臺灣博碩士論文加值系統

PART1: 人體攝取過多的脂肪酸會導致代謝症候群的產生，高血壓、高血糖、高血脂、高密度膽固醇過低以及肥胖都是造成心血管疾病的危險因子，在心臟中飽和脂肪酸的攝取量過多時有助於增加心血管疾病，並且導致心臟產生脂毒性。於先前文獻中指出，脂毒性造成心臟能量代謝異常，並且引起心臟收縮功能異常進而導致心臟衰竭。心臟的能量主要是利用脂肪酸(50-70%)和葡萄糖(20-30%)做為能量代謝來源。脂肪酸是透過Cluster of Differentiation 36 (CD36)進到細胞；葡糖糖則是透過Glucose transporter type 4 (GLUT4)進到細胞。正常心肌細胞中能量代謝有胰島素路徑和肌肉收縮路徑，這兩條途徑分別會影響CD36和GLUT4易位到細胞膜上攝取脂肪酸和葡萄糖，並且已有文獻指出，脂肪酸在心肌細胞中的累積會抑制胰島素路徑Akt/CD36脂肪酸代謝路徑。因此本論文希望研究心肌細胞攝取過多棕櫚酸對於CD36和GLUT4間轉換和代謝的關係。研究中，我們採用了老鼠心臟衍生的H9c2肌原母細胞以及新生鼠初代培養的心室細胞，將其給予長時間的棕櫚酸，結果顯示在心肌細胞脂肪酸累積過多形成脂毒性後，細胞膜上CD36蛋白表現量減少，反之GLUT4則是在細胞膜上大量累積，從不同時間點上也顯示能量代謝從六小時開始從脂肪酸轉為使用葡萄糖，並且影響CD36蛋白表現量下降，而不是影響mRNA。另一方面，我們給予細胞高密度脂蛋白模擬運動是否可以抑制棕櫚酸產生的脂毒性，並且能使已產生毒性的心肌細胞從利用葡萄糖轉回使用脂肪酸，恢復正常能量代謝功能。
PART2: 代謝症候群是指代謝層面的心血管危險因子聚集的現象，已有文獻指出，脂肪酸累積過多會導致心肌細胞產生損傷使心臟衰竭，這樣的現象稱為脂毒性。脂毒性會導致心臟能量帶謝的不平衡，而自噬作用存在細胞產生能量危機的時候，在心臟中，自噬作用扮演非常重要的角色，失調時會牽連許多心血管疾病。AMP-activated protein kinase (AMPK)和自噬作用也有密切的關聯，AMPK是一個energy-sensing kinase，在能量不足的時候會被活化，他會調控自噬作用。已有文獻指出，棕櫚酸會造成心肌細胞產生自噬作用，因此本論文研究棕櫚酸是否透過AMPK調控自噬作用，並且和細胞凋亡間的關係為何。研究中，給予心肌細胞長時間的棕櫚酸，結果顯示在心肌細胞脂肪酸累積過多形成脂毒性並活化自噬作用，Puncta實驗中進一步證明棕櫚酸增加自噬體膜上蛋白LC3的聚集，磷酸化AMPK、自噬作用相關蛋白都有高度表現，並且自噬作用受到抑制劑LY294002、3MA，自噬作用和凋亡相關蛋白皆受到抑制，意外的是，再加入溶酶體的抑制劑BaFA1後，凋亡蛋白Cleavage caspase 3卻大量累積，同樣在溶酶體染劑Lysotracker和流式細胞分析實驗中可以得到相同的結果。綜合上述結果發現，長期攝取脂肪酸的心肌細胞產生脂毒性並導致能量代謝失衡，影響下游AMPK活化mTOR訊息路徑負調控，造成粒線體依賴型細胞凋亡。已有文獻指出，高密度脂蛋白透過活化PI3K/AKT/mTOR訊息傳遞路徑抑制自噬作用並且有抗凋亡的功能。研究中，給予細胞不同濃度的高密度脂蛋白，結果顯示高密度脂蛋白可以抑制棕櫚酸產生的自噬作用和粒線體依賴型細胞凋亡，使用高密度脂蛋白的受體SR-B1的抑制劑證實是透過PI3K/AKT/mTOR訊息傳遞路徑抑制自噬作用和粒線體依賴型細胞凋亡。

