Jinlida granule inhibits palmitic acid induced-intracellular lipid accumulation and enhances autophagy in NIT-1 pancreatic β cells through AMPK activation
Jinlida granule inhibits palmitic acid induced-intracellular lipid accumulation and enhances autophagy in NIT-1 pancreatic β cells through AMPK activation
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金力达颗粒通过AMPK激活抑制棕榈酸诱导的细胞内脂质积累并增强NIT-1胰腺β细胞自噬
DOI：
10.1016/j.jep.2014.12.005
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发表时间：
2015-02-23
影响因子：
5.4
通讯作者：
Lu, Fuer
中科院分区：
文献类型：
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作者：
Wang, Dingkun;Tian, Min;Lu, Fuer
民族药理学相关性：金利达颗粒是由17味中草药组成的治疗糖尿病的常用中药方剂。然而，这种影响的机制仍不清楚。确定JLDG的主要成分，并通过AMP激活的蛋白激酶（AMPK）信号通路探讨JLDG对Poly acid（PA）诱导的NIT-1胰岛β细胞自噬和脂质蓄积的影响。采用超高效液相色谱（UPLC）指纹图谱法对JLDG颗粒中的主要成分进行了鉴定。用含PA的培养基诱导1细胞。油红O染色法观察细胞内脂滴的变化，比色法测定细胞内甘油三酯（TG）含量。透射电镜下观察自噬体的形成。通过Western blot测定AMPK和磷酸化AMPK（pAMPK）蛋白及其下游脂肪酸代谢相关蛋白（脂肪酸合成酶，FAS;乙酰辅酶A羧化酶，ACC;肉毒碱酰基转移酶1，CPT-1）和自噬相关基因（雷帕霉素哺乳动物靶蛋白，mTOR;结节性硬化症复合物1，TSC 1;微管相关蛋白1轻链3，LC 3-II）的表达。真实的RT-PCR检测固醇调节元件结合蛋白1c（SREBP-1c）mRNA的表达。这种作用与NIT-1细胞中pAMPK和AMPK蛋白表达增加有关。JLDG处理还降低AMPK下游脂肪生成基因（SREBP-1c mRNA、FAS和ACC蛋白）的表达，而增加脂肪酸氧化基因（CPT-1蛋白）的表达。此外，JLDG处理的细胞显示出自噬体数量的显著增加，这伴随着mTOR的下调以及TSC 1和LC 3-II蛋白表达的上调。然而，当AMPK磷酸化被化合物C抑制时，JLDG补充对NIT-1细胞中这些AMPK下游分子的表达没有表现出任何影响。结论：结果表明，JLDG可以减少细胞内脂质积聚，增强PA培养的NIT-1胰腺β细胞的自噬。其作用机制可能与AMPK激活有关。(C)2014作者出版社：Elsevier爱尔兰Ltd.
Ethnopharmacological relevance: Jinlida granule (JLDG), composed of seventeen Chinese medical herbs, is a widely used Chinese herbal prescription for treating diabetes mellitus. However, the mechanism underlying this effect remains unclear. To determine the main components in JLDG and to explore the effect of JLDG on autophagy and lipid accumulation in NIT-1 pancreatic beta cells exposed to politic acid (PA) through AMP activated protein kinase (AMPK) signaling pathway.Materials and methods: JLDG was prepared and the main components contained in the granules were identified by ultra performance liquid chromatography (UPLC) fingerprint Intracellular lipid accumulation in NIT-1 cells was induced by culturing with medium containing PA. Intracellular lipid droplets were observed by Oil Red O staining and triglyceride (TG) content was measured by colorimetric assay. The formation of autophagosomes was observed under transmission electron microscope. The expression of AMPK and phospho-AMPK (pAMPK) proteins as well as its downstream fatty acid metabolism-related proteins (fatty acid synthase, FAS; acetyl-coA carboxylase, ACC; carnitine acyltransferase 1, CPT-1) and autophagy-related genes (mammal target of rapamycin, mTOR; tuberous sclerosis complex 1, TSC1; microtubule-associated protein 1 light chain 3, LC3-II) were determined by Western blot. The expression of sterol regulating element binding protein 1c (SREBP-1c) mRNA was examined by real time PCR (RT-PCR).Results: Our data showed that JLDG could significantly reduce PA-induced intracellular lipid accumulation in NIT-1 pancreatic beta cells. This effect was associated with increased protein expression of pAMPK and AMPK in NIT-1 cells. Treatment with JLDG also decreased the expression of AMPK downstream lipogenic genes (SREBP-1c mRNA, FAS and ACC proteins) whereas increased the expression of fatty acid oxidation gene (CPT-1 protein). Additionally, JLDG-treated cells displayed a markedly increase in the number of autophagosomes which was accompanied by the down-regulation of mTOR and the up-regulation of TSC1 and LC3-II proteins expression. However, when AMPK phosphorylation was inhibited by Compound C, JLDG supplementation did not exhibit any effect on the expression of these AMPK downstream molecules in NIT-1 cells.Conclusions: The results suggest that JLDG could reduce intracellular lipid accumulation and enhance the autophagy in NIT-1 pancreatic beta cells cultured with PA. The mechanism is possibly mediated by AMPK activation. (C) 2014 The Authors. Published by Elsevier Ireland Ltd.