CN111944035A - Fgf4及其应用 - Google Patents
Fgf4及其应用 Download PDFInfo
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- CN111944035A CN111944035A CN201910398338.XA CN201910398338A CN111944035A CN 111944035 A CN111944035 A CN 111944035A CN 201910398338 A CN201910398338 A CN 201910398338A CN 111944035 A CN111944035 A CN 111944035A
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- protein
- mice
- rfgf4
- insulin
- glucose
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Abstract
本发明属于蛋白质药物领域，具体涉及成纤维细胞生长因子FGF4及其应用，本发明提供了FGF4蛋白作为一种新型的强效降糖和胰岛素增敏剂，以及它们在治疗糖尿病等疾病中的应用，可快速和持久的调控血糖、增强全身胰岛素敏感性、改进葡萄糖耐受性以及减少肝脏脂肪变性和脂肪组织炎症，且不会引起低血糖或其他明显的负面作用，该蛋白质的给药途径的适应性好，便于成药，拓宽了有效治疗糖尿病等疾病的前景。
Description
技术领域
本发明属于蛋白质药物领域，具体而言，本发明涉及成纤维细胞生长因子4及其变体，以及它们在治疗糖尿病等疾病中的应用。
背景技术
II型糖尿病（T2D）的发病率在全球以惊人的速度上升，并带来巨大的健康和经济负担。遗传易感性和慢性肥胖为T2D发生发展的主要风险因素。在健康个体中，胰岛素通过刺激葡萄糖摄取到包括肌肉、肝脏和脂肪在内的多种组织中，并通过抑制脂肪组织脂肪分解来维持葡萄糖和脂质体内平衡。然而，在T2D患者体内，胰岛素作用的减弱（称为胰岛素耐受性）导致各种代谢紊乱的发生，包括高血糖和血脂异常。这将进一步破坏胰岛素的分泌及其功能，导致严重的器官损伤并最终导致器官衰竭。
胰岛素增敏剂二甲双胍和噻唑烷二酮（TZD）为最常用于改善T2D胰岛素耐受性的处方药。然而，这些药物的主要限制为其作用较为缓慢，可能与其转录介导的作用模式相关。此外，二甲双胍对T2D症状的缓解效果相对较弱，而TZD具有多种不良副作用，包括体重增加、骨质流失和水肿。因此，寻找更安全和更有效的胰岛素增敏剂是临床上迫切需求的。
虽然有报道显示两种内分泌FGF（即，FGF21和FGF19）和一种旁分泌FGF（FGF1）以胰岛素依赖的方式在肥胖和T2D中发挥多种有益效果，包括：强效降糖活性、促进能量消耗和脂质代谢，然而，并没有证据表明FGF4也具备相同的性质。
本发明人凭借长期研究形成的经验，令人意外地发现了本发明的FGF4蛋白是一种新型的强效胰岛素增敏剂，可增强全身胰岛素敏感性、改进葡萄糖耐受性以及减少肝脏脂肪变性和脂肪组织炎症。
发明内容
本发明要解决的技术问题在于提供新的蛋白质、药物组合物以及药用用途，主要用于治疗糖尿病和/或肥胖症等。
具体而言，在第一方面，本发明提供了蛋白质，其氨基酸序列
（a）如SEQ ID NO：1所示；或者，
（b）是对SEQ ID NO：1所示的序列添加、缺失和/或取代一个或几个氨基酸残基而得的氨基酸序列，并且具有氨基酸序列如SEQ ID NO：1所示的蛋白质的功能。在本文中，氨基酸序列如SEQ ID NO：1所示的蛋白质所具备的功能至少包括增强全身胰岛素敏感性、改善葡萄糖耐受性和减少肝脏脂肪变性和脂肪组织炎症之一，优选包括增强全身胰岛素敏感性、改善葡萄糖耐受性和减少肝脏脂肪变性和脂肪组织炎症。
本发明第一个方面的蛋白质的氨基酸序列可以是在SEQ ID NO：1所示的序列的基础上添加、缺失和/或取代一个或几个（优选一个至五个，更优选一个至三个）氨基酸残基而得的氨基酸序列。本领域技术人员知晓，通过改变已知多肽的编码基因序列并将其导入表达载体，可以制备出取代、添加和/或缺失了氨基酸残基的多肽，这些方法广泛记载于《分子克隆实验指南》（北京：科学出版社，2002年）等本领域公知的文献中。例如，在本发明第一个方面的蛋白质中，添加、缺失和/或取代可以是缺失，如缺失第一个氨基酸残基（Met）。在本发明的具体实施方式中，本发明第一个方面的蛋白质的氨基酸序列如SEQ ID NO：1、2、3或4所示。
在第二方面，本发明提供了编码本发明第一方面的蛋白质的多核苷酸。在本文中，多核苷酸可以是DNA形式，也可以是RNA形式，优选DNA形式。DNA形式包括天然cDNA和人工合成的cDNA，DNA可以是编码链或模板链。通过常规技术，如PCR方法、重组法或人工合成的方法，本领域技术人员可以很容易获得编码本发明的突变体的核酸分子多核苷酸或其片段。这些序列一旦获得，就可以将其克隆入载体，再转化或转染入相应的细胞，然后通过常规的宿主细胞进行增殖，从中分离得到大量的核酸分子。
