El-Chaâr D, Gagnon A, Sorisky A
Department of Medicine and Biochemistry, Microbiology & Immunology, University of Ottawa, Ottawa Health Research Institute, Ottawa, Canada K1Y 4E9.
Int J Obes Relat Metab Disord. 2004 Feb;28(2):191-8. doi: 10.1038/sj.ijo.0802554.
Insulin-responsive adipogenic signaling molecules include insulin receptor substrates (IRS)-1 and -2, phosphoinositide 3-kinase (PI3K), and protein kinase B (PKB; also known as Akt). Mammalian target of rapamycin (mTOR) is a PKB substrate, and regulates p70 S6 kinase (p70 S6K). Since p70 S6K is an insulin-responsive kinase downstream of PI3K and PKB, its potential role in adipogenic insulin signaling was investigated.
We measured the effect of rapamycin, a specific inhibitor of mTOR, on insulin-induced 3T3-L1 adipogenesis and on insulin-stimulated p70 S6K activation.
Rapamycin partially reduced differentiation, measured by Oil Red O staining, triacylglycerol accumulation (by up to 46%), and peroxisome proliferator-activated receptor gamma protein expression (by 50%). In contrast, rapamycin completely inhibited insulin-stimulated p70 S6K activation, assessed by phosphorylation of p70 S6K and its substrate, S6. Expression of a constitutively activated form of p70 S6K did not promote 3T3-L1 adipogenesis. The considerable residual differentiation in the presence of rapamycin, despite the complete blockade of p70 S6K activation, prompted us to measure the phosphorylation of another rapamycin-sensitive protein, eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). Insulin-stimulated 4E-BP1 phosphorylation in 3T3-L1 preadipocytes was only partially affected by rapamycin, consistent with the differentiation data. Phosphorylation of eIF4E itself, an expected consequence of 4E-BP1 phosphorylation, was also only partially inhibited.
Our data suggest that adipogenic mTOR signaling occurs via the 4E-BP1/eIF4E pathway, rather than through p70 S6K.
胰岛素反应性脂肪生成信号分子包括胰岛素受体底物（IRS）-1和-2、磷酸肌醇3激酶（PI3K）以及蛋白激酶B（PKB；也称为Akt）。雷帕霉素的哺乳动物靶点（mTOR）是一种PKB底物，并调节p70 S6激酶（p70 S6K）。由于p70 S6K是PI3K和PKB下游的一种胰岛素反应性激酶，因此对其在脂肪生成胰岛素信号传导中的潜在作用进行了研究。
我们测定了mTOR的特异性抑制剂雷帕霉素对胰岛素诱导的3T3-L1脂肪生成以及胰岛素刺激的p70 S6K激活的影响。
通过油红O染色、三酰甘油积累（高达46%）和过氧化物酶体增殖物激活受体γ蛋白表达（50%）来衡量，雷帕霉素部分降低了分化。相反，通过p70 S6K及其底物S6的磷酸化评估，雷帕霉素完全抑制了胰岛素刺激的p70 S6K激活。p70 S6K组成型激活形式的表达并未促进3T3-L1脂肪生成。尽管p70 S6K激活被完全阻断，但在存在雷帕霉素的情况下仍有相当程度的残余分化，这促使我们测定另一种雷帕霉素敏感蛋白真核起始因子4E（eIF4E）结合蛋白1（4E-BP1）的磷酸化。雷帕霉素仅部分影响3T3-L1前脂肪细胞中胰岛素刺激的4E-BP1磷酸化，这与分化数据一致。4E-BP1磷酸化的预期结果即eIF4E本身的磷酸化也仅被部分抑制。
我们的数据表明，脂肪生成的mTOR信号传导是通过4E-BP1/eIF4E途径发生的，而不是通过p70 S6K。
