詹東榮臺灣大學:獸醫學研究所蔡洵洵Tsai, Hsun-HsunHsun-HsunTsai2010-05-042018-07-092010-05-042018-07-092008U0001-2507200816451500http://ntur.lib.ntu.edu.tw//handle/246246/178914肥胖已成為世界普遍的一種新興文明病，而導致肥胖的根本機制乃起於能量的不平衡，此種能量的不平衡會使過多的脂肪和葡萄糖出現在循環中並且以三酸甘油酯的型式積存在脂肪細胞中，脂肪滴中過多的三酸甘油酯會導致脂肪細胞的肥大和脂肪細胞功能的喪失，間接導致高血糖症和高胰島素血症及引發第二型糖尿病，而第二型糖尿病患者則常存在著高腎上腺皮質素血症的情形。瘦體素是脂肪細胞所分泌的細胞激素之一，已知在體質指數(body mass index, BMI)愈高者其血中的瘦體素(leptin)值愈高，且有研究發現瘦體素會抑制人類腎上腺皮質癌細胞株(NCI-H295)的皮質醇分泌和抑制其細胞內與類固醇生成相關的蛋白質表現。在流行病學的研究上發現，有高腰臀比的人對於adrenocorticotropin hormone (ACTH)或corticotrophin releasing hormone (CRH)的刺激會有較低的血清皮質醇；而在另一研究中，經過dexamethasone抑制試驗後，發現肥胖組的男性(BMI>25)其血中皮質醇濃度較正常男性為低，但兩組血中的ACTH濃度卻沒有差異性，這些研究暗示著肥胖者的腎上腺皮質功能可能受到損害。 本次研究主要想藉由in vitro及in vivo兩方面，探討肥胖所引起的血中高瘦體素血症和高血糖症是否會對腎上腺皮質功能造成影響，已有研究顯示槲黃素(quercetin)能抑制和瘦體素細胞訊息傳遞相關的蛋白質激酶之表現。而屬於peroxisome-proliferator activated receptor gamma (PPARγ)致效劑的降血糖藥物rosiglitazonein已被廣泛應用在治療第二型糖尿病，因此本研究也想藉由餵食槲黃素和rosiglitazonein來了解究竟肥胖對於腎上腺皮質功能的影響是由高瘦體素血症或是高血糖症所媒介。研究結果顯示，在in vitro我們發現瘦體素和高濃度的葡萄糖皆能抑制腎上腺細胞的基礎分泌和類固醇生成相關的蛋白質表現，而加入槲黃素後，則能對抗因瘦體素引起的類固醇生成之抑制現象。在in vivo方面，我們發現肥胖組小鼠的腎上腺皮質對於ACTH刺激的反應性不佳，在尿中基礎的皮質酮分泌上，長期追蹤下我們也發現肥胖組小鼠尿中皮質酮量低於對照組小鼠，惟隨著對照組小鼠年齡增加而下降的皮質酮量可能會將兩組的差異性弭平。將小鼠餵食槲黃素14日後，在ACTH刺激試驗下，肥胖組小鼠血中皮質酮濃度的反應性和StAR蛋白質的表現皆比餵食生理鹽水的肥胖組小鼠強；餵食槲黃素30日後也能增加肥胖組小鼠尿中皮質酮量的分泌，顯示槲黃素能改善肥胖組小鼠腎上腺皮質對於ACTH的反應性；在餵食降血糖藥rosiglitazone的實驗中，結果顯示餵食之後肥胖組小鼠的高血糖確實得到改善，而其對腎上腺皮質功能的影響上，發現餵食rosiglitazone後能提升肥胖組小鼠尿中基礎的皮質酮量，惟因後續的ACTH刺激試驗的失敗和StAR蛋白質表現上沒有變化性而無法判斷餵食rosiglitazone後肥胖組小鼠對於腎上腺皮質接受ACTH刺激的反應性是否有所影響。 最後將in vitro和in vivo的各項實驗做一統整，我們由此研究得知瘦體素和高葡萄糖濃度在in vitro的層次上的確會抑制腎上腺細胞類固醇性內泌素的分泌和相關蛋白質的表現；在in vivo的層次上，我們發現肥胖也會對小鼠的腎上腺皮質功能造成影響，但究竟是體內高濃度的瘦體素亦或是高血糖所導致，這個答案需要更多後續且重複性的實驗來釐清才能有更進一步的了解。Obesity is regarded as a kind of chronic metabolic disease and recently the prevalence of obesity is rapidly increasing in the development countries. Obesity is caused by energy imbalance that may lead to storage of excess fat in the form of triglyceride in adipocytes; and make those cells become hyperplasia and dysfunctional. The concequences of dysfunction in adipocytes include hyperglycemia which leads to diabetes mellitus and elevated production of adipokines such as leptin. Serum leptin levels are direct proportion to the body mass index (BMI) in human beings. Several studies have been presented that serum cortisol levels are lower in people with high waist-to-hip