二型糖尿病是目前常見的代謝疾病之一,主要和胰島素阻抗有關。當外源性或內源性胰島素對細胞的刺激不足時,細胞會無法有效的利用胰島素,造成血糖無法進入細胞被利用而滯留在循環中,此時胰島β細胞會代償性增加胰島素的分泌量以便能提高對循環中糖分的處理,當血糖在此機制下長期都無法完全達到正常血糖控制時,會造成胰島素濃度較正常人為高,也就是耐糖障礙的產生,長期惡性循環之下,便產生胰島素阻抗;此階段可能維持數年之久,直到胰島β細胞出現疲乏,細胞功能逐漸衰退,血糖值也會越來越高,即進入所謂第二型糖尿病階段。在進入第二型糖尿病階段前,動物體內葡萄糖的含量漸高,會產生梅納反應,也就是還原糖的羰基(carbonyl group)和蛋白質上的一級胺基 (primary amino group) 進行非酵素性縮合反應,而產生後期糖化終產物 (advanced glycation end products, AGEs),而N-ε-carboxymethyl-lysine (CML) 則是AGEs中的一種。但是目前AGEs和CML與胰島的代償作用機制尚不清楚,因此本實驗目的在主要是探討葡萄糖及胰島素在高濃度時對胰島β細胞所產生的影響,而在高血糖狀態下大量產生的AGEs,是否也是透過誘發RAGE/P38/MAPK/NF-κB以及IRS2/AKT的訊息途徑,繼而造成胰島肥大的因子之一,最後形成胰島β細胞的功能異常而導致胰島素阻抗的產生。 吾人進行的實驗首先是採用糖尿病之對照組小鼠六隻(三隻公的及三隻母的),;糖尿病實驗組則是採用40-50g、8-12周(2∼3月齡)之db/db小鼠四隻(二隻公的及二隻母的)。觀察小鼠的胰島在經過生理上的代償作用後,發現有胰島肥大的現象發生,吾人接著利用葡萄糖濃度值2.8mM、11.1mM及30mM之培養基模擬血糖之狀態,以及外加2nM之胰島素加入培養基中,模擬胰島β細胞周圍含有高濃度胰島素的情形。培養胰島β細胞株(RIN-m5f)後收取細胞總蛋白質(total protein),並利用西方墨點法(western blot)觀察發現,在IRS2/AKT訊息途徑上主要的幾個磷酸化蛋白包括:P-IRS2、P-PDK、P-AKT、P-P70s6k及P-GSK3α/β,皆會隨著葡萄糖及胰島素而造成磷酸化的反應增加;由此結果推測,葡萄糖及胰島素在胰島β細胞的生理環境中,有可能會透過IRS2/AKT之訊息傳遞路徑繼而影響胰島β細胞的增生作用。另外藉由對照組的五隻母小鼠及實驗組五隻db/db母的小鼠犧牲之後取出胰臟組織,經過包埋及切片過程後,使用AGEs及CML antibody進行免疫組織化學染色的結果顯示,AGEs及CML在胰島中有表現增加的趨勢,同樣的採用CML濃度為0、0.1、1、5、20μg/ml進行western blot分析,也發現CML會增加IRS2/AKT的活化作用,最後利用免疫組織化學染色也發現胰島中IRS2/AKT有增加的趨勢,因此判斷這條代償作用的相關路徑可能是經由IRS2的磷酸化作用,活化PI3K、AKT,並抑制GSK-3β,促進cyclin D1的表現而產生。因此推測AGEs及CML在db/db小鼠中,可能是經由IRS2/AKT訊息路徑會造成GSK-3β在細胞中的活性降低,可能會導致cyclin D1的表現增加,進而增加細胞周期的運作而促進細胞的活性,造成胰島的肥大。
Type 2 diabetes is characterized by hyperglycemia due to insulin resistance in peripheral tissues and deficient insulin secretion by pancreatic islet β-cells. Hyperglycemia also fosters the endogenous nonenzymatic glycoxidation of proteins, lipids, and nucleic acids, and results in the accumulation of heterogeneous molecules known as advanced glycation end products (AGEs). Nε-(carboxymethyl)lysine (CML) were modified proteins have been identified as major AGEs. Compelling evidence implicates this accumulation of AGEs in the pathogenesis of diabetic complications. However, their role in β-cell dysfunction is less clear. It has been found that the compensation involves expansion of β cell mass and enhanced insulin biosynthesis in type 2 diabetes mice. In this study, CML treatment of pancreatic β cell line (RIN-m5f) was not arrest proliferation, and highly expressed phosphor IRS2/phosphor AKT, RAGE, and phosphor GSK-3β/cyclin D1, on the protein level when analyzed by Western blotting. Islets exhibited an increased frequency of these proteins staining, indicating a potential role for β-cell differentiation. Moreover we also found that the expansion of islet mass, increased insulin biosynthesis, and IRS2/AKT, and GSK-3β/cyclin D1 expressions in islets of db/db-diabetic mice. Thus, several adaptive mechanisms account for the compensatory growth of β-cells, a combination of enhanced survival and neogenesis as a potential mediator in these processes. It might be suggested that AGEs have identified a key role for the potential regulatory mechanisms whereby IRS2/AKT signaling promotes enhanced β-cell mass in an animal that develops severe insulin resistance in adulthood.