GLP-1 and GLP-1 analogues protect endothelial function against hyperhomocysteinemia and hyperglycemia insults

Ph.D.Hyperhomocysteinemia and type 2 diabetes mellitus are considered as risk factors of cardiovascular disease, one of the leading courses of mortality worldwide. These two factors are closely associated with the onset, progression, and mortality of cardiovascular complications. Both risk factors are closely related to the excessive endoplasmic reticulum (ER) stress and oxidative stress in endothelial cells, the first-line barrier within the vessel wall along the vascular tree, to initiate endothelial dysfunction, which is the early pathological event predisposing to multiple cardiovascular disorders. Therefore, there is an urgent need to unravel deeper cellular mechanisms of cardiovascular dysfunction in relation to hyperhomocysteinemia and diabetes, and to develop novel and effective therapeutic approaches against these two disease conditions. The present investigation aimed to understand (1) whether glucagon-like peptide-1 (GLP-1) analogues restore hyperhomocysteinemia-associated endothelial dysfunction through suppressing ER stress and reactive oxygen species (ROS) over-generation, (2) whether the GLP-1 analogue attenuates ER stress by protecting and enhancing the protein folding machinery, and (3) whether fecal microbiota transplantation alleviates endothelial dysfunction induced by hyperglycemia in diabetes.The first series of experiments show that both ER stress and ER stress-associated oxidative stress serve as crucial contributors to endothelial dysfunction in hyperhomocysteinemia. Ex vivo organ culture of mouse aortae with homocysteine impairs acetylcholine-induced endothelium-dependent vasorelaxations and induces oxidative stress. Acute treatment of the GLP-1 analogues exendin-4 and semaglutide significantly alleviates homocysteine-induced endothelial dysfunction. Feeding a high methionine low folate diet causes diet-induced hyperhomocysteinemia in male C57BL/6 mice, associated with a slight body weight loss and glycose intolerance. Chronic subcutaneous injection of exendin-4 (40 μg kg-1) every other day significantly improves endothelial function in aortae and mesenteric arteries of diet-induced hyperhomocysteinemia mice. Both ex vivo and in vivo treatments of exendin-4 profoundly suppress ROS over-production and ER stress in hyperhomocysteinemia-impaired aortae. The over-production of ROS is likely caused by the build-up of ER stress in mouse aortae during induction of hyperhomocysteinemia. The presence of AMP-activated protein kinase (AMPK) inhibitor compound C pharmacologically ablates both the ER stress- and oxidative stress-lowering effects, and endothelium-protective benefits of the GLP-1 analogue exendin-4, indicating an important role of AMPK in GLP-1 -mediated vascular benefits against hyperhomocysteinemia.The second set of experiments investigated the role of the ER chaperone endoplasmic reticulum oxidoreductase 1α (ERO1α) in AMPK activation-mediated vascular benefits induced by exendin-4. In vitro and in vivo treatment of exendin-4 activates AMPK which in turn promotes the activation of endothelial nitric oxide synthase (eNOS) to increase NO bioavailability in endothelial cells. In addition, exendin-4 up-regulates the expression of ERO1α in human endothelial cells, which is reversed by the treatment of compound C. The siRNA-mediated knockdown of ERO1α abolishes the ER stress-lowering effect of exendin-4 in homocysteine-treated human endothelial cells. Adenoviral overexpression of ERO1α protects against homocysteine-induced elevation of ER stress and endothelial dysfunction in endothelial cells. Moreover, the ERO1α knockdown and overexpression experiments showed that ERO1α