胰高血糖素樣肽-1的研究進(jìn)展

王璠璠，盧海英，劉莉潔

(東南大學(xué) 醫(yī)學(xué)院，江蘇 南京 210009)

1902年Bayliss和Starling在小腸分泌物中發(fā)現(xiàn)了促胰液素(secretin)[1]，創(chuàng)生了“激素”概念[2]。20世紀(jì)30年代，法國學(xué)者La Barre等[3]發(fā)現(xiàn)促胰液素粗制品可使血糖下降，于是提出小腸分泌物中還存在一種不影響胰液分泌卻可降低血糖的因子(intestinal factor)，并稱之為“腸促胰島素(incretin)”。胰島素放射性免疫熒光檢測法出現(xiàn)后，多個(gè)實(shí)驗(yàn)室分別證實(shí)口服葡萄糖對(duì)胰島素分泌的促進(jìn)作用明顯高于等量葡萄糖的靜脈注射[4-7]，腸-胰島軸(entero insular axis)的概念即由此產(chǎn)生[8]，腸促胰島素被認(rèn)為參與其中。Creutzfeldt[9]將“腸促胰島素”的特點(diǎn)歸納為：進(jìn)食后由胃腸細(xì)胞分泌；進(jìn)食后的生理濃度下即可產(chǎn)生促胰島素分泌效應(yīng)；促胰島素分泌效應(yīng)具有葡萄糖依賴性。據(jù)此標(biāo)準(zhǔn)，抑胃多肽(gastric inhibitor polypeptide，GIP)又稱葡萄糖依賴性促胰島素分泌多肽(glucose-dependent insulinotropic peptide，GIP)，成為了第一個(gè)腸促胰島素成員[10-12]。1983年，發(fā)現(xiàn)GIP十多年后，Mclntyre等在分析胰高血糖素原的基因序列時(shí)發(fā)現(xiàn)了另一種效應(yīng)更為強(qiáng)大的腸促胰島素，即胰高血糖素樣肽-1(glucagons like peptide-1，GLP-1)[13]。

GLP-1屬于胰高血糖素原基因的翻譯后組織特異性蛋白水解產(chǎn)物(tissue-specific posttranslational proteolytic product)，進(jìn)食可引起腸L細(xì)胞釋放GLP-1，產(chǎn)生葡萄糖濃度依賴性促胰島素分泌效應(yīng)[14]。近年來，利用GLP-1類似物、GLP-1受體(GLP-1R)激動(dòng)劑及GLP-1R基因靶向破壞等不同手段所進(jìn)行的研究發(fā)現(xiàn)，GLP-1對(duì)于機(jī)體多種組織和器官,尤其是胰島，具有諸多功能調(diào)節(jié)效應(yīng)，GLP-1已成為糖尿病發(fā)病機(jī)制及藥物治療的研究熱點(diǎn)。

1 GLP-1的合成、分泌及降解

1.1 GLP-1的合成

GLP-1是胰高血糖素原基因的編碼產(chǎn)物之一。胰高血糖素原基因位于人類2號(hào)染色體長臂，包括6個(gè)外顯子和5個(gè)內(nèi)含子，GLP-1編碼區(qū)位于第四外顯子[15]。該基因在胰腺α細(xì)胞、腸 L細(xì)胞以及丘腦下部、腦干等處的神經(jīng)元中都有表達(dá)，產(chǎn)生單一的、完全相同的轉(zhuǎn)錄本，進(jìn)而翻譯產(chǎn)生含158個(gè)氨基酸的胰高血糖素原(proglucagon)；此單鏈前體蛋白在不同組織中可加工生成一系列具有不同生物活性的胰高血糖素原衍生肽(proglucagon derived peptides，PGDP)[16]。具有組織表達(dá)特異性的激素原轉(zhuǎn)化酶(prohormone convertases，PCs)PC2和 PC1/3在胰高血糖素原的翻譯后加工過程中具有重要作用。在胰島α細(xì)胞中PC2的作用下，胰高血糖素原的主要翻譯后處理產(chǎn)物是胰島素的主要反調(diào)節(jié)激素(counterregulatory hormone)——胰高血糖素(glucagon)；而在腸黏膜的L細(xì)胞中，PC1/3將胰高血糖素原剪切為胰高血糖素樣肽-1(glucagon like peptide-1，GLP-1 )、胰高血糖素樣肽-2(glucagon like peptide-2，GLP-2)、腸高血糖素(glicentin)、胃泌酸調(diào)節(jié)素(oxyntomodulin)等(圖1)[17-18]。

