、福建省医学创新基金项目(资助号: 2018-CX-23)的资助。

Supplementary Materials

补充资料

Metformin improved beta cell function independent of weight and insulin resistance. (Supplementary Materials)

二甲双胍改善 β 细胞功能独立于体重和胰岛素抵抗

References

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