鼠模式中高脂飲食、肥胖與脂質調控基因之表現

Abstract

過氧化體增殖劑活化受器 (peroxisome proliferators-activated receptor, PPAR)，是固醇核受器的成員。PPAR有α、β、γ三種isoform，其中PPARα與PPARγ分別對脂肪代謝、運輸、生合成、儲存與血糖恆定具有調節作用。脂肪酸是PPARα ligands，脂肪酸的濃度、種類與結構透過PPARα調節脂肪代謝有其不同的影響。本論文研究，主要目的在探討飲食油脂、肥胖、PPAR及脂質代謝相關基因表現之關聯性。 已知18:1 (n-9) 是PPARα ligands，能活化PPARα傳訊途徑。本論文研究採用富含高18:1 (n-9) 的紅花籽油作為實驗用油。第一部份，將含5%、20%或30%紅花籽油實驗飼料 (5S、20S、30S) 分別餵食 Wistar大鼠13週或C57BL/6J小鼠22週。發現此兩種齧齒類對此二種高油脂飲食反應不同：30S使大鼠肥胖，但20S使小鼠肥胖，30S對小鼠則否。高脂飲食雖有增進PPARα下游基因表現之趨勢，但以肥胖鼠 （30S大鼠與20S小鼠） 肝中PPARα與其下游相關基因的表現增高最明顯。推測高脂飲食確實可能因提供較多PPARα ligands，而使 PPARα下游基因表現增加。但造成肥胖後，因PPARα本身的表現也增加，使得整個PPARα傳訊途徑的增加最為明顯。此外，血清leptin濃度反應脂肪量，但不能反應攝食量。而30S造成Wistar大鼠肥胖與20S造成C57BL/6J小鼠肥胖，均同時呈現明顯之血糖與血脂異常現象。 第二部份實驗探討餵食不同飽和度油脂是否會因體內脂肪酸作為PPARα ligands的種類不同，進而影響PPARα傳訊途徑。將含5B (4% 奶油與1%紅花籽油)、30B (29%奶油與1%紅花籽油) 、5S (5%紅花籽油) 與30S (30%紅花籽油) 飼料分別餵食大鼠13週後，可見30B組鼠之葡萄糖耐受度顯著劣於對照組，且血清三酸甘油酯濃度亦顯著較30S組高。此時將30B組隨機分成二組，一組維持原飼料，另一組則更換為30%紅花籽油 (30B/S)，繼續飼養。至30B/S組葡萄糖耐受度顯著改善，即進行動物犧牲，共歷時15週。研究結果顯示不論飽和或不飽和之高油脂飲食均造成鼠肥胖，且伴有肝臟PPARα傳訊途徑表現增加，但此表現增加量在30B組低於30S組，顯示不飽和油脂對PPARα下游基因表現之促進活性比飽和油脂強。有趣的是，在脂肪組織觀察到30S組比30B組有較低的lipoprotein lipase, adipocyte P2、fatty acid synthase、acetyl-CoA carboxylase與sterol regulatory element binding protein -1c的mRNA表現，且腹脂重量亦較低。在肝臟更觀察到30S組比30B組有較高的acyl-CoA oxidase (ACO), carnitine palmitoyl-transferance 1A, fatty acid binding protein (FABP) 及 mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase的mRNA表現。暗示不飽和度高之紅花籽油使鼠脂肪組織中有較低之脂肪生合成與肝臟中有較高的脂肪酸氧化。在30S組的肝臟與脂肪組織中皆發現有顯著較高的18:2 (n-6) 與20:4 (n-6) 含量，約為30B組的2倍。這些結果指出高不飽和油脂飲食抑制體內生脂作用及脂肪堆積或許是與組織中有較高的18:2 (n-6) 與20:4 (n-6) 含量有關。此外，血清leptin濃度與肝臟PPARα mRNA呈正相關。肥胖鼠具有hyperleptinemia特徵，且同時具有顯著較高的肝臟PPARα與其下游基因表現。是否leptin會促進肝臟PPARα之表現? C57BL/6J小鼠經腹腔注射recombinant mouse leptin (1 mg/kg body weight/day)，長期14天與短期1.5小時。腹腔注射leptin會降低小鼠攝食量，因此增加一組對飼育 (pair feeding) 組 (PF組)，控制其攝食量與leptin組相同。leptin投予14天，小鼠肝臟中PPARα、ACO與FABP的mRNA表現顯著較PF組增加 (P < 0.05)。實驗證明leptin濃度調控PPARα並增加其下游基因表現。 本論文建立一個飲食誘發肥胖與胰島素抗性的動物模式且提供了支持性的證據：飲食脂肪酸之質與量，可能透過影響體內脂肪酸組成，而藉由PPAR與SREBP等轉錄因子調控其下游基因的表現，來影響脂質代謝與生合成。另外，肥胖鼠呈現hyperleptinemia及肝臟PPARα與其下游基因表現增加之現象。由於外源性投予leptin也會增加PPARα轉錄因子本身的表現，推測高脂飲食造成肥胖，促使脂肪組織釋出leptin，進而增進肝臟PPARα表現。而高不飽和油脂飲食提供不飽和脂肪酸作為PPARα之ligands，可更有效提高PPARα傳訊途徑，增進脂肪酸氧化，降低血脂、肝脂與腹脂。