在第三方面，本发明提供了一种载体，其含有本发明第二个方面的多核苷酸。在本文中，载体包括表达载体和克隆载体，是指本领域中常用的细菌质粒、粘粒、噬菌粒、酵母质粒、植物细胞病毒、动物病毒及其它各种病毒载体。本发明中适用的载体包括但不限于：在细菌中表达用的载体（原核表达载体）、在酵母中表达用的载体（如毕赤酵母载体、汉逊酵母载体等）、在昆虫细胞中表达的杆状病毒载体、在哺乳动物细胞中表达用的载体（痘苗病毒载体、逆转录病毒载体、腺病毒载体、腺伴病毒载体等）、在植物中表达用的植物病毒载体以及在哺乳动物乳腺中表达用的各种载体。总之，只要能在宿主细胞中稳定复制，任何质粒和载体都可使用。优选表达载体包含选择标记基因，如细菌的氨苄青霉素抗性基因、四环素抗性基因、卡那霉素抗性基因、链霉素抗性基因、氯霉素抗性基因；酵母菌的新霉素抗性基因、Zeocin抗性基因，酵母菌的缺陷选择标志，如His, Leu, Trp等；真核细胞的新霉素抗性基因、Zeocin抗性基因、二氢叶酸还原酶基因及荧光蛋白标记基因等。在本发明的具体实施方式中，使用的是已经商品化的pET质粒。
在第四方面，本发明提供了一种细胞，如宿主细胞，其含有本发明第二个方面的多核苷酸。本发明的细胞可以含有本发明第三方面的载体，也可以用本发明第二方面的多核苷酸转化或转染。宿主细胞可以是原核细胞，也可以是真核细胞，如，细菌细胞、酵母细胞、植物细胞、昆虫细胞、哺乳动物细胞等。宿主细胞在转化或转染含本发明所述编码蛋白的基因序列后，即构成工程化细胞或细胞株，可用于生产所需融合蛋白。本领域技术人员能够恰当地选择适当的载体、宿主细胞，并熟知如何将载体高效地转化或转染入宿主细胞中，所用方法包括但不限于：氯化钙法、电穿孔法用于细菌细胞，电穿孔法和原生质体融合法用于酵母细胞，脂质体包裹、磷酸钙共沉淀、电融合法以及显微注射法用于哺乳动物细胞等真核细胞。优选本发明的宿主细胞是大肠杆菌BL21（DE3）。
在第五方面，本发明提供了制备本发明第一方面的蛋白质的方法，其包括在适宜的条件下使用本发明第四方面的细胞表达本发明第一方面的蛋白质，然后纯化。获得的宿主细胞可以通过常规方法培养、诱导来表达所需要的融合蛋白，包括发酵过程和纯化工艺。上述表达的蛋白可在细胞内、细胞膜上或分泌到细胞周质、细胞外。根据需要，可利用融合蛋白的物理的、化学的以及其它生物学特性，进行分离纯化。分离纯化的方法包括但不限于：裂菌（超声波裂菌、渗透压裂菌），离心，盐析，分子筛色谱（又称为分子尺寸排阻色谱），离子交换色谱，吸附色谱（亲和层析、金属鏊合层析），反向色谱，高效液相色谱，毛细管电泳，制备性等电聚焦以及常规的变性、复性处理等，这些方法均是本领域技术人员所熟知的。为了能够高效地获得人肝再生增强因子，优选在本发明制备方法的方面的方法中，宿主细胞为含有构建了表达本发明第一方面的蛋白质的pET载体的大肠杆菌BL21（DE3）。
在第六方面，本发明提供了药物组合物，其包含本发明第一方面的蛋白质以及药学上可接受的载体。本发明的药物组合物能治疗糖尿病、降血糖、治疗或预防肥胖症、抗炎。在本文中，药学上可接受的载体指无毒的填充剂、稳定剂、稀释剂、佐剂或其他制剂辅料。根据本领域的公知技术，可以根据治疗目的、给药途径的需要将药物组合物制成各种剂型，包括干粉剂（如冻干粉剂）、片剂、胶囊、滴剂、乳液剂、注射剂和喷剂等。该药物组合物优选是注射剂。
在第七方面，本发明提供了本发明第一方面的蛋白质在制备用于增强胰岛素敏感性的药物中的应用。相应地，本发明提供了增强胰岛素敏感性的方法，包括给有需要的个体给药有效量的本发明第一方面的蛋白质。优选在本发明第七方面的应用中，药物是本发明第六方面所述的药物组合物，个体优选是人。
在第八方面，本发明提供了本发明第一方面的蛋白质在制备用于降血糖的药物中的应用。相应地，本发明提供了降血糖的方法，包括给有需要的个体给药有效量的本发明第一方面的蛋白质。优选在本发明第八方面的应用中，药物是本发明第六方面所述的药物组合物，个体优选是人。
在第九方面，本发明提供了本发明第一方面的蛋白质在制备用于改善葡萄糖耐受性的药物中的应用。相应地，本发明提供了改善葡萄糖耐受性的方法，包括给有需要的个体给药有效量的本发明第一方面的蛋白质。优选在本发明第九方面的应用中，药物是本发明第六方面所述的药物组合物，个体优选是人。
在第十方面，本发明提供了本发明第一方面的蛋白质在制备用于减少肝脏脂肪变性和/或脂肪组织炎症的药物中的应用。相应地，本发明提供了减少肝脏脂肪变性和/或脂肪组织炎症的方法，包括给有需要的个体给药有效量的本发明第一方面的蛋白质。优选在本发明第十方面的应用中，药物是本发明第六方面所述的药物组合物，个体优选是人。
在第十一方面，本发明提供了本发明第一方面的蛋白质在制备用于治疗糖尿病的药物中的应用。相应地，本发明提供了治疗糖尿病的方法，包括给有需要的个体给药有效量的本发明第一方面的蛋白质。优选在本发明第十一方面的应用中，药物是本发明第六方面所述的药物组合物，个体优选是人，糖尿病是II型糖尿病。
在第十二方面，本发明提供了本发明第一方面的蛋白质在制备用于治疗肥胖症的药物中的应用。相应地，本发明提供了治疗肥胖症的方法，包括给有需要的个体给药有效量的本发明第一方面的蛋白质。优选在本发明第十二方面的应用中，药物是本发明第六方面所述的药物组合物，个体优选是人。
