植多酚作用於小鼠C2C12骨骼肌細胞中抑制由游離脂肪酸所引起之胰島素抗性機制探討

Suppression of free fatty acid induced insulin resistance by phytopolyphenols in C2C12 mouse skeletal muscle cells

張采文(Tsai-Wen Chang)

指導教授 ： 林仁混

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.03332

摘要

在過去已被報導胞漿中游離脂肪酸的提高與肌肉中所發展出的胰島素抗性有很大的相關性，並且也在肥胖及第二型糖尿病所發展出的胰島素抗性中扮演相當重要的角色。肌肉細胞中的胰島素受體基質-1 (Insulin receptor substrate-1, IRS-1) 被認為是游離脂肪酸作用的目標之一，許多研究已推斷高濃度的游離脂肪酸會活化一些絲胺酸/蘇胺酸激酶，包含蛋白質激酶C (protein kinase C, PKC)、c-jun 胺基端激酶 (c-jun N-terminal kinase, JNK) 及I kappaB激酶 (I kappaB kinase, IKK) 去增加胰島素受體基質-1絲胺酸307位置上的磷酸化，此為胰島素抗性中的一個重要特徵，經由此磷酸化修飾，會導致IRS-1上酪胺酸磷酸化下降，從而降低了其對磷脂酰肌醇3-激酶(phosphatidylinositol-3 kinase, PI3K) 的結合親和力，最後造成葡萄糖運送受到抑制。
表没食子兒茶素没食子酸酯 (epigallocatechin gallate, EGCG) 以及薑黃素 (curcumin) 兩種分別由綠茶及薑黃中萃取出來之植物多酚在過去已被推測具有抗糖尿病及抗肥胖的潛力。兩者皆可防止不正常的葡萄糖代謝，並且藉由去抑制一些細胞周期相關的激酶，包含細胞外信號調節激酶 (extracellular signal-regulated kinase 1/2, ERK1/2) 及p38有絲分裂活化蛋白質 (p38 mitogen activated protein kinase, p38 MAPK) 進而抑制脂肪細胞的增生。此外也可藉由活化 AMP-活化蛋白激酶 (AMP-activated protein kinase, AMPK) 路徑去促進能量的恆定調節。而表兒茶素没食子酸酯 (epicatechin gallate, ECG) 為另一種茶多酚，做過去已被報導其抗氧化及抗發炎的功效與EGCG相當，故推斷其可能具有抗糖尿病的效果。
骨骼肌為由胰島素促進葡萄糖吸收的主要場所，並且參與在能量的恆定調節中，因此，骨骼肌在由游離脂肪酸所引起之胰島素抗性中扮演重要的角色。在過去的報導中指出EGCG及curcumin具有改善肝臟細胞、脂肪細胞及胰臟細胞中胰島素抗性的情形，故肌肉細胞會是相當好的研究對象。
在本篇研究中我們使用了一種PKC的促效劑12-O-十四烷酰佛波醇-13-醋酸酯(12-O-tetradecanoylphorbol 13-acetate, TPA) 以及棕櫚酸去引起C2C12小鼠骨骼肌細胞中的胰島素抗性。實驗結果顯示EGCG及curcumin皆可抑制由TPA所引起之 IRS-1上的絲胺酸307位置上的磷酸化，並且促進Akt的磷酸化。此外我們發現在經過5小時棕櫚酸的培養之下，ECG可以去顯著的提高Akt, ERK1/2, p38 MAPK 以及 AMPK的活化。然而在長時間棕櫚酸的培養之下，IRS-1蛋白表現量明顯降低，但在經由EGCE, ECG 及 curcumin的處理之下可防止其被降解，並且促進Akt, MAPK路徑的活化，改善葡萄糖在肌肉細胞中吸收的情形，特別是curcumin及ECG。此外此三者多酚類亦可抑制乙醯輔酶A羧化酶 (acetyl-CoA carboxylase, ACC)，但僅EGCG可顯著抑制細胞內脂肪的堆積。最後，本篇研究推測相較於其他兩者，curcumin對於改善由游離脂肪酸所引起之胰島素抗性具有最好的效果，而ECG則較EGCG來的好。