胰高血糖素原在胰腺、腸和腦組織中翻譯后處理機(jī)制及產(chǎn)物不同。圖中數(shù)字代表了各胰高血糖素原衍生肽在胰高血糖素原160個(gè)氨基酸序列中的對(duì)應(yīng)位點(diǎn)。GRPP，腸高血糖素相關(guān)胰多肽;IP-1，中間肽-1；IP-2，中間肽-2

圖1胰高血糖素原基因的組織特異性翻譯后加工[18]

1.2 GLP-1的分泌

GLP-1由腸L細(xì)胞分泌。腸L細(xì)胞可見于大鼠及人類的全段腸黏膜上皮，遠(yuǎn)端回腸(distal ileum)和結(jié)腸(colon)中分布密度相對(duì)較高[19]。腸L細(xì)胞是一類開放型上皮內(nèi)分泌細(xì)胞(open-type intestinal epithelial endocrine cell)，其頂端暴露于腸腔，底端與腸壁內(nèi)神經(jīng)叢及微循環(huán)系統(tǒng)相臨近，因此L細(xì)胞GLP-1的分泌可受到食物營養(yǎng)成分、腸壁內(nèi)神經(jīng)信號(hào)和激素信號(hào)的共同調(diào)控[20]。攝食，尤其是富含脂肪和碳水化合物的食物，是GLP-1迅速釋放的主要生理性刺激因素[21]。在嚙齒動(dòng)物和人類，攝食后GLP-1呈雙相分泌：10～15 min內(nèi)的早期分泌相,繼以30～60 min的較長期二相分泌。由于L細(xì)胞主要分布于遠(yuǎn)端小腸，營養(yǎng)物對(duì)L細(xì)胞的直接作用不太可能是引起GLP-1早期分泌的因素。大量研究表明，迷走神經(jīng)、乙酰膽堿以及GIP等多種神經(jīng)及體液因素在攝食后GLP-1的早期分泌中發(fā)揮重要作用，而GLP-1的二相分泌則很可能是由營養(yǎng)物對(duì)L細(xì)胞的直接作用所引起。L細(xì)胞內(nèi)PKA、PKC、鈣離子以及MAPK等信號(hào)的激活參與了GLP-1的分泌[14]。

1.3 GLP-1的活性形式及降解

體內(nèi)存在多種形式的GLP-1，其中GLP-1(1-37)和GLP-1(1-36)酰胺被認(rèn)為無生物活性,它們再經(jīng)兩步酶切，去除N端6個(gè)氨基酸和形成C端酰胺化，生成具有高度生物活性的GLP-1(7-37)多肽和GLP-1(7-36)酰胺。人類循環(huán)系統(tǒng)中的活性GLP-1主要是GLP-1(7-36)酰胺。研究表明，C末端酰胺化不但大幅提高了GLP-1的生物活性，也提高了GLP-1的體內(nèi)穩(wěn)定性[22]。

GLP-1(7-36)酰胺可被蛋白水解酶二肽基肽酶4(dipeptidyl peptidase-4，DPP-4)快速降解失活[23]。DPP-4是一種廣泛表達(dá)于細(xì)胞表面的絲氨酸蛋白酶，部分以可溶形式存在于循環(huán)血液中。小腸絨毛毛細(xì)血管內(nèi)皮細(xì)胞高表達(dá)DPP-4，肝臟、胰腺等組織也有DPP-4表達(dá)，因而腸L細(xì)胞分泌的GLP-1在進(jìn)入門脈循環(huán)之前就已有半數(shù)以上被DPP-4降解失活。腎臟是GLP-1及其代謝產(chǎn)物的主要清除場所[24]。

2 GLP-1的生物學(xué)作用

2.1 GLP-1受體

GLP-1通過與細(xì)胞膜表面的GLP-1受體(GLP-1R)結(jié)合發(fā)揮作用。GLP-1R是由463個(gè)氨基酸組成的7次跨膜螺旋G蛋白偶聯(lián)受體，其氨基端胞外段在GLP-1與GLP-1R的結(jié)合中發(fā)揮重要作用，而第三段胞內(nèi)環(huán)中的結(jié)構(gòu)域則為受體與G蛋白的有效偶聯(lián)所必須。GLP-1與GLP-1R結(jié)合，激活胞內(nèi)腺苷酸環(huán)化酶、磷脂酶C，進(jìn)而增加胞漿內(nèi)Ca2+濃度,激活PKA、PKC、PI-3K以及MAPK等信號(hào)通路[25-26]。GLP-1R在胰島、腎臟、肺、心臟、腸、迷走神經(jīng)節(jié)和下丘腦、垂體、海馬、大腦皮層等神經(jīng)組織均有表達(dá)[27-28]。

