缺乏ACE2 的小鼠引起葡萄糖耐受性不良和降低蘭氏小島β細胞體積相關性之研究

Abstract

隨著科技發展、經濟成長與人類生活型態改變，肥胖已經成為全世界關注的慢性疾病。研究發現，肥胖會促進腎素-血管收縮素系統(Renin-Angiotensin system)過度活化，導致肥胖相關的慢性疾病產生。腎素-血管收縮素系統(Renin-Angiotensin system)在人體內負責調控體液與血液的恆定。血管收縮素轉化酵素2 (Angiotensin converting enzyme2 (ACE2))是腎素-血管收縮素系統的組成之一，血管收縮素轉化酵素2在腎素-血管收縮素系統內主要的作用為分解血管收縮素II(Angiotensin II)，將血管收縮素II分解成血管收縮素1-7(Angiotensin 1-7)，血管收縮素1-7會和其下游的Mas receptor結合之後誘導一連串的反應發生，例如:抗細胞增生、抗氧化性壓力、促使血管舒張等反應發生。而近年來的研究發現一種新的賀爾蒙，纖維母細胞生長因子21(FGF21)，已經證實FGF21可以調節葡萄糖與脂質的恆定與改善胰島素阻抗的問題。由於過度活化的腎素-血管收縮素系統(Renin-Angiotensin system)會增加葡萄糖耐受性不良(glucose intolerance)與胰島素阻抗(insulin resistance)，因此，我們想要研究缺乏Ace2的小鼠(Ace2-y)是否會引起胰島素阻抗，如果假設成立的話，我們接著會研究FGF21是否在缺乏Ace2的小鼠引起血漿內血管收縮素II濃度增加的過程中扮演著重要的調控角色。 為了想要確認缺乏Ace2的小鼠是否會引起胰島素阻抗，我們將Ace2-/y小鼠與野生型(WT)小鼠分別給予高脂肪飲食(HFD)與一般正常飲食(NC) 4個月，我們發現在高脂肪飲食的組別中發現，Ace2-/y小鼠與野生型(WT)小鼠比較，Ace2-/y小鼠的體重有顯著的減少(31.13±0.73g vs. 42.79±1.6g, p< 0.01)。然而在一般正常飲食的組別中，兩種小鼠的體重是相似的(26.12g±1.65 vs. 26.93±0.17g)。我們比較Ace2-/y和野生型小鼠，發現在Ace2-/y小鼠中脂肪酸合成相關的基因Fatty Acid Synthase (FASN)、Acetyl-CoA carboxylase (ACC2)、Sterol regulatory element-binding protein 1c (SREBP1c)表現減少，而脂肪酸氧化相關的基因Peroxisome proliferator-activated receptor alpha (PPARα),and Carnitine palmitoyltransferase-1 α (CPT1α) 表現有顯著的增加。 有趣的是，雖然Ace2-/y小鼠在高脂肪飲食後的體重較輕，但我們卻發現它有比較嚴重的葡萄糖耐受性不良，此結果與糖質新生作用顯著增加符合。因此， 在高脂肪飲食的組別中，比較Ace2-/y和野生型小鼠，發現Ace2-/y小鼠在高脂肪飲食後肝臟會製造比野生型小鼠還要多的葡萄糖。 由於胰島素在葡萄糖恆定中扮演重要的角色，而我們的實驗結果中發現高脂肪飲食的Ace2-/y小鼠血漿中胰島素的濃度較野生型小鼠低(0.24 ug/L vs.4.87 μg/L p< 0.01)，因此我們認為胰島素在Ace2-/y小鼠中扮演關鍵的角色。 接著我們想要確認是否血管收縮素II在Ace2-/y小鼠中扮演重要的角色，我們使用血管收縮素II第1型接受器的抑制劑(Angiotensin II type I receptor inhibitor) Losartan以及血管收縮素轉化酵素的抑制劑 Imidapril，在高脂肪飲食同時給予Ace2-/y小鼠Losartan或是Imidapril，而實驗的結果中發現，Imidapril顯著的改善Ace2-/y小鼠葡萄糖耐受性不良的問題，但是給予Losartan的Ace2-/y小鼠葡萄糖耐受性不良的問題確沒有明顯的獲得改善。重要的是，我們發現高脂肪飲食的Ace2-/y小鼠注射FGF21一個月之後，葡萄糖耐受性不良的問題獲得改善以及糖質新生作用明顯的減少，因此，這也暗示了FGF21在Ace2-/y小鼠中扮演重要的角色。 接著，為了確認血管收縮素II是否可以調控FGF21，以及探討FGF21在肝臟的表現，我們發現不論是在高脂肪飲食或是一般飲食的Ace2-/y小鼠中，FGF21的表現都比野生型的小鼠少，而在給予Imidapril和FGF21的Ace2-/y小鼠中發現FGF21的表現有回升的現象，但是在給予Losartan的Ace2-/y小鼠小鼠中沒有改變。此實驗結果暗示血管收縮素II可能抑制FGF21的表現以及可能進一步的影響Ace2-/y小鼠的生理表現。 由於insulin由β cell製造，而在Ace2-/y小鼠胰島素不足的原因可能為β cell 變少。另我們驚訝的是我們發現高脂肪飲食的Ace2-/y小鼠的β cell比野生型小鼠還要小，這暗示了缺乏胰島素可能是造成Ace2-/y小鼠產生葡萄糖耐受性不良的主要原因。然而，我們還不能確定胰島素不足是否會造成FGF21表現量降低，因此，FGF21在Ace2-/y小鼠中扮演著重要的角色，並且FGF21可能影響β cell的增生作用。