本发明的有益效果在于：提供了一种新的药物蛋白质，拓宽了有效治疗糖尿病等疾病的前景；该蛋白质可增强全身胰岛素敏感性、改进葡萄糖耐受性以及减少肝脏脂肪变性和脂肪组织炎症，且不会引起低血糖或其他明显的副面作用；该蛋白质的给药途径的适应性好，便于成药。
为了便于理解，以下将通过具体的实施例和附图对本发明进行详细地描述。需要特别指出的是，这些描述仅仅是示例性的描述，并不构成对本发明范围的限制。依据本说明书的论述，本发明的许多变化、改变对所属领域技术人员来说都是显而易见的。
另外，本发明引用了公开文献，这些文献是为了更清楚地描述本发明，它们的全文内容均纳入本文进行参考，就好像它们的全文已经在本文中重复叙述过一样。
附图说明
图1显示了FGF4的氨基酸序列。
图2显示了在db/db小鼠体内rFGF4的急性和慢性降糖效果。其中，（A）在急性注射rFGF4（0.25 mg/kg、0.5 mg/kg和1.0 mg/kg体重）6小时后，db/db小鼠体内血糖水平呈剂量依赖性降低。数据呈现为平均值+/- SEM（n=6）；与 db/db相比，**p<0.01，***p<0.001；与同窝db/m对照相比，###p<0.001。（B）在急性腹腔注射rFGF4（1.0 mg/kg体重）或缓冲液对照后，在db/db小鼠体内测量不同时间的血糖水平。数据呈现为平均值+/-SEM（n=6）；与db/db相比，*p<0.05，**p<0.01，***p<0.001。（C、D）在急性腹腔注射rFGF4（1.0 mg/kg体重）或缓冲液对照后，在饮食诱导的肥胖（DIO）（C）或ob/ob（D）小鼠体内测量不同时间的血糖水平。数据呈现为平均值+/- SEM（n=6）；与缓冲液对照相比，*p<0.05，**p<0.01，***p<0.001。（E）在皮下（s.c.）注射rFGF4（1.0 mg/kg体重）12小时或在静脉内（i.v.）注射rFGF4（1.0 mg/kg体重）3小时后，测量db/db小鼠的血糖水平。数据呈现为平均值+/- SEM（n=6）；与db/db相比，***p<0.001。（F、G）经受连续37天注射rFGF4（1.0 mg/kg体重）或缓冲液对照的db/db小鼠的血糖（F）和体重（G）。同窝db/m小鼠用作另外的对照。数据呈现为平均值+/- SEM（n=8）；与db/db相比，***p<0.001。
图3显示了rFGF4改进db/db小鼠葡萄糖耐受不良和胰岛素耐受性。其中，（A）左：单次腹腔注射rFGF4（1.0 mg/kg体重）3小时后，在db/db小鼠体内进行口服葡萄糖耐受性测试（OGTT）。右：血糖水平变化的曲线下积分面积（AUC）。数据呈现为平均值+/- SEM（n=6）；与db/db相比，*p<0.05，**p<0.01，***p<0.001。（B、C）OGTT期间经急性rFGF4（1.0 mg/kg体重）预处理db/db小鼠t=0和t=15分钟时的胰岛素水平。数据呈现为平均值+/- SEM（n=6）；与db/ db相比，*p<0.05，***p<0.001。（C）急性腹腔注射rFGF4或对照缓冲液后，db/db小鼠在不同时间测量的胰岛素水平。数据呈现为平均值+/- SEM（n=6）；与db/db相比，*p<0.05。（D）左：db/db小鼠慢性腹腔注射rFGF4（1.0 mg/kg体重）37天后，在小鼠体内进行OGTT。右：血糖水平变化的积分AUC。数据呈现为平均值+/- SEM（n=6）；与db/db相比，*p<0.05，**p<0.01，***p<0.001；与同窝db/m对照相比，###p<0.001。（E、F）db/db小鼠慢性腹腔注射rFGF4（1.0 mg/kg体重）10天后，在高胰岛素正常血糖钳夹实验持期间的血糖水平（E）和葡萄糖输注速率（GIR）（F）。数据呈现为平均值+/- SEM（n=5）；与db/db相比，*p<0.05，**p<0.01。（G）通过蛋白质印迹分析（左图）和使用ImageJ软件（右图）定量测定的慢性rFGF4（1.0 mg/kg体重）处理37天后db/db小鼠的骨骼肌、肝脏和附睾脂肪组织中AKT的磷酸化水平。同窝db/m小鼠用作对照。数据呈现为平均值+/- SEM（n=6）；与db/db相比，**p<0.01，***p<0.001。（H）急性腹腔注射rFGF4（1.0 mg/kg体重）或对照缓冲液后，I型（T1D）糖尿病小鼠的血糖水平。（I）进行ITT实验之前和期间T1D糖尿病小鼠的血糖水平，小鼠事先腹腔注射胰岛素（1 U/kg），或胰岛素与rFGF4 6小时（1.0 mg/kg体重）。数据呈现为平均值+/- SEM（n=6）；与缓冲液对照相比，*p<0.05，**p<0.01，***p<0.001。