PART1: Metabolic syndrome is a group of risk factors that increase heart disease and other health problems. The risk factors for development of metabolic syndrome include hyperglycemia, hyperlipidemia, hypertension, low levels of HDL and high levels of triglyceride. Acute toxicity from accumulation of long-chain fatty acids lead to cardiovascular diseases, a phenomenon known as lipotoxicity. Lipoptoxicity leads to imbalance of energy metabolism and result in cardiac dysfunction and contractile dysfunction. Cardiomyocytes use fatty acid (50-70%) and glucose (20-30%) for ATP production. Fatty acids and glucose are uptake by cells through via cluster of differentiation 36 (CD36) and glucose transporter type 4 (GLUT4). In health cardiomyocyte, there are two metabolic pathways, one is muscle contraction pathway and the other one is insulin pathways. They affect CD36 and GLUT4 translocated to cell membrane. In addition, there is convincing evidence that when fatty acids accumulate, the pathway from Akt to CD36 would be inhibited. In this study, we aim to investigate the effect of CD36 and GLUT4 switch and metabolism. In the present study, heart-derived H9c2 cells and neonatal rat ventricular myocytes (NRVMs) were treated with palmitic acid for 24 h. Results showed that CD36 was decreased in cell membrane. Conversely, GLUT4 was accumulated. Palmitic acid also affects energy to switch from CD36 (fatty acid) to GLUT4 (glucose) in a time dependent manner and only affects CD36 protein level but not transcription level. On the other hand, exercise can increase level of high-density lipoprotein (HDL). We want to know whether HDL could inhibit palmitic acid-induced lipoptoxicity and energy switch.
PART2:Metabolic syndrome is a group of risk factors that increase heart disease and other health problems. Recent study showed that acute toxicity from accumulation of long-chain fatty acids lead to cardiovascular diseases and result in heart failure, a phenomenon known as lipotoxicity. Lipoptoxicity leads to imbalance of energy metabolism. Autophagy is critical for cell survival during energy crisis state. Its dysregulation has been implicated in a wide variety of cardiovascular pathologies. AMPK related with autophagy. AMPK is an energy-sensing kinase which was activated by metabolism stress. It can regulate autophagy. Recent study showed that palmitic acid-induced autophagy in H9c2 cell, but mechanism still unclear. Our aim to know whether the palmitic acid-induced autophagy via AMPK regulated and the relationship with apoptosis. The H9c2 cardiomyoblast cells were treated with palmitic acid. The result showed that palmitic acid-induced lipoptoxicity and autophagy, and further, we founded that palmitic acid increased LC3 in autophagosome by puncta assay. In western blot, PT172AMPK and other autophagy-related proteins were increased. After added LY294002 and 3-MA, autophagy and apoptosis-related proteins were inhibited. Specially, after added BaFA1 which was lysosome inhibitor, cleavage caspase 3 was accumulated. The same result showed in lysotracker assay and flow cytometry. Taken together, our present results confirmed that palmitic acid-induced lipotoxicity and result in energy imbalance to activate AMPK, mTOR-indepentent signaling pathway which caused autophagic cell death. Recent study showed that high-density lipoprotein inhibited autophagy via PI3K/Akt/mTOR signaling pathway and had anti-apoptosis. The H9c2 cardiomyoblast cells were treated with different dosage of HDL. The result showed that HDL could inhibit palmitic acid-induced autophagy and autophagic cell death. After combined with BLT-1 which was inhibitor of HDL receptor SR-B1, we proved that HDL inhibited autophagy and autophagic cell death via PI3K/Akt/mTOR signaling pathway.

學位考試委員審定書 I
誌謝 II
Contents V

Part I:
中文摘要 2
Abstract 4
Abbreviation 5
Introduction 7
1. Metabolic Syndrome 7
2. Cardiac energy metabolism pathways 8
3. AMP-activated kinase (AMPK) 10
4. Cluster of differentiation 36 (CD36) 12
5. Glucose transporter type 4 (GLUT4) 13
6. High-density lipoprotein (HDL) 13
Aims 15
Materials and Methods 16
1. Cell culture 16
2. Western blot 16
3. ELISA assay of membrane protein 17
4. Semi quantitative RT-PCR 17
5. Co-immunoprecipitation (Co-IP) 18
6. Lipoprotein separation 19
7. MTT assay 19
8. Primary cardiomyocytsotrlture 19
9. Statistical analysis 20
Results 21
Discussion 24
Reference 27
Figures 35