2.2 GLP-1的胰腺內(nèi)作用及機(jī)制

2.2.1 GLP-1可促進(jìn)胰島素的分泌 對(duì)于培養(yǎng)的胰島、在體或離體胰腺，GLP-1都具有葡萄糖依賴性促胰島素分泌效應(yīng)[29]。

GLP-1與β細(xì)胞膜表面GLP-1R結(jié)合，可激活腺苷酸環(huán)化酶，提高胞內(nèi)cAMP水平，進(jìn)而激活PKA、鳥苷酸轉(zhuǎn)化因子Ⅱ(cAMP-regulated guanine nucleotide exchange factor Ⅱ，Epac2)，通過下列機(jī)制促進(jìn)葡萄糖刺激下的胰島素分泌(圖2)：(1) 抑制KATP通道，導(dǎo)致β細(xì)胞膜去極化；(2) 增加Ca2+內(nèi)流，動(dòng)員胞內(nèi)Ca2+存儲(chǔ)庫，增加β細(xì)胞內(nèi)Ca2+濃度；(3) 增加線粒體ATP的合成，以進(jìn)一步促進(jìn)細(xì)胞膜去極化；(4) 關(guān)閉電壓依賴性K+通道，阻礙β細(xì)胞膜復(fù)極化；(5) 直接促進(jìn)胰島素囊泡的胞吐[30-33]。

圖2GLP-1葡萄糖依賴性促胰島素分泌效應(yīng)的細(xì)胞內(nèi)分子機(jī)制[18,34-35]

GLP-1可還上調(diào)β細(xì)胞內(nèi)葡萄糖感應(yīng)元件葡萄糖轉(zhuǎn)運(yùn)體以及葡萄糖激酶的表達(dá)，從而增加β細(xì)胞對(duì)葡萄糖的敏感度以及反應(yīng)性。

2.2.2 GLP-1可促進(jìn)胰島素的合成 GLP-1可與葡萄糖協(xié)同作用促進(jìn)胰島素基因的表達(dá)、維持其mRNA穩(wěn)定性并促進(jìn)胰島素生物合成，具有補(bǔ)充β細(xì)胞胰島素儲(chǔ)量、防止β細(xì)胞胰島素耗竭的作用[36-37]。β細(xì)胞內(nèi)cAMP-PKA依賴性及非依賴性信號(hào)通路、Ca2+濃度的增加都參與了此生物過程[37]。胰腺十二指腸同源異形盒1(panereatic duodenum homeobox-1，PDX-l)是一種對(duì)胰腺發(fā)育及β細(xì)胞功能都非常重要且必需的轉(zhuǎn)錄因子，它在GLP-1的促進(jìn)胰島素基因轉(zhuǎn)錄及胰島素分泌中具有核心作用。GLP-1能夠促進(jìn)PDX-1的表達(dá)，并增加PDX-1結(jié)合胰島素啟動(dòng)子的能力。PDX-1的表達(dá)對(duì)于整合GLP-1R依賴的信號(hào)通路，調(diào)節(jié)β細(xì)胞胰島素的分泌、β細(xì)胞生長、功能分化和細(xì)胞生存是必需的[38]。

2.2.3 GLP-1可增加胰島β細(xì)胞數(shù)量 細(xì)胞增殖與凋亡之間的平衡維持著β細(xì)胞數(shù)量的穩(wěn)定。在體情況下，GLP-1能夠促進(jìn)β細(xì)胞的增殖分化、促進(jìn)胚胎胰島細(xì)胞的分化與成熟；可提高β細(xì)胞的葡萄糖耐受性、抑制糖尿病模型動(dòng)物β細(xì)胞的凋亡[39]。 GLP-1類似物Exendin-4可促進(jìn)內(nèi)質(zhì)網(wǎng)應(yīng)激狀態(tài)下β細(xì)胞的功能及存活[40]；還可延緩自發(fā)糖尿病模型G-K大鼠、db/db小鼠以及實(shí)驗(yàn)誘導(dǎo)宮內(nèi)發(fā)育遲緩大鼠成年糖尿病的發(fā)生[39,41-42]。離體實(shí)驗(yàn)中，培養(yǎng)基中加入GLP-1可使大鼠及人類胰腺導(dǎo)管細(xì)胞分化為胰島素分泌細(xì)胞[43-45]。GLP-1還有助于保持游離胰島的形態(tài)結(jié)構(gòu)、促進(jìn)其葡萄糖刺激的胰島素分泌并抑制其細(xì)胞凋亡[46]，增加原代胰島對(duì)細(xì)胞毒性因子的耐受力[47]。

