探討CPR-1基因在第二型糖尿病之治療與應用

Abstract

第二型糖尿病（Type 2 diabetes）為不可逆之慢性代謝疾病，占總糖尿病人口之90%。根據國際糖尿病聯盟統計，全世界至2015年有將近四億人口受到影響。其成因主要來自胰島素抗性（insulin resistance）增加，影響體內葡萄糖、脂質與蛋白質代謝，進而增加體內氧化壓力最終使胰臟細胞衰竭（β-cell failure）與失能。目前治療糖尿病的諸多方式均無法有效保護而遏止胰島細胞的死亡，進而造成不可逆的傷害而使得糖尿病成為無法治癒之疾病。 實驗室先前研究發現，CPR-1基因為蛋白質雙硫異構酶家族成員之一，主要表現於胰臟與胰島細胞，且CPR-1基因剔除在第二型糖尿病模式鼠 ( CPR-1-/- Leprdb/db ) 可保護胰島細胞功能、降低血糖、減少體內活性氧化物含量、增加血液胰島素濃度進而改善糖尿病。甚至使58%的小鼠血糖完全控制在正常不發病的範圍內；而在本篇研究也發現，若給予高脂飼料與極低劑量之常見胰島專一性毒化物STZ處理可誘發CPR-1基因過度表現B6小鼠 ( CPR-1 tg / B6) 產生糖尿病，但同樣處方式卻不會影響控制組血糖表現，證實CPR-1基因確實為第二型糖尿病之重要指標。在另一方面，先前研究同時發現在42%無法有效控制血糖而形成邊緣型糖尿病（borderline diabetes）之CPR-1基因剔除糖尿病鼠中，小鼠血糖趨勢與攝食量及體重具高度相關。於是本篇研究中利用這42%發病小鼠處理熱量限制（CR）、運動訓練（Ex）與Metformin（Met）複合治療，藉由同時保護β細胞與增加週邊組織對胰島素敏感性達到改善糖尿病的效果。實驗結果顯示，持續的CR與階段性Ex和Met處理，可透過活化週邊組織AMPK路徑改善葡萄糖使用效率，降低胰島素抗性，並同時影響胰臟之胰島素分泌，最終翻轉第二型糖尿病。 在機制實驗中發現，CPR-1基因剔除與野生型糖尿病鼠週邊及胰臟磷酸化AMPK的活化表現相反，因此推論CPR-1的有無可能影響AMPK的活化，且證實兩者間存在交互作用。此篇研究結果也提供了新的方向與可行性在未來治療第二型糖尿病的應用上。

Type 2 diabetes (T2D) affects 400 million people in the world, and causes 1.5 million deaths annually. T2D is characterized by a failure of functional β-cells to adapt insulin secretion to compensate for increasing insulin resistance, driving diabetes development. Thus, pancreatic β-cell failure is central to diabetes development. Accumulating data suggest that preserving a portion of functional β-cells can change clinical outcome of diabetes. However, none of current anti-diabetic drugs is clinically effective for this preservation. Therefore, identification of the key players in β-cell dysfunction and death helps understand β-cell pathogenesis and diabetes development and invent new strategies for diabetes treatment. CPR-1, one of the protein disulfide isomerases (PDI), is primarily expressed in pancreata and islets. Our unpublished data showed that 58% of diabetic mice were diabetes-free in CPR-1-/-db/db mice. In this thesis, I found that the treatment with B6 mice with high fat diet (HFD) and STZ induced T2D in B6 mice expressing CPR-1 transgene. In contrast, the same treatment failed to induce T2D in wild-type B6 littermates. In sharp contrast, 42% of CPR-1-/-db/db mice apparently developed borderline T2D. This dichotomy seemed to be relevant to food intake and body weight. This observation prompted me to investigate the effect of calorie restriction, exercise and metformin treatment in CPR-1-/-db/db mice with diabetic onset. These treatments reversed T2D of the CPR-1-/-db/db mice with diabetic onset. AMP-activated protein kinase (AMPK) is a key enzyme of energy metabolism. Generally, activated AMPK stimulates catabolic pathways (glycolysis, fatty acid oxidation and mitochondrial biogenesis) and inhibits anabolic pathways (gluconeogenesis, glycogen, fatty acid and protein synthesis). In this thesis, mechanistic studies demonstrated that the combination therapy increased insulin sensitivity in peripheral tissues and co-regulated insulin secretion in pancreas via AMPK pathway. Furthermore, the results showed that there are some protein-protein interactions between CPR-1 and AMPK, and CPR-1 could be involved in AMPK activity. In conclusion, a combination of CPR1 deletion with metformin treatment, calorie restriction and exercise can reverse T2D through AMPK pathway, making CPR-1 a novel target of T2D therapy.