血清澱粉狀蛋白A對脂質代謝基因產生之調控

Abstract

血清澱粉狀蛋白（serum amyloid A, SAA）為載脂蛋白的一種，在發炎反應時可大量表現，並且具有置換另一個位於高密度脂蛋白（high density lipoprotein, HDL）上的載脂蛋白A1的能力。當以富含二十二碳六烯酸（docosahexaenoic acid, DHA）之飼料餵飼豬隻時，可提高肝臟中SAA的表現。本試驗之目的在於釐清豬隻SAA對於代謝相關基因的調控。首先為了得知SAA所調控的基因為何，以人類HepG2細胞株培養於含有2 μM人類SAA重組蛋白之培養液中72小時，抽取RNA進行微陣列基因晶片分析。試驗發現，SAA會降低許多脂肪酸代謝基因的表現，包括轉錄因子過氧化小體增生活化受體α（peroxisome proliferator-activated receptor α, PPARα, P<0.01）、肝細胞核因子4α（hepatocyte nuclear factor 4α, HNF4α, P<0.01），而固醇調節因子結合蛋白1c（sterol regulatory element binding protein 1c, SREBP1c）則未受影響。脂肪酸合成酶（Fatty acid synthase）、acyl-CoA oxidase及liver type fatty acid binding protein（L-FABP）則會受SAA的抑制。因此推測人類SAA可以藉由抑制過氧小體增生活化受體α的表現而達到調控脂質代謝的目的。 為了瞭解豬隻SAA所扮演的角色，本研究首先證明SAA大量的表現於豬隻的肝臟組織，所以從肝臟cDNA中選殖該基因進行後續研究。序列分析之結果指出豬SAA蛋白質N端具有一段分泌的訊息序列，顯示SAA為一具有外泌能力的蛋白質。而將此基因架接於pQE-31表現載體後，成功產製保有SAA活性的重組蛋白，豬隻的SAA可刺激豬脂肪前身細胞介白素-6（interleukin 6）的表現（P<0.05），此結果符合在人類脂肪前身細胞所觀察到的現象。當以2 μM豬SAA重組蛋白處理脂肪細胞後可抑制PPARα及PPARγ的表現（分別降低40％及60％），SAA亦會增加發炎反應激素基因表現（如介白素-6）及細胞內甘油的釋出，顯示細胞的脂質分解作用增加，同時也發現SAA處理時保護脂肪堆積的蛋白質perilipin的表現受到抑制，而且由於perilipin的抑制會增加內泌素敏感解脂酶（hormone sensitive lipase, HSL）活性，所以推測SAA可藉由降低perilipin的表現而增加脂質分解作用。由於PPARγ的標的基因，adipocyte FABP與脂蛋白解脂酶（lipoprotein lipase）也受到抑制，推測細胞內的脂肪酸攝入會隨之下降。 綜言之， SAA於HepG2細胞株中具有調控脂質代謝基因表現的能力。在豬隻的試驗中，由DHA刺激產生的SAA可能參與抑制轉錄因子PPARs及其下游基因的表現，因此我們推測SAA在DHA調控體內脂質代謝中扮演了重要的角色。

Serum amyloid A protein (SAA) is an apolipoprotein, whose concentration increases during acute inflammation, and can replace apolipoprotein A1 (apoA1), the major apolipoprotein of HDL. Porcine SAA mRNA is increased by dietary docosahexaenoic acid (DHA) treatment. The purpose of this study was to investigate the role of SAA protein in regulating gene expression related to lipid metabolism in pigs. In the first experiment, human cell line, HepG2, was treated with 2 μM SAA for 3 days. Microarray technology was utilized to explore candidate genes regulated by the SAA treatment. The SAA down-regulated several genes involved in fatty acid metabolism, including transcription factors peroxisome proliferator-activated receptor α (PPARα, P<0.01) and hepatocyte nuclear factor 4α (HNF4α, P<0.01). Fatty acid synthase, acyl-CoA oxidase, and liver type fatty acid binding protein were also repressed by the SAA treatment, suggesting that SAA may exert its effects by suppressing the expression of PPARα to modify the expression of aforementioned genes. In order to further extend understandings about the role of SAA in pigs, porcine liver was first demonstrated abundant SAA gene expression. In the next study, porcine SAA gene was cloned from the liver showing that there is a signal peptide sequence, a strong evidence for porcine SAA as a secretory protein. Construction of the recombinant protein expression system in pQE-31 vector to express porcine SAA in E. coli. (M15 strain) was conducted. The addition of porcine SAA recombinant protein (pSAA) in porcine preadipocyte culture stimulated interleukin-6 (IL-6) mRNA expression (P<0.05), indicating a similar biological function of porcine SAA as compared with that in human. Peroxisome proliferator-activated receptor α (PPARα) and PPARγ mRNA was decreased by 40% and 60%, respectively, in differentiated adipocytes when treated with 2 μM pSAA. In addition, the SAA treatment caused a dramatic increase of inflammatory cytokine gene expressions (e.g., IL-6), and glycerol release, indicating an increase of lipolysis. Because the expression of perilipin, a lipid droplet-protective protein, was reduced by the SAA treatment, and the reduced perilipin would increase hormone sensitive lipase (HSL) activity, the increase of lipolysis by the SAA treatment may be mediated by decreasing perilipin. In conclusion, the present study demonstrated that SAA can affect the expression of lipid metabolism related transcription factors in HepG2 cells. In pigs, the DHA-induced SAA gene expression decrease PPAR expression and therefore, down-regulates the target genes expression. Accordingly, SAA may play a critical role in mediating the function of dietary DHA on lipid metabolism.