图4显示了用rFGF4长期治疗db/db小鼠改进肝脏脂肪变性、糖原含量和葡萄糖产生。其中，（A至K）用rFGF4（1.0 mg/kg体重）治疗db/db小鼠37天；同窝db/m小鼠用作对照。（A）具有代表性的苏木精和曙红（H＆E）和油红O染色。比例尺，100 μm。（B）通过蛋白质印迹法测定的FAS、SCD-1和SREBP1的表达以及肝脏ACC的磷酸化水平（左图）；在右边显示使用ImageJ软件定量的结果。（C至F）肝脏质量（C）、肝脏提取物中的TG水平（D）和ALT、AST（E）、NEFA（F）的血清水平。（G、H）具有代表性过碘酸-希夫染色（PAS；品红色表示糖原）（G）和db/ db肝脏糖原含量（H）。（I）通过蛋白质印迹（左图）和使用ImageJ软件（右图）定量测定的GSK3β、GS和FOXO1的磷酸化水平以及Pepck和G6Pase的蛋白表达水平。（J、K）通过实时PCR分析测定的G6pase和Pepck mRNA水平。（L、M）实时PCR分析测定的C57BL/6J小鼠肝脏中G6pase和Pepck的mRNA水平。在L、M中，将小鼠禁食6小时进行rFGF4（1.0 mg/kg体重）单次腹腔给药。所有数据均以n=6获得，并呈现为平均值+/- SEM。在（B至K）中，与db/db相比，*p<0.05，**p<0.01，***p<0.001；与db/m相比，#p<0.05，##p<0.01，###p<0.001；在（L、M）中，与禁食小鼠相比，*p<0.05；与食物喂养的（对照）小鼠相比，##p<0.01，###p<0.001。
图5显示了通过代谢笼测量的rFGF4对db/db小鼠的能量消耗和体力活动的影响。其中，在代谢笼中测量的经rFGF4慢性治疗的（1.0 mg/kg体重）db/db小鼠（用缓冲液对照（红色符号）或rFGF4（蓝色符号）治疗37天）耗氧量（VO2）（A、B）、二氧化碳产量（VCO2）（C、D）、呼吸交换率（RER）（E、F）、产热量（G、H）、食物摄入量（I、J）、总活动量（K、L）和走动活动量（M、N）。数据呈现为平均值+/- SEM（n=4）；与db/db相比，*p<0.05，**p<0.01。
图6显示了rFGF4对db/db小鼠的附睾脂肪组织形态和身体成分的效果。其中，在用rFGF4（1.0 mg/kg体重）治疗37天后，在db/db小鼠中测量各种参数。（A、B）附睾脂肪组织的重量（A）和（B）及其苏木精和曙红（H＆E）染色。（C、D）总体重、脂肪和瘦体重（C）和体脂百分比（D）。（E、F）骨矿物质密度（BMD）（E）和骨矿物质含量（BMC）（F）。在所有情况下，数据呈现为平均值+/- SEM（n=4）。
图7显示了rFGF4不会使野生型或db/db小鼠产生低血糖。其中，（A、B）在正常小鼠（C57BL/6J）（A）或db/db小鼠急性腹腔注射1.0 mg/kg rFGF4或高剂量rFGF4（3.0 mg/kg体重）后血糖水平的变化情况。在A和B中，数据呈现为平均值+/- SEM（n=6）。
具体实施方式
以下通过实施例进一步说明本发明的内容。如未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段和市售的常用仪器、试剂，可参见《分子克隆实验指南（第3版）》（科学出版社）、《微生物学实验（第4版）》（高等教育出版社）以及相应仪器和试剂的厂商说明书等参考。
实施例1 实验材料和方法
1，重组FGF4（rFGF4）的表达和纯化
编码FGF4的cDNA片段（Ala67-Leu206）经PCR扩增，并经由NcoI和XhoI位点亚克隆到细菌表达载体pET-15b中，经测序，其编码的蛋白质的氨基酸序列如SEQ ID NO：1所示（图1）。使用FGF4表达构建体转化的感受态大肠杆菌BL-21（DE3）细胞在37℃和200 rpm下的培养箱摇床中培养。培养基含有2%葡萄糖和30 mg/mL卡那霉素（LB培养基）。在λ600下光密度为0.8至1.0时，通过加入异丙基-L-硫代-β-D-吡喃半乳糖苷（IPTG）至1 mM来诱导重组蛋白表达，并在37℃下进一步生长4小时。收获细胞并使用Emulsiflex-C3（Avestin, Inc., Ottawa,Ontario, Canada）高容量匀浆器将其在含有300 mM NaCl的25 mM Na/K磷酸盐缓冲液（pH7.5）中裂解。发现重组FGF4主要存在于可溶性级分中。通过在4℃下以15,000 x g离心30分钟分离裂解物，随后使用在缓冲液A（150 mM NaCl，25 mM HEPES，pH 7.5）中平衡的肝素亲和柱（Hitrap Heparin HP, GE Healthcare, Piscataway, NJ，柱体积（CV）= 5 mL）纯化。使用在25 mM HEPES（pH 7.5）中的NaCl线性梯度洗脱结合的rFGF4。经由12% SDS-PAGE分析测定目的蛋白质，并使用在缓冲液C（1M NaCl，25 mM Tris-HCl，PH 8.0）中运行的凝胶过滤柱（SuperdexTM-75 GE Healthcare, Piscataway, NJ）浓缩蛋白。重组蛋白的纯度为98%。
2，实验动物
