丙戊酸(Valproic acid)造成肝臟細胞脂質堆積機制探討

Valproic acid induces lipid accumulation in hepatocytes by regulating lipid transportation

洪立盈(Li-Ying Hung)

指導教授 ： 邱智賢

共同指導教授 ： 陳億乘(Chen-Yi Chen)

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(2013年)

https://doi.org/10.6342/NTU.2013.01167

摘要

Valproic acid (VPA) 是現今臨床治療癲癇的處方用藥，為人工合成之支鏈脂肪酸，使用在臨床治療已長達35年以上。除了用於治療癲癇外，VPA在臨床上的使用非常廣泛，例如治療躁鬱症、預防偏頭痛、穩定情緒、治療神經退化相關疾病如阿茲海默症等，且因其為組織蛋白去乙醯酶抑制劑，可能具有抗癌潛力。此外，相較於其他抗癲癇藥物，VPA對病患而言耐受度較高、副作用較少，種種優點使得VPA在臨床上具有相當的優勢。然而，其仍具有副作用如導致病患體重增加、肝臟脂質浸潤 (hepatic steatosis) 及致死性肝衰竭 (fatal liver injury) 等。根據臨床統計指出，約有六成病患服用VPA而造成hepatic steatosis，然而其機制仍未完全明瞭。因此，本研究的目的為探討VPA 可能造成肝臟脂質浸潤的機制。
本研究使用小鼠肝臟細胞株FL83B細胞，以in vitro模式探討VPA造成脂質堆積的可能機制。以real-time PCR進行基因表現量分析結果顯示，VPA與18碳單鍵不飽和脂肪酸 (oleic acid, OA) 共處理組之Perilipin2 (為合成油滴必須之表面蛋白) 表現量增加。脂肪酸轉位酶Cd36在VPA單處理及共處理組別中表現量均增加，顯示此基因可能受到VPA調控。此外，合成三酸甘油脂 (triacylglycerol，TAG) 最後步驟所需之限制速率酵素Dgat2, 表現量也在VPA處理下顯著提升。而與脂肪酸合成相關酵素Accα 及 Fasn, 在VPA處理組的表現量則受到抑制。顯示VPA可能是透過增加胞外攝入的脂肪酸含量，促使其合成TAG，並增加Perilipin2表現，使油滴大量堆積在細胞質中。儘管調控CD36表現的轉錄因子脂小體增生活化受體γ (peroxisome proliferator-activated receptor γ, PPARγ) 基因及蛋白質表現量變化發現並無顯著差異；但螢光免疫染色結果觀察到其表現位置大幅轉移至細胞核中，可能暗示其被活化後進入細胞核中而造成下游基因被轉錄。CD36表現量在共處理及VPA單處理的情況下均有所增加，而部分蛋白質則從細胞質內轉至細胞膜上，推測其可能增加細胞膜上的脂肪酸運輸。
綜上所述，VPA增加肝臟細胞脂質堆積的可能機制為透過PPARγ轉移至細胞核內，造成下游CD36基因表現量增加，並進而造成脂肪酸攝入量大幅提升；同時藉由Dgat2表現量增加使得TAG產量增加，致使油滴大量堆積在肝臟細胞質內。然而，其中詳細的調控機制，仍須更進一步試驗來釐清。

關鍵字

丙戊酸 ； 脂肪酸轉位酶脂小體增生活化受體γ ； 肝臟脂質浸潤 ； 脂質運輸

並列摘要

Valproic acid (VPA) is an antiepilepsy drug with broadest spectrum used in all types of epileptic seizures for more than 40 years in human medicine. It’s also commonly prescribed to treat bipolar disorder, neuropathic pain, migraine headache, and central nervous system (CNS) diseases. Although VPA is successfully applied to clinics for decades, it has been reported that VPA treatment also cause some adverse effects such as weight gain, hepatic steatosis and fatal liver injury. About 60 % of patients who received VPA monotherapy gained hepatic steatosis with higher BMI values. In this study, we aimed to explore the possible mechanisms of VPA-induced hepatic steatosis.
To figure out the possible mechanisms of VPA-induced hepatic steatosis, mouse hepatocyte FL83B cell line was used to study the mechanism of VPA-induced hepatic steatosis in vitro. As the results of real-time PCR, VPA treatment increased the expression of Perilipin2, which encodes a lipid droplet protein. VPA also significantly enhanced Fat/Cd36 which transports fatty-acid across the cell membrane; moreover, it elevates Dgat2, an enzyme that catalyzes the final step of triacylglycerol synthesis. Meanwhile, genes related to fatty-acid synthesis such as Accα and Fasn, were both down-regulated compared to OA-treatment which may imply that the fatty-acid synthesis does not contribute to VPA-enhanced hepatic steatosis. Since CD36 is a direct target of peroxisome proliferator-activated receptor gamma (PPARγ), a transcriptional factor that regulates lipogenesis, protein expressions of CD36 and PPARγ were analyzed by flow cytometry. Consequently, protein expression of CD36 was enhanced by VPA; whereas both mRNA and protein expression of PPARγ were unchanged. However, immunocytochemistry was used to evaluate the protein location of PPARγ. The results showed that PPARγ translocated from cytoplasm to nucleus in OA, VPA and co-treatment group. Additionally, CD36 translocated from cytoplasm to membrane in OA and co-treatment group.
In summary, VPA may enhance lipid accumulation in hepatocytes through increasing fatty-acid uptake by enhancing PPARγ translocation and CD36 expression; simultaneously, DGAT2 was elevated to increase triacylglycerol synthesis. However, further studies need to perform to elucidate the mechanisms of VPA-induced hepatic steatosis in detail.

並列關鍵字

valproic acid ； CD36 ； PPARγ ； hepatic steatosis ； lipid transportation

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