二十二碳六烯酸媒介PKA增加血清澱粉狀蛋白A之機制

Docosahexaenoic acid enhances hepatic serum amyloid A expression via protein kinase A-dependent mechanism

戴甄真(Chen-Chen Tai)

指導教授 ： 丁詩同

共同指導教授 ： 李宣書

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10254

摘要

血清澱粉狀蛋白A ( serum amyloid A, SAA) 能減少人類肝細胞和脂肪細胞中脂肪堆積並且SAA在這些細胞的表現會因二十二碳六烯酸 ( docosahexaenoic acid, DHA) 處理而增加。藉由了解DHA是否媒介SAA以及以何種機制來減少脂肪堆積將有助於發展對抗肥胖和脂肪肝的新策略。本試驗先對數種常用的肝癌細胞株進行篩選，結果顯示DHA處理後SAA1在SK-HEP-1表現會增加，在PLC/PRF/5和Hep3B會減少。因為先前發現富含DHA的飼糧會增加豬隻肝臟SAA的表現，所以SK-HEP-1便被用於下列的實驗。SK-HEP-1細胞處理100及200 μM的DHA會增加SAA1和其上游調控因子CCAAT/enhancer binding protein beta ( C/EBPβ ) mRNA的表現。利用啟動子刪除分析法可得知在SAA1啟動子上為於-242 and -102的片段對DHA所調控的SAA1表現很重要，而此片段包含有C/EBPβ結合點。當突變C/EBPβ結合點上的DNA序列使其失去與C/EBPβ結合的能力後，因DHA處理所增加的promoter activity就會受到抑制，這顯示出此C/EBPβ結合點參與DHA對SAA1的調控。本試驗亦發現因DHA處理所增加SAA和C/EBPβ蛋白質表現會因PKA抑制劑 (H89) 的添加而受到抑制，此可顯示C/EBPβ是經由PKA所媒介而參與DHA對SAA1的調控。除此之外對於DHA減少脂肪細胞和肝細胞中脂肪堆積的機制也進行相關研究。DHA處理會增加脂肪細胞甘油的釋出。為了解DHA對脂肪肝的效應，SK-HEP-1在5 mM oleic acid處理2天後，細胞內脂質堆積明顯增加。以100 μM DHA處理這些脂肪肝細胞後，其細胞內的三酸甘油脂會減少。DHA對抑制人類肝細胞和脂肪細胞中脂肪堆積的效應亦會因H89的添加而消失，顯示DHA對脂肪堆積的效應也是由PKA所媒介。綜言之，由於DHA對SAA1的正向調控和脂肪堆積的負向調控皆經由PKA這條路徑，所以可以推論DHA可能是藉由SAA1減少脂肪堆積。這些發現對於了解DHA所調控的脂肪代謝機制提供了新線索，此將有助於DHA在改善人類健康上的應用。

關鍵字

血清澱粉狀蛋白A ； 二十二碳六烯酸 ； C/EBPβ ； 脂解作用 ； 脂肪肝

並列摘要

Serum amyloid A (SAA) reduces fat deposition in adipocytes and hepatocytes. Human SAA1 mRNA is increased by docosahexaenoic acid (DHA) treatment in human cells. These studies asked whether DHA decreases fat deposition through SAA1 and explored the corresponding mechanisms. We demonstrated that DHA increased human SAA1 and C/EBPβ mRNA expression in human hepatoma cells, SK-HEP-1. Utilizing promoter deletion assay, we found that a C/EBPβ binding site in the SAA1 promoter region between -242 and -102 bp is critical for DHA-mediated SAA1 expression. When we mutated the putative C/EBPβ binding site, the DHA-induced SAA1 promoter activity was suppressed, suggesting that this binding sequence was very important for the DHA regulated transcription activation. The addition of the protein kinase A inhibitor, H89, negated the DHA-induced increase in C/EBPβ protein expression. The up-regulation of SAA1 mRNA and protein by DHA was inhibited by H89 treatment, as well. We further demonstrated that DHA increased protein kinase A (PKA) activities. These data suggest that C/EBPβ is involved in the DHA-regulated increase in SAA1 expression via PKA-dependent mechanisms. Furthermore, the suppressive effect of DHA on triacylglycerol accumulation was abolished by H89 treatment in hepatocytes and adipocytes indicating that DHA reduces lipid accumulation via PKA as well. The phenomena of increased SAA1 expression coupled with reduced fat accumulation mediated by DHA via PKA suggest that SAA1 is involved in DHA-induced triacylglycerol breakdown. These findings provide new insights into the complicated regulatory network in DHA-mediated lipid metabolism and may be useful in developing new therapeutic approaches to reduce body fat deposition and fatty liver.

並列關鍵字

C/EBPβ ； DHA ； fatty liver ； SAA

參考文獻

Akashi, S. et al. 2000. Cutting edge: Cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-md-2 complex on mouse peritoneal macrophages. J. Immunol. 164: 3471-3475.

Al-Khalili, L. et al. 2006. Signaling specificity of interleukin-6 action on glucose and lipid metabolism in skeletal muscle. Mol. Endocrinol. 20: 3364-3375.

Andersen, G., K. Harnack, H. F. Erbersdobler, and V. Somoza. 2008. Dietary eicosapentaenoic acid and docosahexaenoic acid are more effective than alpha-linolenic acid in improving insulin sensitivity in rats. Ann. Nutr. Metab. 52: 250-256.

Anthonsen, M. W., L. Ronnstrand, C. Wernstedt, E. Degerman, and C. Holm. 1998. Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro. J. Biol. Chem. 273: 215-221.

Baeuerle, P. A., and T. Henkel. 1994. Function and activation of nf-kappa b in the immune system. Annu. Rev. Immunol. 12: 141-179.