ratio (WHR) after adrenocorticotropin (ACTH) or corticotrophin releasing hormone (CRH) stimulation test compared with those with normal WHR. These studies imply that obesity may influence the function of adrenal cortex. Some studies observed that leptin was able to decrease the secretion of steroid hormones and inhibit the expressions of steroidogenic proteins in NCI-H295 cells. Recently, it was observed that diabetic patients have elevated serum/urine cortisol levels and the activities of their hypothalamo-pituitary-adrenocortical axis are out of control. This study is going to detect the effect of obesity on the adrenal cortex and the effects of the key factors associated with obesity, leptin and glucose on regulation of steroidogenesis.uercetin is a kind of flavnoid and has been found playing as a broad protein kinase inhibitors such as JAK2 or PI3K which are involved in leptin signal transduction pathway. Rosiglitazone, a kind of PPARγ agonist is used as an antidiabetic drug recently in human beings. We fed mice with quercetin to study if it could influence the adrenal cortex function in obese mice by blocking the signal transduction pathway of leptin and fed mice with rosiglitazone to study if it could influence the adrenal cortex function in obese mice by normalizing their blood glucose levels.he results of our in vitro studies showed that leptin and high-level glucose decreased the secretion of steroid hormones and depressed the expression of steroidogenic proteins, both StAR and P450scc in adrenal cells, but quercetin could reverse these effects of leptin on steroidgenesis in cells. Our in vivo studies indicated that after ACTH stimulation test the serum corticosterone levels of obese mice were lower than which of normal-weighted ones. Besides this, the basal corticosterone levels during 24 hours in urine of obese mice were lower than normal-weighted ones, although which of normal-weighted mice were decreased by age. Feeding mice with quercetin for 14 days or for 30 days, using ACTH stimulating tests, we found that corticosterone levels and the expression of steroidogenic protein, StAR in obese mice were increased by quercetin. After feeding mice with rosiglitazone for 30 days, the corticosterone levels in urine of obese ones were elevated.n conclusions, this