關鍵字

植多酚 ； 胰島素抗性

並列摘要

It was reported that increased plasma levels of free fatty acids (FFA) is associated with profound insulin resistance in skeletal muscle and may also play a critical role in the insulin resistance of obesity and type 2 diabetes mellitus. Insulin receptor substrate (IRS), such as IRS-1, has been suggested as a molecular target of FFA. Various studies implicated that high level of FFA activates many serine/ threonine kinases, including PKC, JNK and IKK, and increases IRS-1 serine phosphorylation especially on 307 residue, a possible hallmark of insulin resistance in insulin- responsive cells or tissues. Such modification on IRS-1 leading to decrease IRS-1 tyrosine phosphorylation and PI3K binding affinity results in the stppression of insulin-stimulated glucose transport activity.
EGCG and curcumin, phytopolyphenol compounds derived from green tea and spice turmeric, respectively, have been suggested that they have potential of anti-diabetes and anti-obesity. Both of them prevent abnormal changes in glucose metabolism and induce a decrease phosphorylation on ERK1/2 and p-38 MAPK, two cell cycle control kinases, inhibiting adipocyte proliferation. Besides, which can also activate AMPK cascade promote energy homeostasis. ECG, another tea polyphenol, has been recently reported that was as effective as EGCG on anti-oxidation and anti-inflammation, but the potential of anti-diabetes is unknown.
Skeletal muscle is the major site for insulin-stimulated glucose uptake and involved in energy regulation and homeostasis. Therefore, skeletal muscle plays an important role in FFA-induced insulin resistance. In present studies, EGCG and curcumin have display an improvement on insulin resistance in hepatocyte, adipocyte and pancreatic beta cells. Therefore, skeletal muscle cell will be a good model for investigation.
In this study, we used TPA, a PKC activator, and palmitate to induce insulin resistance in C2C12 mouse skeletal muscle cells. Our data show that EGCG and curcumine treatment reduce IRS-1 Ser307 phosphorylation, and curcumine have more potent to increase Akt phosphorylation in TPA induction. Moreover, we found that after 5-h palmitate incubation, ECG can suppress IRS-1 Ser307 phosphorylation, and significantly promote Akt, ERK1/2, p38 MAPK and AMPK activation. With a longer incubation, IRS-1 exhibited a dramatically depletion, and treatment with EGCE, ECG and curcumin can reverse IRS-1 expression, Akt phosphorylation and MAPK signaling cascade activation, improving glucose uptake in C2C12 skeletal muscle cells, especially ECG and curcumin. Besides, treatment with these polyphenols can suppress ACC activation, but only EGCG could inhibit lipid accumulation in intracellular site. These findings may suggest that curcumin show the best capacity to improve FFA-induced insulin resistance than the other two, and ECG was more effective than EGCG in against insulin resistance.

並列關鍵字

phytopolyphenol ； insulin resistance

參考文獻

1. White MF. 1997 The insulin signaling system and the IRS proteins. Diabetologia. 40(2):S2-S17.

2. Pessin JE, Saltiel AR. 2000 Signaling pathways in insulin action: molecular targets of insulin resistance. J Clin Invest. 106(2):165-169.

3. Bhattacharya S, Dey D, Roy SS. 2007 Molecular mechanism of insulin resistance. J. Biosci. 32(2):405–413.

4. Saltiel AR, Kahn CR. 2001 Insulin signalling and the regulation of glucose and lipid metabolism. Nature. 414(6865):799–806.

5. Loddick SA, Rothwell NJ. 1999 Mechanisms of tumor necrosis factor alpha action on neurodegeneration: interaction with insulin-like growth factor-1. Proc Natl Acad Sci U S A. 96(17):9449–9451.