Obesity has truly become a worldwide problem, affecting countries rich and poor. The Renin-angiotensin system (RAS) is a hormone system that regulates blood pressure and fluid homeostasis. Overactive RAS has been associated with obesity. Angiotensin converting enzyme2 (ACE2) is one of components of RAS. ACE2 can catalyze the conversion of Angiotensin II to Angiotensin (1-7), which in turn binds to Mas receptor, and induces vasodilation, anti-oxidative stress, anti-inflammation, and anti-proliferation. Recently, FGF21, a novel hormone, has demonstrated to regulate glucose and lipid homeostasis and improve insulin resistance. Because overactive RAS has shown to increase glucose intolerance and insulin resistance, we therefore would like to investigate whether Ace2-/y mice cause glucose intolerance. If so, we next investigate whether FGF21 plays an important in the Ace2-/y mice due to increased plasma angiotensin II concentration.

To determine whether Ace2-/y mice cause glucose intolerance, we kept Ace2-/y mice on normal chow (NC) or high fat diet (HFD) for 4 months. We found that Ace2-/y mice had significantly decreased body weight compared to WT fed HFD (31.13±0.73g vs. 42.79±1.6g, p< 0.01), whereas they had the similar body weight treated with normal chow (26.12g±1.65 vs. 26.93±0.17g). We also found that fatty acid synthesis related genes Fatty Acid Synthase (FASN), Acetyl-CoA carboxylase (ACC2), and Sterol regulatory element-binding protein 1c (SREBP1c) were lower in Ace2-/y mice treated HFD, whereas fatty acid oxidation relate genes Peroxisome proliferator-activated receptor alpha (PPARα),and Carnitine palmitoyltransferase-1 α (CPT1α)had significantly increased as compared to WT mice treated HFD.

Interestingly, although Ace2-/y mice treated HFD had a lower body weight, we also observed Ace2-/y mice had glucose intolerance after challenged HFD 4 months. This data was consistent with pyruvate tolerance test (PTT) that was markedly increased in Ace2-/y mice treated HFD. Thus, Ace2-/y mice treated HFD produce more glucose from livers than that of WT treated HFD. Because insulin plays a key role in glucose hemostasis, we found that insulin levels of Ace2-/y mice treated HFD had lower than those of WT treated HFD (0.24 ug/L vs.4.87 μg/L p< 0.01). Thus, insulin may play a crucial key in the Ace2-/y mice phenotype. Moreover, the triglyceride (64.66±7.44 mg/dl vs. 94.71±2.02 mg/dl p<0.01, 69.90±6.5mg/dl p<0.01) was lower in Ace2-/y mice treated with NC or HFD.

Next, we determine whether angiotensin II effects on the Ace2-/y mice. We used losartan, angtensin II type 1 inhibitor, and imidapril, ACE inhibitor, for four months The imidapril significantly improved glucose intolerance and insulin levels, whereas losartan did not rescue phenotype of Ace2-/y mice. Importantly, we found that Ace2-/y mice with HFD treated FGF21 for one month also improved glucose intolerance and decreased PTT, suggesting FGF21 plays an important role in the phenotype of Ace2-/y mice. To determine whether FGF21 is regulated by angiotensin II, we also investigate FGF21 in liver which is predominately expression of FGF21. We observed that Ace2-/y mice had a lower intensity in NC and HFD as compared to WT with NC and HFD, while Ace2-/y mice treated with imidapril and FGF21 restored FGF21 levels, but not losartan. This data implicate angiotensin II may inhibit FGF21 expression and then affect the phenotype of Ace2-/y mice.

Since β cell is produce insulin and insufficient insulin levels of Ace2-/y mice may due to lower β cell mass, we also investigate this. Surprise to us, we found that Ace2-/y mice treated HFD had lower β cell mass than WT mice treated HFD, suggesting that glucose intolerance in Ace2-/y mice is causative of insulin deficiency. However, it is not sure whatever lower insulin levels cause decreased FGF21 expression. Thus, role of FGF21 may play an important role in the phenotype of Ace2-/y mice and may also involve in β cell proliferation.