雄性db/db（C57BLKS/J-leprdb/leprdb）小鼠及其非糖尿病db/m同窝小鼠、雄性ob/ob（C57BL/6J-lepob/lepob）小鼠、雄性EGFP转基因（STOCK-Tg（CAG-EGFP）/Nju）小鼠和雄性C57BL/6J小鼠购自南京大学模型动物研究中心（中国南京（Nanjing, China））。我们研究中使用的所有方案均经中国温州医科大学动物护理与使用委员会批准。所有动物在使用前均已适应我们的实验室环境，并饲养在环境受控（22±2℃，50%至60%湿度，12小时光照/黑暗循环，早上7点光照）且无特定无病原体（SPF）的动物设施中，小鼠可自由获取食物和水。通过用高脂肪饮食（60%脂肪； Research Diets, Inc, New Brunswick, NJ）喂养3周龄雄性C57BL/6J小鼠来建立饮食诱导的肥胖（DIO）小鼠。标准食物或高脂肪饮食喂养12周后，通过体重和血糖水平（使用FreeStyle全血糖监测仪（Abbott Diabetes Care Inc., Alameda,CA）测定）来随机分配小鼠。通过在5周龄C57BL/6J小鼠单次腹腔注射溶解在柠檬酸盐缓冲液（pH 4.5）中的STZ（150 mg/kg），来诱导I型（T1D）糖尿病；对照小鼠接受相同体积的柠檬酸盐缓冲液。七天后，血糖≥300 mg/dl（16.7 mM）的小鼠被视为T1D造模成功。
3，rFGF4在糖尿病小鼠中的功能评估
在每次研究之前，基于体重和血糖水平将小鼠随机分组。除非另有说明，所有药物和对照均以腹腔注射方式注射入小鼠体内。为了分析rFGF4对血糖水平的剂量依赖性作用，将db/db小鼠分成4组，每组6只小鼠：对照（PBS）、rFGF4（低剂量：0.25 mg/kg体重）、rFGF4（中剂量：0.5 mg/kg体重）和rFGF4（高剂量：1.0 mg/kg体重）。用PBS作为另外的对照。注射6小时从尾静脉采集血液样品，并使用FreeStyle全血糖监测仪（Abbott Diabetes Care Inc.,Alameda, CA）测量葡萄糖含量。
对于时间过程研究，在单次注射rFGF4（1.0 mg/kg体重）或缓冲液后，如上所述在不同的时间点从db/db、DIO或ob/ob小鼠尾静脉采集血液样品。如上所述测量血糖水平；根据制造商的说明，使用大鼠/小鼠胰岛素ELISA试剂盒（Merck Millipore, Darmstadt,Germany）测定db/db小鼠体内的胰岛素水平。小鼠禁食过夜（12小时）后进行口服葡萄糖耐受性测试（OGTT）；db/db小鼠在OGTT之前3小时接受单次注射rFGF4（1 mg/kg体重）或缓冲液，并口服给予右旋糖溶液（1.5 g/kg体重）。如上所述测定葡萄糖和胰岛素水平。使用GraphPad Prism 7软件（San Diego, California, GraphPad Software），通过葡萄糖耐受性曲线的梯形法则计算OGTT的曲线下面积（AUC）。
为了评估rFGF4给药途径对血糖水平的潜在效果，将db/db小鼠分成3组，每组6只小鼠：对照（PBS）、rFGF4（皮下注射，s.c.）和rFGF4（静脉注射，i.v.）组。静脉注射后3小时或皮下注射12小时后从尾静脉采集血液样品，并且如上所述测定葡萄糖水平。为了评估rFGF4对正常C57BL/6J小鼠或高剂量rFGF4对db/db小鼠血糖的影响，在单次注射rFGF4（1.0 mg/kg或3.0 mg/kg体重）后采集血液样品。Db/m小鼠注射PBS作为对照。为了评估rFGF4对T1D小鼠体内血糖的影响，在单次注射rFGF4（1.0 mg/kg体重）6小时和12小时后采集血液样品，并如上所述测量葡萄糖水平。在T1D小鼠体内按如下步骤进行胰岛素耐受性测试（ITT）：注射对照PBS或1.0 mg/kg rFGF4的STZ小鼠在注射1.0 U/kg胰岛素（Humulin R，Eli Lilly,Indianapolis, IN）前禁食4小时，并在注射后0小时、0.5小时、1小时、2小时、4小时和6小时监测血糖。为了评估根皮素对FGF4介导的降糖效应的影响，在单次注射rFGF4（1 mg/kg体重）前1小时给予db/db小鼠注射根皮素或对照溶剂（200 mg/Kg体重）。
对于慢性功效评估，每隔一天用缓冲液（PBS）或rFGF4（1.0 mg/kg体重）注射db/db小鼠，持续37天；db/m小鼠作为对照。通过尾部采集血液样品，并如上所述测量葡萄糖水平。在最后一次注射后，如上所述进行OGTT实验。使用自动生化分析仪（日立自动分析仪 7020，Hitachi Co. Ltd., Tokyo, Japan）测量小鼠血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）和非酯化脂肪酸（NEFA）水平。为了测量肝脏甘油三酯（TG）水平，用氯仿/甲醇（2/1，v/v）提取100 mg 至200 mg肝脏组织。将提取物在氮气流中冻干并溶解在5%不含脂肪酸的BSA中，并使用VET测试化学分析仪（抗体在线，德国）测量提取物TG含量。使用MILLIPLEX MAP小鼠代谢激素磁珠小组-代谢多重测定法（德国达姆施塔特默克密理博公司）测量TNF-α和IL-6水平。使用双能X射线吸光测定法（PIXImus2， GE lunar, Madison,WI）测量骨矿物质密度（BMD）、骨矿物质含量（BMC）和身体成分。
4，代谢笼测定
在用缓冲液（PBS）和db/m小鼠作为对照的30天内，每隔一天用rFGF4（1.0 mg/kg体重）腹腔注射db/db小鼠。在监测之前，使小鼠在代谢笼中适应24小时。所有动物的光照周期和黑暗周期测量均以49分钟间隔记录四天。通过间接测热法（Oxymax， ColumbusInstruments, Columbus, OH）测量耗氧量（VO2）、二氧化碳产量（VCO2）、呼吸交换率（RER）、产热量、食物摄入量以及总活动量和走动活动量。