Part II:
中文摘要 43
Abstract 45
Abbreviation 47
Introduction 49
1. Metabolic Syndrome 49
2. Autophagy 49
3. Role of autophagy in heart 52
4. Autophagic cell death (ACD) 52
5. AMP-activated kinase (AMPK) and autophagy 53
6. High-density lipoprotein (HDL) 55
Aims 57
Materials and Methods 58
1. Cell culture 58
2. Western blot 58
3. Transient-transfected GFP-LC3 and autophagy detection 59
4. Annexin V-FITC/PI staining 59
5. Lysosome staining 60
6. Animals model 60
7. Lipoprotein separation 61
8. Statistical analysis 61
Results 62
Discussion 66
References 68
Figures 76

PART1:
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5.Beeharry, N., J.A. Chambers, and I.C. Green, Fatty acid protection from palmitic acid-induced apoptosis is lost following PI3-kinase inhibition. Apoptosis, 2004. 9(5): p. 599-607.

6.Warensjo, E., et al., Markers of dietary fat quality and fatty acid desaturation as predictors of total and cardiovascular mortality: a population-based prospective study. Am J Clin Nutr, 2008. 88(1): p. 203-9.

7.Katsoulieris, E., et al., alpha-Linolenic acid protects renal cells against palmitic acid lipotoxicity via inhibition of endoplasmic reticulum stress. Eur J Pharmacol, 2009. 623(1-3): p. 107-12.

8.Sparling, P.B., B.A. Franklin, and J.O. Hill, Energy balance: the key to a unified message on diet and physical activity. J Cardiopulm Rehabil Prev, 2013. 33(1): p. 12-5.

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PART2:
1.Kirshenbaum, L.A., Regulation of autophagy in the heart in health and disease. J Cardiovasc Pharmacol, 2012. 60(2): p. 109.

2.Rask-Madsen, C. and C.R. Kahn, Tissue-specific insulin signaling, metabolic syndrome, and cardiovascular disease. Arterioscler Thromb Vasc Biol, 2012. 32(9): p. 2052-9.

3.Zhou, Y.T., et al., Lipotoxic heart disease in obese rats: implications for human obesity. Proc Natl Acad Sci U S A, 2000. 97(4): p. 1784-9.

4.Buchanan, J., et al., Reduced cardiac efficiency and altered substrate metabolism precedes the onset of hyperglycemia and contractile dysfunction in two mouse models of insulin resistance and obesity. Endocrinology, 2005. 146(12): p. 5341-9.

5.Beeharry, N., J.A. Chambers, and I.C. Green, Fatty acid protection from palmitic acid-induced apoptosis is lost following PI3-kinase inhibition. Apoptosis, 2004. 9(5): p. 599-607.

6.Warensjo, E., et al., Markers of dietary fat quality and fatty acid desaturation as predictors of total and cardiovascular mortality: a population-based prospective study. Am J Clin Nutr, 2008. 88(1): p. 203-9.

7.Katsoulieris, E., et al., alpha-Linolenic acid protects renal cells against palmitic acid lipotoxicity via inhibition of endoplasmic reticulum stress. Eur J Pharmacol, 2009. 623(1-3): p. 107-12.

8.Sparling, P.B., B.A. Franklin, and J.O. Hill, Energy balance: the key to a unified message on diet and physical activity. J Cardiopulm Rehabil Prev, 2013. 33(1): p. 12-5.

9.Stanley, W.C. and M.P. Chandler, Energy metabolism in the normal and failing heart: potential for therapeutic interventions. Heart Fail Rev, 2002. 7(2): p. 115-30.

10.Lopaschuk, G.D., et al., Myocardial fatty acid metabolism in health and disease. Physiol Rev, 2010. 90(1): p. 207-58.
11.Goodwin, G.W., C.S. Taylor, and H. Taegtmeyer, Regulation of energy metabolism of the heart during acute increase in heart work. J Biol Chem, 1998. 273(45): p. 29530-9.

12.Cetrullo, S., et al., Antiapoptotic and antiautophagic effects of eicosapentaenoic acid in cardiac myoblasts exposed to palmitic acid. Nutrients, 2012. 4(2): p. 78-90.

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