茶鹼衍生物經由調控PPARγ路徑影響 脂肪細胞之分化及脂解作用

Xanthine derivative inhibits the differentiation and increases the lipolysis through PPARγ pathway

張雃庭(Chain-Ting Chang)

指導教授 ： 陳英俊

高雄醫學大學/醫學院/藥理學科研究所/碩士(2014年)

摘要

摘要
　　本研究主要探討茶鹼衍生物 KMUP-1 在3T3-L1前驅纖維母細胞(3T3-L1 pre-adipocyte)分化為脂肪細胞過程中，是否能抑制其油滴脂質累積 (adipogenesis)及促進成熟脂肪細胞的脂解作用 (lipolysis)，並探討此作用在人類脂肪組織 (Human adipose tissue)之基因表現。本研究使用新合成之茶鹼衍生物 KMUP-1，探討其經由eNOS/sGC/ cGMP/PKG (protein kinase G) / HSL及PPAR-γ等路徑影響脂肪組織或細胞之脂肪累積、代謝狀況。
　　此次實驗使用3T3-L1前驅纖維母細胞(3T3-L1 pre-adipocyte)，以insulin(10 ?慊/ml), dexamethasone (0.25 ?嵱), 3-isobutyl-1 methylxanthine (0.5 mM)之細胞分化促進劑(合稱IDM)，給予48小時，使細胞進行分化作用，而後在IDM處理後之第5至16天，加入本茶鹼衍生物KMUP-1培養48hr，觀察油滴脂質累積之情形，並使用油紅染色法(oil red-O stain)觀察細胞內油滴之分佈及大小，接著使用聚合酶連鎖反應(Polymerase chain reaction, PCR)測定基因表現量。
　　另使用人類脂肪組織所分離的纖維母細胞，將其培養密度至106/440mm2，以insulin (10 μg/ml), dexamethasone (0.25 μM), 3-isobutyl-1-methylxanthine (0.5 mM)之細胞分化促進劑(合稱IDM)，給予4天，促進其分化成為脂肪細胞，後在IDM處理後之第10、12天給予本茶鹼衍生物，模擬人體脂肪組織之情況。
　　本茶鹼衍生物之作用，係經過不同時間之細胞培養，再以qPCR分析細胞PPAR-γ、eNOS、GLUT4之基因表現量；在細胞進行脂解作用期間，利用免疫細胞化學(Immunocytochemistry)顯現脂肪細胞中Hormone sensitive lipase (HSL) 之影像，以評估其促進脂解作用的能力。
　　實驗結果顯示，本茶鹼衍生物在不同濃度下會降低3T3-L1脂肪細胞油滴脂質之累積，其在PPAR-γ基因之表現量，也隨藥物濃度之增加而改變；本茶鹼衍生物可增加細胞之Hormone sensitive lipase (HSL)產生，以促進脂解作用。由以上研究結果顯示，本茶鹼衍生物可經由eNOS/PKG (protein kinase G)/HSL及PPAR-γ之作用路徑，減少3T3-L1脂肪細胞油滴脂質之累積，及促進細胞之脂解作用。

關鍵字

脂肪生成 ； 脂解 ； 脂肪細胞 ； 過氧化物酶增生因子活化受體 ； 脂解酶

並列摘要

Abstract
Background:
To investigate whether the xanthine derivative KMUP-1 regulates peroxisome proliferator-activated receptor gamma (PPAR-γ) in adipogenesis and lipolysis of adipocytes, and if so, examines the mechanism of action in 3T3-L1 pre-adipocyte and associated adipocytes to expose the contribution of adipocytes involved in their lipids metabolism.

Materials and Methods:
3T3-L1 pre-adipocytes cell line and adipocytes isolated from fat tissue were used to study adipogenesis and lipolysis. Adipogenesis is stimulated by IDM, a standard hormonal combination of insulin (10 μg/ml), dexamethasone (0.25 μM), and isobutyl-methylxanthine (0.5 mM). Oil-Red O staining was used to quantify the accumulation of lipids in oil droplets of adipocytes. Immunocytochemistry (ICC) technique was employed to examine the hormone sensitive lipase (HSL) protein expression in adipocytes. Expression of HSL was used to determine the stimulation of lipolysis. MTT assay was used to examine the cytotoxicity of adipocytes. Lastly, gene expression of PPARγ and eNOS in adipocytes were measured by real-time PCR.

Results:
In 3T3-L1 pre-adipocytes, the xanthine derivative inhibited IDM-induced PPAR-γ1, PPAR-γ2 in the stage of adipogenesis, but increased PPAR-γ in lipolysis stage. There was a significant HSL protein expression after treated with compound. Enhanced HSL was found in the stage of lipolysis. There was no difference in MTT assay between control and KMUP-1 (1-40 μM). The Oil-Red O staining showed that lipid droplet size of adipocytes were decreased.

Conclusion:
The xanthine derivative KMUP-1 inhibits adipogenesis and induces lipolysis of adipocytes, thus, it could be a potent candidate for body-weight control and obesity-driven inflammation.

並列關鍵字

adipogenesis ； lipolysis ； PPAR ； adipocyte ； 3T3-L1 ； eNOS ； HSL

參考文獻

Chung, H.H., Dai, Z.K., Wu, B.N., Yeh, J.L., Chai, C.Y., Chu, K.S., Liu, C.P., and Chen, I.J. (2010). The xanthine derivative KMUP-1 inhibits models of pulmonary artery hypertension via increased NO and cGMP-dependent inhibition of RhoA/Rho kinase. British journal of pharmacology 160, 971-986.

Liou, S.F., Hsu, J.H., Lin, I.L., Ho, M.L., Hsu, P.C., Chen, L.W., Chen, I.J., and Yeh, J.L. (2013). KMUP-1 suppresses RANKL-induced osteoclastogenesis and prevents ovariectomy-induced bone loss: roles of MAPKs, Akt, NF-kappaB and calcium/calcineurin/NFATc1 pathways. PloS one 8, e69468.

Yeh, J.L., Liu, C.P., Hsu, J.H., Tseng, C.J., Wu, P.J., Wang, Y.Y., Wu, J.R., and Chen, I.J. (2012). KMUP-1 inhibits hypertension-induced left ventricular hypertrophy through regulation of nitric oxide synthases, ERK1/2, and calcineurin. The Kaohsiung journal of medical sciences 28, 567-576.

Davies, G.F., Khandelwal, R.L., Wu, L., Juurlink, B.H., and Roesler, W.J. (2001). Inhibition of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by troglitazone: a peroxisome proliferator-activated receptor-gamma (PPARgamma)-independent, antioxidant-related mechanism. Biochemical pharmacology 62, 1071-1079.

Enerback, S. (2009). The origins of brown adipose tissue. The New England journal of medicine 360, 2021-2023.