GLP-1抵抗胰臟β細胞凋亡的分子機制

Abstract

胰臟β細胞(pancreatic β cells)發生自體凋亡(apoptosis)是糖尿病的指標。眾多引起β細胞apoptosis的因子中，體內累積大量糖解作用之中間產物2含氧丙醛(methylglyoxal; MG)會對細胞形成慢性的葡萄糖毒性(glucotoxicity)，最後導致apoptosis。類升糖素胜肽1 (glucagon-like peptide-1; GLP-1)是由人體腸道中所分泌，具有保護β細胞免於apoptosis。然而，GLP-1影響MG的分子機制尚未完全明瞭。尤其在GLP-1促進胰島素分泌機制扮演重要角色之胰臟的ATP敏感性鉀離子通道 (ATP-sensitive potassium; KATP channel)， Kir6.2是否也參與在GLP-1對MG影響β細胞凋亡的分子機制是本研究致力於探討的。 本研究利用大鼠胰島細胞瘤細胞RIN-m5做為材料，以1 mM MG處理經過隔夜靜置培養後，我們發現MG會降低細胞的存活率，且增加apoptosis的現象，包括細胞膜外翻，DNA斷裂，活化的胱天蛋白酶-3（caspase-3），以及磷酸化的c-jun 胺基端蛋白激酶(c-jun N-terminal kinase, JNK)。此外，我們發現JNK和eIF2的磷酸化增加，顯示MG會引起細胞的內質網壓力( ER stress); 我們也發現β細胞氧耗率和ATP含量降低，顯示MG也造成細胞粒線體 (mitochondria)的功能受創。但是，若以GLP-1前處理，則β細胞仍可維持高的存活率，抑制上述與apoptosis和ER stress相關的現象，可以保護mitochondria的功能。而當細胞的Kir6.2受到shRNA抑制表現後，GLP-1卻仍可對抗apoptosis，ER stress，及保護mitochondria的能力。由於MG可以長期活化AMP-活化蛋白激酶 (AMP-activated protein kinase; AMPK)，但AMPK的活化會被GLP-1所抑制。因此，我們總結GLP-1可能是透過降低AMPK的長期活化而來調節MG引起的β細胞apoptosis，且此路徑不需要Kir6.2的參與。

Pancreatic β-cell apoptosis is the hall marker of diabetes. Among various potential factors involved, the accumulation of methylglyoxal (MG), an intermediate of glycolysis which contributes to glucotoxicity, is a promising factor leading to β-cell apoptosis. Glucagon-like peptide-1 (GLP-1), secreted from small intestine, possesses the protective ability against β-cell apoptosis. However, the molecular mechanism by which GLP-1 prevents MG-induced β-cell apoptosis remains unclear. ATP-sensitive potassium channels (KATP channels) has a tissue-specific expression, similar to GLP-1 receptor, plays a crucial role on GLP-1’s insulinotropic function. We aim to study molecular mechanisms of protective effect of GLP-1 on β-cell apoptosis and examine whether KATP channels is involved in the effects of GLP-1 on MG-induced β-cell apoptosis. In the present study, we use rat insulinoma cell line, RIN-m5f cells as a model. We found that MG-treated cells showing decreased cell viability and increased apoptotic markers, including externalization of phosphatidylserine (PS), DNA fragmentation, activation of caspase 3 and increased phosphorylation of JNK. Besides, MG-treated cells showed a higher ER stress as phosphorylation of JNK and eIF2α was enhanced. We also found mitochondria dysfunction in the MG-treated cells, as was documented decline in oxygen consumption rate and ATP production. Importantly, we found GLP-1 pretreatment can protect β cells from MG-induced apoptosis. This effect is independent of Kir6.2 as shown by the shRNA experiments. Finally, GLP-1 prevented the MG-induced prolonged activation of AMPK and mitochondria dysfunction. Taken together, we conclude that GLP-1 can protect β cells from MG-induced apoptosis via ameliorating ER stress and mitochondrial dysfunction. This effect is independent of Kir6.2. The role of prolonged AMPK activation on β-cell apoptosis deserves further studies.