5，高胰岛素-正常血糖钳夹研究
在单独用缓冲液作为对照的情况下， db/db小鼠每隔一天接受rFGF4（1.0 mg/kg体重）治疗连续10天后，进行高胰岛素-正常血糖钳夹实验。简而言之，在小鼠右颈静脉内植入双导管（MRE-025， Braintree Scientific）后，将导管从皮下隧穿到颈后部并外露。在钳夹实验前，使小鼠恢复3天。将手术前体重减少<6%的小鼠纳入以下测试中。小鼠禁食6小时后，在t=-10和t=0分钟时对血液取样以确定基础胰岛素水平。然后给小鼠注射人胰岛素（500 mU/kg，Humulin R，印第安纳州印第安纳波利斯礼来制药厂）预注剂。通过输注葡萄糖（50% D-葡萄糖，可变输注速率）和胰岛素（用于实验的剩余部分，20 mU/kg/min）在t=0分钟开始钳夹。每10分钟从尾静脉收集血液并分析葡萄糖浓度。在t=110和t =120（实验结束）分钟收集的血液用于确定稳态下的胰岛素水平。当葡萄糖输注和血浆葡萄糖水平保持恒定至少30分钟时，认为钳夹已达到稳态条件（120 mg/dl ± 5 mg/dl）。在达到接近稳定状态的最后30分钟内测量GIR。
6，RNA提取、cDNA合成和定量RT-PCR
用TRIzol试剂（Thermo Fisher Scientific, Waltham, MA）从小鼠组织中提取总RNA，并使用RNeasy微型试剂盒（Qiagen， Valencia, CA）纯化总RNA。使用两步式M-MLVPlatinum SYBR Green qPCR Super Mix-UDG试剂盒（马萨诸塞州沃尔瑟姆赛默飞世尔科学公司）进行逆转录和定量PCR。GAPDH或β-肌动蛋白（对于g6pase和pepck）用作内源对照以标准化加入到每个反应中的总RNA量的差异。引物均购自Invitrogen。
7，小鼠组织的病理学、组织病理学、免疫组织化学和免疫荧光评估
在每隔一天给小鼠腹腔注射PBS（作为对照）或rFGF4（以1.0 mg/kg体重）37天后，从db/ m（对照）和db/db小鼠（每组6只）中切除肝脏和附睾脂肪组织，并且将其称重。将肝脏和附睾脂肪组织在4%多聚甲醛中固定过夜，并将其在石蜡中或使用组织-Tek OCT化合物（Sakura,Tokyo, Japan）进行包埋。脱石蜡和再水化后，使用标准方法用苏木精和曙红（H＆E）试剂对石蜡切片（5 μm）染色。通过肝冷冻切片的油红O染色或通过肝脏石蜡切片的过碘酸-希夫（PAS）染色分别可视化肝脏脂肪变性或肝糖原含量。我们在补充实验程序中描述了石蜡切片的免疫组织化学和免疫荧光分析的相关细节。根据制造商的说明书（Abcam, Cambridge,UK）测定用PBS（作为对照）或rFGF4治疗37天db/db小鼠肝脏组织的糖原含量（100 mg 至200mg）。
8，蛋白质印迹分析
将原代小鼠肝细胞、L6肌肉细胞、骨髓来源的巨噬细胞或小鼠组织在含有蛋白酶和磷酸酶抑制剂（均来自马萨诸塞州沃尔瑟姆赛默飞世尔科学公司）的RIPA裂解缓冲液（25 mMTris，pH 7.6，150 mM NaCl，1% NP-40，1%脱氧胆酸钠，0.1% SDS）中进行匀浆和裂解。使用BCA试剂盒（Protein Assay Kit，中国上海贝奥蒂姆生物技术公司）测定蛋白质浓度。使用8%至12% SDS-PAGE分离等量的可溶性蛋白质（40 μg）并将其电转移到硝酸纤维素膜上。用一抗探测蛋白质印迹。通过与缀合有辣根过氧化物酶的二抗（Santa Cruz Biotechnology,Dallas, TX；或，中国合肥Biosharp）一起孵育来检测免疫反应带，并使用增强化学发光（ECL）试剂（Bio-Rad, Hercules, CA）进行条带可视化。使用ImageJ图像软件（版本1.38e，NIH, Bethesda, MD）分析免疫印迹的光密度，并将其标准化为它们各自对照的扫描信号。
9，统计分析
体外实验至少重复三次以上。结果表示为平均值±SEM。通过使用来自SPSS公司（SPSSInc.）的统计软件NASDAQ：SPSS进行统计学处理，用单因素或双因素方差分析（ANOVA）和Student t-检验进行统计学分析。P<0.05被视为具有统计学意义。
实施例2 实验结果和结论
1，rFGF4可诱导产生显著的降糖效果
我们通过向高血糖db/db小鼠体内腹腔注射单剂量（i.p.，0.25 mg/kg、0.5 mg/kg或1mg/kg体重）FGF4来检查其治疗潜力，并发现血糖水平呈剂量依赖性减少（图2A）。在1 mg/kg体重的剂量下，rFGF4在注射3小时后引发显著的降血糖效应，并在6小时达到其最大效果。这种效果可持续长达36小时（图2B）。我们在饮食诱导的肥胖（DIO）小鼠（图2C）以及第二糖尿病小鼠模型（ob/ob）中进行的平行实验中也观察到了相似的降糖效果（图2D）。更为重要的是，皮下（s.c.）或静脉（i.v.）注射rFGF4也可以相类似的方式降低血糖水平（图2E）。因此，我们得出结论：rFGF4的降糖效果与给药途径无关。