is protective against ER stress-induced oxidative stress in hyperhomocysteinemia.Although the comprehensive underlying mechanisms remain unclear, lifestyle modifications such as regular exercise training brings about metabolic and cardiovascular benefits. However, disabled persons and people with a busy schedule may not enjoy the benefits of physical exercise; the development of exercise mimetics is a needed effort to reproduce the health benefit of exercise. The third group of experiments explored the vascular benefits of fecal microbiota transplantation (FMT) from exercised donors in sedentary diabetic db/db recipients. FMT from exercised mice slightly attenuates weight gain in sedentary diabetic mice. More importantly, FMT improves glucose homeostasis and thermoregulation in sedentary diabetic mice. In terms of vascular benefits, FMT improves acetylcholine-induced endothelium-dependent relaxations in both conduit and resistance arteries of sedentary diabetic mice. However, FMT does not cause lowering of the resting heart rate and resting respiratory rate, which are common benefits of exercise, in recipient mice. Moreover, FMT does not improve the lipid profile of the recipient mice. Interestingly, FMT elevates the level of circulatory native GLP-1 in the recipient diabetic mice, which may account for the improved endothelial function in diabetic mice. These results reveal that FMT from an exercised donor partially confer the metabolic and vascular benefits of exercise in the recipients.To conclude, the current investigation provided several lines of previously unidentified clues in supporting the protective effect of AMPK-dependent ERO1α activation and ER stress attenuation as a therapeutic target to counteract hyperhomocysteinemia-related vasculopathy. The present study also provides new insights into suggesting FMT as another potential strategy to mimic the exercise-induced vascular protection in diabetic patients.心血管疾病是威脅人類健康的世界難題。高同型半胱氨酸血症和2型糖尿病作為心血管疾病的危險因子，與心血管疾病的發生、發展和死亡息息相關。兩種危險子都與血管的內皮細胞的內質網應激和氧化應激的積聚密切相聯，從而誘發內皮功能障礙。而內皮功能障礙是誘發多種心血管疾病的早期起因。因此，揭示高同型半胱氨酸血症和糖尿病導致心血管疾病的細胞機制，發展更有效針對這兩種危險因子的治療方針是目前刻不容緩的課題。本論文主要就以下方面進行探討研究：第一，GLP1類似物是否通過抑制內質網應激和活性氧自由基(ROX)的過度產生以抑制同型半胱氨酸血症引起的內皮功能障礙；第二，GLP-1類似物是否通過增強蛋白質折疊以抑制內質網應激；第三，糞便菌群移植是否可以緩解糖尿病高血糖所引起的內皮功能障礙。論文的第一部分揭示了內質網應激及其相關的氧化應激在高同型半胱氨酸血症中內皮功能障礙的關鍵作用。離體組織培養實驗結果顯示，高半胱氨酸可以顯著地損傷乙酰膽鹼誘導的內皮依賴性血管舒張，而且誘導氧化應激。用GLP-1類似物exendin-4和semaglutide處理小鼠血管，可顯著地減輕高半胱氨酸所引起的內皮功能障礙。高甲硫氨酸低葉酸飲食在C57BL/6小鼠成功誘導高同型半胱氨酸血症，同時伴隨輕微體重減少和糖耐量異常。慢性的exendin-4皮下注射治療，顯著改善飲食誘發的高同型半胱氨酸血症小鼠中的主動脈及小動脈的內皮功能障礙。Exendin-4的離體和體內治療均能顯著抑制高同型半胱氨酸血症在主動脈裡引致的氧化應激及內質網應激。高同型半胱氨酸血症引發的內質網應激促成活性氧自由基的過量產生。AMP活化的蛋白激酶（AMPK)的抑製劑化合物C消除了GLP-1類似物exendin-4對於內皮的保護作用，使內質網應激及氧化應激再次增加。這些結果表明透過激活AMPK，GLP-1類似物可保護血管免受高同型半胱氨酸血症的損害。論文的第二部分研究了內質網分子伴侶內質網氧化還原酶1α(ERO1α)在GLP-1類似物exendin-4誘導的AMPK激活中對血管功能的作用。exendin-4的體外和體內治療可激活AMPK，從而促進內皮型一氧化氮合酶（eNOS）的活化，增加內皮細胞的一氧化氮(NO)的產量。此外，exendin-4上調人類內皮細胞中ERO1α的表達，而AMPK抑製劑化合物C的運用會抵銷ERO1α的上調。在siRNA介導的ERO1α敲低實驗中，exendin-4引發的內質網應激降低作用被抵銷。相反，在由腺病毒感染致使的ERO1α過表達實驗中，成功防止由高半胱氨酸誘導的內質網應激及內皮功能障礙。另外，無論是ERO1α的敲低或過表達實驗均表明ERO1α對於內質網應激引起的氧化應激起到關鍵保護作用。儘管現時對於運動所帶來益處的詳細機制尚有含糊之處，但定期的運動和訓練可起到改善血管及代謝的作用。但是，工作繁忙的人及殘障人士未必能輕易享受到運動帶來的好處。因此，研發運動的替代治療方案或者可以讓這類人士感受到運動帶來的部分益處。論文的第三部分探討了糞便微生物群移植（FMT)能否為欠缺運動的糖尿病病患受眾，帶來改善血管功能的益處。當把有運動習慣的小鼠的糞便微生物群移植至欠缺運動的db/db糖尿病小鼠時，其體重增長、溫度調節及葡萄糖穩態都得到了改善。在血管功能方面，FMT改善了糖尿病小鼠主動脈及腸系膜動脈的內皮功能。但是，FMT起不到降低靜息心率及呼吸率的作用。此外，FMT不能改善糖尿病小鼠的脂質水平。有趣的是，FMT提高了糖尿病小鼠血液循環內GLP-1的水平，這或許解釋了為何糖尿病小鼠的血管功能得到了改善。這些研究結果均表明，FMT可以起到部分運動在代謝和血管方面所帶來的益處。總而言之，論文的研究提供了一系列新穎的證據和機制。本論文提供了基礎證明AMPK的激活上調了ERO1α，從而抑制了內質網應激，起到保護內皮細胞免於高同型半胱氨酸血症損害。另外，本論文也提供了證據證明運動相關的FMT可改善糖尿病患者的血管功能，對糖尿病的治療起到新的啟示。Cheng, Chak Kwong.Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 115-136).Abstracts also in Chinese.Title from PDF title page (viewed on February 10, 2022)