Fatty acids are known to regulate lipid metabolism at the level of gene expression. Peroxisome proliferator activated receptors (PPAR) is the first transcription factor identified as a mediator of fatty acid-regulated gene expression. PPARs regulate the metabolism, transportation, and storage of fat and glucose homeostasis. Common fatty acids are ligands of PPARs. High-fat diets provide more fatty acids that presumably could enhance lipid catabolism through an up-regulation of PPARα signaling. However, high intake of fat could also lead to obesity. This study is aimed at examining the mRNA expressions of PPARα and related genes involved in lipid metabolism in high-fat diets feeding and the resulted obesity.The first experiment examined the hepatic mRNA expression of PPARα and some of its target genes in Wistar rats and C57BL/6J mice fed 2 levels (20% or 30% wt/wt, the 20S or 30S diets) of high oleic acid-rich safflower oil (ORSO) diets until animals showed significantly higher body weight (13 wk for rats and 22 wk for mice) than those of control groups fed a 5% ORSO diet. At the end of these respective feeding periods, only the 30% SFO fed rats and the 20% SFO fed mice among the two high fat fed groups showed significantly higher body weight, white adipose tissue weight, serum leptin concentration and the mRNA expression of PPARα (P < 0.05) compared to the respective control group. Despite of the elevated acyl-CoA oxidase (ACO, a PPARα target gene) protein and activity in both of the two high fat fed groups, the mRNA expression level of most PPARα target genes examined correlated mainly to PPARα mRNA levels and not to fat intake or liver lipid levels. The second experiment compared a high ORSO diet and a high butter diet for their effect on adipose mass and expressions of genes regulated by PPAR and SREPB-1c. Four groups of Wistar rats were respectively fed 30S (30% ORSO), 5S (5% ORSO), 30B (29% butter plus 1% ORSO), or 5B (4% butter plus 1% ORSO) diets for 15 weeks. Compared to the 30B group, the 30S group had significantly less retroperitoneal adipose (RWAT) mass and lower mRNA expressions of lipoprotein lipase, adipocyte P2, fatty acid synthase and sterol regulatory element protein-1c (SREBP-1c) in the RWAT, higher mRNA expressions of ACO, carnitine palmitoyl-transferance 1A, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase and fatty acid binding protein (FABP) in the liver (P < 0.05). The 30% fat diets significantly increased mRNA expressions of PPARα and SREBP-1c in the liver, decreased expressions of SREBP-1c in the RWAT (P < 0.05). The linoleic and arachidonic acids content in liver and RWAT lipids of the 30S group was more than 2 fold those of the 30B group (P < 0.05). These results implied that the smaller RWAT mass in rats fed the high ORSO diet might be related to the effect of higher tissue linoleic and arachidonic acids that might increase the expressions of fatty acid catabolic genes through the activation of PPARα in the liver and reduce the expression of lipid storage and lipogenic gene expressions through the suppression of SREBP-1c in the RWAT.The third experiment examines the role of hyperleptinemia in the higher expressions of heaptic PPARα and some of its target genes. Leptin was administrated i.p. to C57BL/6J mice for 14 days at the dose of 1 mg/kg/day-1. A group of mice were pair-fed (the PF group) to the leptin treated mice. The leptin treated mice showed significantly higher mRNA expressions of PPARα, ACO and FABP in the liver relative compared to the PF group (P < 0.05).In conclusion, the roles of lipid-regulated transcription factors, PPARα and SREBP-1c, in the regulation of fat metabolism and storage were demonstrated by comparing the obesitogenesis of a high ORSO diet and a high butter diet. With the accumulation of more unsaturated fatty acid in tissues, the less obesitogenic high ORSO diet resulted in higher expressions of fatty acid catabolic genes through the activation of PPARα in the liver and lower expression of lipid storage and lipogenic gene expressions through the suppression of SREBP-1c in the RWAT. It is also demonstrated that the hyperleptinemia in obesity might further up-regulated the hepatic PPARα mRNA expression.