接下来，我们通过在37天每隔一天用rFGF4腹腔注射db/db小鼠来探讨FGF4在改善高血糖症方面的长期效果。此慢性rFGF4治疗可产生持续的降葡萄糖效果（图2F），而体重没有发生显著的变化（图2G）。与这些数据一致，rFGF4并不影响小鼠如氧气消耗速率（VO2）、二氧化碳产生速率（VCO2）、产热量、食物摄入量或总活动量等能量代谢指标（图5A至图5N）。在用rFGF4治疗db/db小鼠期间，其附睾脂肪（Epi-WAT）质量、脂肪细胞大小、瘦肉质量和脂肪质量并没有发生变化（图6A至图6D）。更为重要的是，长期使用rFGF4并不影响小鼠的骨矿物质密度（BMD）或骨矿物质含量（BMC）（图6E、图6F）。而且，以1mg/kg体重单次腹腔注射rFGF4对野生型小鼠的血糖水平没有效果（图7A），而更高剂量的rFGF4（3 mg/kg体重）在db/db小鼠体内没有产生低血糖（图7B）。我们得出结论，rFGF4作为降糖剂具有显著的潜力，且不会引起低血糖或其他明显的副面作用。
2，rFGF4可改善葡萄糖耐受和胰岛素耐受性
为了进一步评估rFGF4平衡体内葡萄糖的效果，我们在腹腔注射rFGF4 3小时后对db/ db小鼠进行口服葡萄糖耐受性试验（OGTT）。我们发现在2小时内， rFGF4可产生显著持续的降糖效果（图3A），且在0和15分钟时，血清胰岛素水平也相应减少（图3B）。此外，单剂量的rFGF4可明显缓解db/db小鼠体内的高胰岛素血症，该效果持续至少12小时（图3C）。在用rFGF4进行慢性治疗后，可明显改善胰岛素耐受性（图3D）。这些结果经由高胰岛素/正常血糖钳夹试验（图3E、图3F）证实（期间db/db小鼠用rFGF4治疗10天）：在高胰岛素条件下，rFGF4治疗小鼠需要更多的外源性葡萄糖来维持正常血糖，如葡萄糖输注速率（GIR）所示（图3F）。通过检测蛋白激酶B（AKT）的磷酸化水平，我们发现：rFGF4改进的胰岛素耐受性伴随着骨骼肌、肝脏和附睾脂肪组织中胰岛素信号的增强（图3G）。我们得出结论，慢性rFGF4治疗可改进胰岛素靶组织中的胰岛素耐受性。
为了确定rFGF4的降葡萄糖效果是否为胰岛素依赖型，我们进一步检查了FGF4在链脲佐菌素（STZ）诱导的I型糖尿病（T1D）小鼠体内的效果。我们发现rFGF4在这些小鼠体内没有显著的降糖活性（图3H）。然而，联合注射rFGF4与胰岛素可明显增强rFGF4在降糖和改善胰岛素耐受方面的效果（图3I）。我们得出结论，FGF4以胰岛素依赖型方式调节体内糖代谢平衡。
3，长期rFGF4给药可改进T2D小鼠肝脏脂肪变性、促进肝脏糖原合成和抑制肝脏糖异生
肝脏脂肪变性为众所周知的肥胖后遗症。重要的是，我们发现在rFGF4治疗37天后，db/ db小鼠中肝脏脂肪积累已基本消除（图4A）。与此观察结果一致，我们发现rFGF4可明显下调生脂基因-脂肪酸合成酶（FAS）、硬脂酰辅酶A去饱和酶-1（SCD-1）和固醇调节元件结合蛋白-1（SREBP-1）的表达水平，同时可增强乙酰辅酶A羧化酶（ACC）的磷酸化水平（调节肝脏脂肪酸β-氧化的关键酶）（图4B）。此外，通过rFGF4治疗，肝脏质量和甘油三酯（TG）水平显著降低（图4C、图4D），丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）和非酯化脂肪酸（NEFA）的血清水平显著减少（图4E、图4F）。
在T2D中，由于严重的胰岛素耐受，肝脏调节葡萄糖水平的正常功能逐渐受损。这导致肝脏葡萄糖产生增加和糖原储存减少，而这两者都会加剧代谢异常。我们发现在db/db小鼠体内，通过用rFGF4慢性治疗，肝糖原含量显著增加（图4G、图4H），其与GSK3β磷酸化水平的上调和 GS蛋白表达水平的下调密切相关（图4I）。与急性给药实验中rFGF4对糖异生的抑制效果一致，在用rFGF4慢性治疗的db/db小鼠体内，PEPCK和G6Pase的mRNA和蛋白水平受到抑制（图4I至图4K）。此外，rFGF4介导了FOXO1转录因子的磷酸化和失活（PEPCK和G6pase表达的关键调节因子，并且其活化被胰岛素/AKT信号传导消除）（图4I）。在禁食C57BL/6J小鼠体内，rFGF4亦可下调PEPCK和G6pase mRNA水平（图4L、图4M）。因此，我们得出结论，肝脏为FGF4调节血糖稳态的主要靶器官之一，其可通过增强肝脏糖原合成和抑制糖异生来降低T2D血糖水平。
4，结论
通过这些实验（图1至图3）发现FGF4是一种新型强效的胰岛素增敏因子。rFGF4通过促进肌肉和肝脏的葡萄糖摄取来发挥其作用，同时它不会导致低血糖或骨质流失。此外，rFGF4可直接作用于巨噬细胞以阻断肝脏、肌肉和脂肪组织中的炎性反应。鉴于炎性细胞因子可阻断胰岛素信号并直接诱导胰岛素耐受，因此，rFGF4对巨噬细胞浸润和炎症的抑制作用是其长期给药增强胰岛素敏感性的主要原因。由此可见，在临床上，rFGF4作为治疗T2D潜在的药剂具有惊人的潜力。
我们的数据显示，rFGF4的代谢效果为胰岛素和GLUT2依赖性。然而，肝脏中GLUT2向细胞膜的转运与胰岛素无关。对这些不同发现的可能解释在于以下事实：在肝细胞中，细胞外葡萄糖被GLUT2吸收，而细胞内葡萄糖代谢（即， 糖酵解和糖原生成）为胰岛素信号依赖。如果抑制胰岛素信号传导，则细胞内葡萄糖浓度将不可避免地升高。一旦细胞内葡萄糖浓度变得与循环系统中血液中浓度相同，细胞内外葡萄糖梯度就会消失，梯度依赖性的GLUT2功能也会丧失。因此，在这种情况下，尽管胰岛素不直接调节GLUT2的转运，但胰岛素信号仍然为介导GLUT2降糖效果所必需。