與葡萄糖依賴性的促胰島素分泌效應(yīng)不同，GLP-1的促β細(xì)胞增殖、分化作用不依賴于葡萄糖的濃度，在正常的血糖情況下就能產(chǎn)生。GLP-1促進(jìn)β細(xì)胞增殖、分化及對(duì)β細(xì)胞營養(yǎng)、保護(hù)功能的實(shí)現(xiàn)有GLP-1R下游多種信號(hào)通路的參與。GLP-1與GLP-1R結(jié)合,激活腺苷酸環(huán)化酶產(chǎn)生cAMP，通過cAMP激活磷脂酰肌醇3-激酶(PI3K)及其絲裂原活化蛋白激酶(MAPK/ERK)、蛋白激酶A(PKA)、蛋白激酶B(PKB/Akt)等信號(hào)轉(zhuǎn)導(dǎo)途徑，加強(qiáng)促凋亡蛋白(如caspase-3)的降調(diào)節(jié)和抗凋亡蛋白(如bcl-2)的升調(diào)節(jié)機(jī)制，從而調(diào)節(jié)胰島β細(xì)胞的增殖和凋亡[39,48]。轉(zhuǎn)錄因子PDX-1的激活是這些信號(hào)通路的共享組件(a shared component)，在PDX-1基因敲除的小鼠，GLP-1及其類似物的促β細(xì)胞增殖、抑制β細(xì)胞凋亡的效應(yīng)均消失[36]。

2.2.4 GLP-1可抑制胰高血糖素分泌、刺激生長抑素釋放 GLP-1通過直接與胰腺δ細(xì)胞上的GLP-1R結(jié)合而發(fā)揮促進(jìn)生長抑素釋放的效應(yīng)。GLP-1抑制胰腺α細(xì)胞胰高血糖素釋放的機(jī)制尚不清楚，可能是通過促進(jìn)胰島素和生長抑素釋放間接抑制胰高血糖素釋放[49-50]。GLP-1對(duì)胰高血糖素釋放的抑制效應(yīng)也具有葡萄糖依賴性，因此不會(huì)增加低血糖發(fā)生率。

2.3 胰腺外組織的作用

2.3.1 GLP-1對(duì)于胃排空和胃酸分泌的作用 GLP-1可抑制胃部平滑肌的活動(dòng)，從而抑制胃排空、延緩胃部營養(yǎng)物質(zhì)向小腸的運(yùn)輸，減緩由進(jìn)食引起的血糖升高[51]，這也是GLP-1控制餐后血糖一個(gè)重要原因。GLP-1抑制胃排空和胃酸分泌的作用機(jī)制較為復(fù)雜，胃腸黏膜壁細(xì)胞、迷走神經(jīng)傳入纖維以及中樞神經(jīng)系統(tǒng)中的GLP-1R可能發(fā)揮了主要作用[52]。

2.3.2 GLP-1對(duì)于神經(jīng)系統(tǒng)的影響 中樞神經(jīng)系統(tǒng)中，與攝食、胃腸道運(yùn)動(dòng)、血糖調(diào)節(jié)、心血管活動(dòng)等多項(xiàng)生理活動(dòng)有關(guān)的調(diào)節(jié)區(qū)域，都有GLP-1R及GLP-1的表達(dá)；GLP-1R還分布于向腦干孤束核投射的腹腔迷走傳入神經(jīng)的結(jié)狀神經(jīng)節(jié)。

動(dòng)物研究顯示，外周或中樞給予GLP-1R激動(dòng)劑可減少食物攝入、降低體重。對(duì)正常個(gè)體、肥胖及糖尿病患者，GLP-1R激動(dòng)劑都可使其產(chǎn)生飽腹感、降低食欲、減少食物攝入并引起體重的持久降低，因而有助于糖尿病人體重和血糖的控制。GLP-1及Exendin-4都是小分子物質(zhì)，可以自由通過血腦屏障。GLP-1對(duì)下丘腦攝食中樞GLP-1R的直接作用以及對(duì)胃腸運(yùn)動(dòng)及胃酸分泌的抑制作用，共同導(dǎo)致了GLP-1對(duì)攝食行為的調(diào)節(jié)[53-56]。有趣的是，GLP-1 R-/-小鼠并無顯著肥胖，這可能是由于GLP-1不是調(diào)節(jié)體重的關(guān)鍵必需因素，或者是由于發(fā)生了某些代償機(jī)制的上調(diào)[57]。