study demonstrated that the secretion of steroid hormones and the expression of steroidogenic proteins, both StAR and P450scc were inhibited by leptin and high-level of glucose in adrenal cells and the function of adrenal cortex in obese mice induced with high-fat-diet was impaired. However, the mechanisms of the effects of hyperleptinemia and hyperglycemia on causing dysfunction of adrenal cortex in obese mice still remains unclear and requires more detailed studies.摘要……………………………………………………………………Ibstract………………………………………………………………III目錄…………………………………………………………………XI目錄…………………………………………………………………XVII一章、文獻探討……………………………………............01一節、肥胖…………………………………………………………01、肥胖的定義………………………………………………………01、脂肪細胞功能異常…………………………………………....02、代謝性症候群……………………………………………………03、糖尿病……………………………………………………………04、糖尿病診斷標準…………………………………………………05二節、腎上腺結構和生理功能……………………………………06三節、下視丘-腦垂腺-腎上腺軸( H-P-A axis)……………… 07四節、類固醇內泌素生成的途徑…………………………………07五節、類固醇內泌素生成的分子機制……………………………09六節、肥胖和腎上腺皮質功能……………………………………10七節、瘦體素對於腎上腺皮質功能的影響………………………11八節、糖尿病對於腎上腺皮質功能的影響………………………12九節、槲黃素…………………………………………………… 13十節、Peroxisome proliferator activated receptors,PPARs14二章、實驗材料與方法………………………………………… 17一節 樣本收集…………………………………………………… 17-1、血清樣本收集和保存………………………………………… 17-2、尿液樣本收集和保存………………………………………… 17二節、實驗方法………………………………………………… 17-1、細胞培養……………………………………………………… 17-2、細胞加藥處理及後續培養液的收集和保存………………… 18-3、酵素連結免疫吸附分析法( Enzyme-linked immunosorbent assay, ELISA)………………………………… 19-4、西方墨點法 (Western blotting)………………………… 23-5、小鼠腎上腺初代培養細胞製備……………………………… 24-6 、MTT 分析…………………………………………………… 25-7、小鼠空腹血糖及葡萄糖耐受性試驗………………………… 25-8、小鼠血清中瘦體素濃度測試………………………………… 25-9、小鼠尿液中24小時內之皮質酮基礎量測量………………… 26-10、小鼠促腎上腺皮質素(ACTH)刺激試驗…………………… 26-11、小鼠ACTH刺激試驗後，以西方墨點法分析腎上腺細胞內類固 醇生成蛋白StAR的表現量………………………………… 26三節、化學藥品………………………………………………… 27四節、統計……………………………………………………… 27三章、實驗設計與結果………………………………………… 28驗一、瘦體素對小鼠腎上腺皮質癌細胞株 (Y1 cells)的影響 28-1、ACTH對Y1細胞分泌助孕酮的刺激作用…………………… 28-2、瘦體素受體 (OBRb)在Y1細胞的表現……………………… 30-3、Y1細胞因ACTH刺激而增加的助孕酮分泌可被瘦體素所抑制 ………………………………………………………………… 33 -4、瘦體素可抑制因ACTH刺激而增加的Y1細胞內類固醇生成蛋 白 StAR和 P450scc的蛋白質表現………………………… 35驗二、 槲黃素對腎上腺皮質功能的影響……………………… 39-1、霍亂毒素 (cholera toxin)對人類腎上腺皮質癌細胞株 (NCI- H295)的分泌皮醇的刺激作用……………………………… 39-2、NCI-H295細胞於不同濃度槲黃素下的MTT分析……………. 