通常认为，由胰岛素介导的高达75%的葡萄糖利用发生在骨骼肌中，而其他组织则占剩余25%。与先前研究一致，我们发现rFGF4的急性降糖作用为胰岛素依赖性，并且由此介导的葡萄糖摄取增加在骨骼肌中最为显著，而在肝脏中则不明显。而肝脏和脂肪组织为内分泌FGF19和FGF21降糖作用的主要靶器官。这些发现与rFGF4的发现形成对比。这种矛盾的一种可能解决方案在于以下事实：FGF19和FGF21的生物学效应为β-klotho依赖性，而FGF4的效果为HS依赖性。而βklotho的表达局限于代谢器官，包括BAT、WAT、肝脏、胰腺、大脑和肠道，但不限于骨骼肌。因此，由FGF19或FGF21介导的葡萄糖摄取活性局限于表达β-klotho的器官，而FGF4不受此约束。由于骨骼肌表达FGFR1，FGF4可在此器官中介导其强效降糖效果。
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序列表
<110> 温州医科大学
<120> FGF4及其应用
<130> CN
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<170> PatentIn version 3.5
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Thr Arg Asp Ser Leu Leu Glu Leu Ser Pro Val Glu Arg Gly Val
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Val Ser Ile Phe Gly Val Ala Ser Arg Phe Phe Val Ala Met Ser
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Ser Lys Gly Lys Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys
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Ser Tyr Lys Tyr Pro Gly Met Phe Ile Ala Leu Ser Lys Asn Gly
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Thr Phe Lys Glu Ile Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu
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Val Ser Ile Phe Gly Val Ala Ser Arg Phe Phe Val Ala Met Ser
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Claims (10)
1.蛋白质，其氨基酸序列如SEQ ID NO：1、2、3或4所示。
2.多核苷酸，其编码权利要求1所述的蛋白质。
3.载体，其包含权利要求2所述的多核苷酸。
4.细胞，其包含权利要求2所述的多核苷酸。
5.制备权利要求1所述的蛋白质的方法，其包括在适宜的条件下使用权利要求4所述的细胞表达权利要求1所述的蛋白质，然后纯化。
6.药物组合物，其包括权利要求1所述的蛋白质以及药学上可接受的载体。
7.权利要求1所述的蛋白质在制备用于增强胰岛素敏感性的药物中的应用。
8.权利要求1所述的蛋白质在制备用于降血糖和/或改善葡萄糖耐受性的药物中的应用。
9.权利要求1所述的蛋白质在制备用于减少肝脏脂肪变性和/或脂肪组织炎症的药物中的应用。
10.权利要求1所述的蛋白质在制备用于治疗糖尿病（尤其是II型糖尿病）和/或肥胖症的药物中的应用。
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| CN115515969A (zh) * | 2020-11-04 | 2022-12-23 | 温州医科大学 | FGF4和Fgfr1结合配体在糖尿病中的应用 |
| CN115785248A (zh) * | 2022-12-08 | 2023-03-14 | 杭州生物医药创新研究中心 | 靶向FGFR1c的FGF变构体及其应用 |
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| CN115515969B (zh) * | 2020-11-04 | 2025-02-18 | 温州医科大学 | FGF4和Fgfr1结合配体在糖尿病中的应用 |
| CN115785248A (zh) * | 2022-12-08 | 2023-03-14 | 杭州生物医药创新研究中心 | 靶向FGFR1c的FGF变构体及其应用 |
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