GLP-1的促胰島素分泌效應(yīng)也可能有神經(jīng)機(jī)制的參與。前已述及，GLP-1由L細(xì)胞分泌后，50%以上即迅速被DPP-4降解失活；所剩具有活性的GLP-1在流經(jīng)肝臟過程中也大量失活，因而只有不到10%的GLP-1能以活性形式到達(dá)胰腺。實(shí)驗(yàn)證實(shí)，GLP-1R表達(dá)于腹腔迷走傳入神經(jīng)的結(jié)狀神經(jīng)節(jié)；肝靜脈給予GLP-1引起肝迷走傳入沖動(dòng)增多，并反射性引起胰腺迷走傳出沖動(dòng)的增多；肝靜脈注射GLP-1所引起的促胰島素分泌效應(yīng)可被神經(jīng)節(jié)阻斷劑所取消。因此，局部神經(jīng)環(huán)路很可能參與介導(dǎo)了腸分泌的GLP-1對(duì)胰腺的作用[58]。中樞神經(jīng)系統(tǒng)中GLP-1R的激活還可反射性抑制外周肌肉組織的糖利用、增加胰島素分泌促進(jìn)肝糖原合成，提示中樞神經(jīng)系統(tǒng)中的GLP-1在整體血糖穩(wěn)態(tài)的調(diào)節(jié)中具有重要作用[59]。

表1羅列了GLP-1對(duì)多個(gè)器官系統(tǒng)所具有的生物學(xué)作用，其中很多作用的生理和治療意義還有待于進(jìn)一步研究。

表1GLP-1對(duì)多組織器官的生物學(xué)作用

器 官GLP-1的作用胰腺促進(jìn)胰島素分泌,促進(jìn)胰島素原的合成,促進(jìn)β細(xì)胞增殖及分化,抑制凋亡,抑制胰高血糖素分泌和刺激生長抑素釋放胃抑制胃排空,抑制胃酸分泌[51]中樞神經(jīng)增進(jìn)飽腹感,降低食欲,升高體溫,促進(jìn)學(xué)習(xí)和記憶[60]周圍神經(jīng)刺激肝迷走神經(jīng)的輸入端[60]心臟增加心率,升高血壓[61];保護(hù)缺血心肌及再灌注損傷,改善心肌能量代謝[62]肝臟促進(jìn)糖原合成,抑制肝糖元的分解,增加肌肉組織糖代謝,調(diào)節(jié)葡萄糖和脂肪在脂肪細(xì)胞中的代謝[63]肺刺激肺表面活性物質(zhì)的分泌,舒張肺血管[64]腎利尿排鈉,可能與抗高血壓有關(guān)[65]

3 展 望

GLP-1的多重生物學(xué)作用賦予了其在2型糖尿病治療中的廣闊應(yīng)用前景。由于天然GLP-1在體內(nèi)的半衰期非常短，因此基于GLP-1的糖尿病藥物研發(fā)主要有兩種策略：一種是GLP-1類似物的研發(fā)，采用氨基酸替換、肽鏈的延伸以及修飾，延長GLP-1的作用時(shí)間；另一種是減緩體內(nèi)GLP-1的降解，主要是抑制DPP-4以維持體內(nèi)的活性GLP-1水平。GLP-1類似物Exendin-4和Liraglutide分別于2005年和2010年通過美國FDA認(rèn)證，現(xiàn)已用于2型糖尿病的臨床治療[66-67]。繼DPP-4抑制劑Sitagliptin于2006年通過美國FDA認(rèn)證之后，Saxagliptin和Vildagliptin也已被相繼發(fā)現(xiàn)。

同樣由于GLP-1以及DPP-4的多重生物學(xué)作用，其相關(guān)藥物對(duì)2型糖尿病的長遠(yuǎn)療效以及對(duì)其它系統(tǒng)器官功能的影響還有待時(shí)間的進(jìn)一步考驗(yàn)。相信，隨著研究的不斷深入，GLP-1及其相關(guān)信號(hào)通路將為2型糖尿病的治療提供更多有效靶點(diǎn)。

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