41-3、NCI-H295細胞被瘦體素所抑制的皮質醇分泌可被槲黃素所提升 ……………………………………………………………………43-4、NCI-H295細胞被瘦體素抑制的類固醇生成蛋白StAR和P450scc 的表現量可因槲黃素而增加…………………………………46驗三、瘦體素與槲黃素對小鼠腎上腺初代培養細胞(adrenal primary culture cells)的影響………………………………………51-1、小鼠腎上腺初代培養細胞對ACTH刺激的反應性……………51-2、槲黃素對小鼠腎上腺初代培養細胞的影響…………………54-3、小鼠腎上腺初代培養細胞被瘦體素抑制的助孕酮和皮質酮分泌 可被槲黃素所提升…………………………………………57驗四、高濃度葡萄糖對腎上腺皮質功能的影響………………62-1、NCI-H295細胞在高濃度葡萄糖下的MTT分析………………62-2、於高濃度葡萄糖下，NCI-H295細胞的皮質醇分泌會受到抑制 ……………………………………………………………… 64-3、小鼠腎上腺初代培養細胞於高濃度葡萄糖下，其基礎助孕酮和 皮質酮分泌會受到抑制……………………………………67-4 、NCI-H295細胞於高濃度葡萄糖下，細胞的類固醇生成蛋白StAR 和P450scc的表現會受到抑制…………………………… 71驗五、Rosiglitazone對腎上腺皮質功能的影響…………… 76-1、NCI-H295細胞在不同濃度rosiglitazone下的MTT分析… 76-2 、Rosiglitazone可增加NCI-H295細胞的皮質醇分泌…… 78-3、Rosiglitazone可增加小鼠腎上腺初代培養細胞的皮質酮分泌..80驗六、肥胖小鼠模式建立………………………………………82驗七、A組小鼠任飼非常高脂肪飼糧或對照飼糧後的腎上腺皮質功 能、 空腹血糖、葡萄糖耐受性試驗…………………… 87-1 、A組小鼠任飼非常高脂肪飼糧或對照飼糧25週後進行ACTH 刺激試驗，肥胖組小鼠的腎上腺皮質對於ACTH刺激後所反 應的血清中皮質酮濃度不如對照組小鼠…………………87-2、 A組小鼠任飼非常高脂肪飼糧或對照飼糧36週後，再餵食槲 黃素14天之空腹血糖、葡萄糖耐受性試驗………………90-3 、A組小鼠於任飼非常高脂肪飼糧或對照飼糧第42週之瘦體素 濃度及腎上腺皮質功能……………………………………95-4 、A組小鼠於任飼非常高脂肪飼糧或對照飼糧第42週，餵食槲 黃素14日後之瘦體素濃度及腎上腺皮質功能……………99驗八、B組小鼠任飼高脂肪飼糧或對照飼糧連續過程過程中於不同 週數分析24小時尿中之皮質酮量及任飼第36週餵食槲黃素30 日後的血清中瘦體素濃度、空腹血糖、葡萄糖耐受性試驗、腎腺皮質功能………………………………………………….. 113-1 、B組小鼠任飼高脂肪飼糧或對照飼糧的連續過程中於不同週 數，24小時內尿中之皮質酮基礎量………………………113-2 、B組小鼠於任飼高脂肪飼糧或對照飼糧的第36週，血清中的 瘦體素濃度、空腹血糖、葡萄糖耐受性試驗、腎上腺皮質功能 ………………………………………………………………117-3、B組小鼠於任飼高脂肪飼糧或對照飼糧的第36週，餵食槲黃素 30日後，血清中的瘦體素濃度、空腹血糖、葡萄糖耐受性試驗 和腎上腺皮質功能…………………………………………126驗九、C組小鼠於任飼高脂肪飼糧或對照飼糧第25週，餵食槲黃 素或rosiglitazone 30日後的血清中瘦體素濃度、空腹血糖、 葡萄糖耐受性試驗、腎上腺皮質功能……………………142-1、C組小鼠任飼高脂肪飼糧或對照飼糧連續過程中的空腹血糖測 試……………………………………………………………142-2、C組小鼠於任飼高脂肪飼糧或對照飼糧第25週，測試血清中瘦 體素濃度、空腹血糖、葡萄糖耐受性試驗、腎上腺皮質功145-3、C組小鼠任飼高脂肪飼糧或對照飼糧第25週，餵食槲黃或 rosiglitazone 30日後的血清中瘦體素濃度、空腹血糖、葡萄糖耐 受性試驗、腎上腺皮質功能………………………………154驗十、-Leprdb/-Leprdb小鼠(肥胖組)和C57BL/6 (對照組)在28週齡 時連續餵食rosiglitazone 30日後的血清中瘦體素濃度、空腹血 糖和腎上腺皮質功能………………………………………1790-1 -Leprdb/-Leprdb小鼠 (db)和C57BL/6 (B6)在10週齡時，ACTH刺 激試驗後顯示db小鼠已開始出現高腎上腺皮質素血症的現象 ………………………………………………………………1790-2 -Leprdb/-Leprdb小鼠 (db)和C57BL/6 (B6)在28週齡時血清中的瘦 體素濃度、空腹血糖和腎上腺皮質功能…………………1820-3 -Leprdb/-Leprdb小鼠(db)和C57BL/6 (B6)在28週齡時餵食rosiglitazone 30日 後血清中的瘦體素濃度、空腹血糖和腎上腺皮質能……188四章、討論………………………………………………………204考文獻……………………………………………………………215錄一………………………………………………………………223錄二………………………………………………………………226錄三………………………………………………………………228錄四………………………………………………………………229application/pdf2300894 bytesapplication/pdfen-US肥胖瘦體素高血糖腎上腺皮質功能槲黃素obesityleptinhyperglycemiaadrenocortical functionquercetin[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178914/1/ntu-97-R95629020-1.pdf