探討致糖尿病因子 Galectin-3 調控胰臟癌誘發新生糖尿病的機制

To investigate the mechanisms of galectin-3-mediated insulin resistance in pancreatic cancer-associated new-onset diabetes (PCDM).

張晏慈(Yen-Tzu Chang)

指導教授 ： 周綠蘋

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202002343

摘要

胰臟癌由於缺乏明顯病徵，導致85 %的病人在確診時已為晚期，且根據報導有將近40 %的胰臟癌病患伴隨著新生糖尿病的症狀。而這些新生糖尿病（PCDM）被認為是胰臟癌釋放的致糖尿病因子所導致，因此鑑定出這些致糖尿病因子可以作為區分第二型糖尿病與PCDM的生物標記，並提供胰臟癌早期診斷的新方向。
根據實驗室先前發表的paper，galectin-3被認為是PCDM的致糖尿病因子。且利用ELISA分析病人血清中的galectin-3濃度，發現galectin-3可以作為有效的區分第二型糖尿病以及PCDM病人的潛力biomarker。
為了更進一步釐清galectin-3在PCDM中扮演的角色，實驗室利用nickel column 純化出重組蛋白galectin-3進行細胞實驗，結果顯示在胰島素刺激下galectin-3可以抑制肌肉細胞的葡萄糖攝取，且是透過活化IRS-1的抑制型磷酸化位點ser 307及降低AKT的磷酸化來抑制胰島素訊息傳遞路徑。而TLR4已被報導與胰島素抗性有關，且下游的IKK會活化IRS-1 ser307以抑制胰島素訊息傳遞路徑。實驗結果顯示，galectin-3抑制的胰島素訊息傳遞路徑在TLR4及IKK抑制劑分別處理下都有回升的情形，代表galectin-3會透過活化TLR4/ IKK路徑來抑制胰島素訊息傳遞路徑，來促進C2C12細胞的胰島素抗性。
另一方面，實驗室前人發現胰臟癌細胞株的條件培養基以及galectin-3都可以使胰島β細胞INS-1存活率下降，進一步探討其中機制發現，galectin-3處理下IKK及ERK的磷酸化會增加、apoptotic marker BAX/Bcl-2 ratio及cleavage caspase-3都有上升。除此之外，在TLR4及IKK抑制劑分別處理下，pERK、BAX/Bcl-2 ratio及cleavage caspase-3表現量都有回升的情形，說明了galectin-3會透過活化TLR4/IKK路徑來促進ERK誘導的細胞凋亡。
綜上所述，本篇論文了解到galectin-3可以作為PCDM的潛力生物標記，並且在機制上，會活化TLR4/IKK進而抑制胰島素訊息傳遞路徑來造成肌肉細胞C2C12的胰島素抗性；在胰島β細胞INS-1方面則是透過活化TLR4/IKK/ERK路徑來誘導細胞凋亡。

關鍵字

胰臟癌誘發之新生糖尿病 ； 胰島素阻抗 ； 細胞凋亡 ； 半乳糖凝集素-3 ； 生物標記 ； 胰島β細胞

並列摘要

Without evident cancer-specific symptom, 85% pancreatic cancer patients are diagnosed at advanced stage. It is reported that nearly 40% patients are followed with pancreatic cancer-associated new-onset diabetes mellitus (PCDM). PCDM are mediated by some unknown tumor-secreted diabetogenic factors. Therefore, identifying these diabetogenic factors could help us find new biomarker for distinguishing PCDM from type 2 diabetes, and provide a new approach to early detection for pancreatic cancer.
In our previous study, galectin-3 played a diabetogenic role in PCDM. Also, galectin-3 was identified as the biomarker for distinguishing the PCDM from T2DM patients by analyzing the patients’ serum sample.
To further understand the role of galectin-3 in PCDM, recombinant galectin-3 protein was purified. Our result showed that galectin-3 could inhibit glucose uptake in C2C12 cell upon insulin stimulation. In mechanism, they could inhibit insulin signaling pathway by upregulating the inhibitory phosphorylation of IRS-1 and downregulating the phosphorylation of AKT. Next, the IRS-1 activity and AKT phosphorylation was partially rescued upon TLR4 and IKK inhibitor treatment, respectively. It suggested that galectin-3 inhibited insulin signaling pathway through TLR4/IKK pathway, which induce insulin resistance in C2C12 cell.
In addition, the role of galectin-3 in β cell was also investigated. We found that the conditioned medium of pancreatic cancer cell line and galectin-3 could inhibit INS-1 cell viability. Data showed that galectin-3 triggered IKK and ERK phosphorylation and induced BAX/Bcl-2 ratio upregulation and caspase 3 activity. Furthermore, galectin-3 induced-ERK phosphorylation, BAX/Bcl-2 and caspase-3 activation was rescued by TLR4 and IKK inhibitor treatment, respectively. It showed that galectin-3 induced ERK/caspase-3 activation through TLR4/IKK pathway, which caused INS-1 cell apoptosis.
Until now, these data have showed that galectin-3 is a diabetogenic factor in PCDM. It promoted insulin resistance through TLR4/IKK pathway to inhibit insulin signaling in C2C12 cell, and induced INS-1 cell apoptosis through TLR4/IKK/ERK pathway to upregulate the expression of BAX/Bcl-2 and caspase 3.

並列關鍵字

PCDM ； insulin resistance ； cell apoptosis ； galectin-3 ； biomarker ； β cell

參考文獻

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