Protein targeting to glycogen overexpression results in the specific enhancement of glycogen storage in 3T3-L1 adipocytes
Protein targeting to glycogen overexpression results in the specific enhancement of glycogen storage in 3T3-L1 adipocytes
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DOI：
10.1074/jbc.m303846200
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发表时间：
2003-08-15
影响因子：
4.8
通讯作者：
Brady, MJ
中科院分区：
文献类型：
--
作者：
Greenberg, CC;Meredith, KN;Brady, MJ
蛋白磷酸酶-1(PP1)在胰岛素调节糖原合成中起重要作用。针对糖原的蛋白质靶向(PTG)通过增加PP1对抗糖原代谢酶的活性来促进糖原积累。然而，PTG对细胞去磷酸化和葡萄糖代谢的影响的特异性尚不清楚。使用多西环素可控的腺病毒载体在3T3-L1脂肪细胞中过表达PTG，导致PTG水平增加10-20倍，糖原水平增加8倍。在培养液中加入1µg/ml的多西环素可抑制PTG的表达，并完全逆转所有依赖PTG的效应。PTG腺病毒感染3T3-L1脂肪细胞后，糖原合成酶明显去磷酸化和活化。PTG的作用似乎是特定的，因为基础和胰岛素刺激的各种信号蛋白的磷酸化不受影响。在P-32标记的细胞中，糖原合成酶是主要的蛋白质，其磷酸化状态降低。PTG过表达不改变PP1蛋白水平，但在体外使PP1抗磷酸化酶活性增加6倍。相反，使用髓鞘碱性蛋白测量的PP1活性没有变化，这表明PTG的过度表达特异性地引导PP1活性对抗糖原代谢酶。为了研究PTG水平改变对代谢的影响，测定了3T3-L1脂肪细胞对葡萄糖的摄取和储存。PTG过表达不影响基础细胞和胰岛素刺激细胞的2-脱氧葡萄糖转运率，但显著提高两种条件下的糖原合成率。尽管PTG过表达导致细胞葡萄糖流量大幅增加，但基础葡萄糖和胰岛素刺激下的葡萄糖并入脂质中的情况没有变化。综上所述，这些数据表明，PTG在3T3-L1脂肪细胞中的过表达离散地刺激PP1活性，对抗糖原合成酶和磷酸化酶，导致显著和特异性地增加葡萄糖的摄取和作为糖原的储存。
Protein phosphatase-1 (PP1) plays an important role in the regulation of glycogen synthesis by insulin. Protein targeting to glycogen (PTG) enhances glycogen accumulation by increasing PP1 activity against glycogen-metabolizing enzymes. However, the specificity of PTG's effects on cellular dephosphorylation and glucose metabolism is unclear. Overexpression of PTG in 3T3-L1 adipocytes using a doxycycline-controllable adenoviral construct resulted in a 10-20-fold increase in PTG levels and an 8-fold increase in glycogen levels. Inclusion of 1 mug/ml doxycycline in the media suppressed PTG expression, and fully reversed all PTG-dependent effects. Infection of 3T3-L1 adipocytes with the PTG adenovirus caused a marked dephosphorylation and activation of glycogen synthase. The effects of PTG seemed specific, because basal and insulin-stimulated phosphorylation of a variety of signaling proteins was unaffected. Indeed, glycogen synthase was the predominant protein whose phosphorylation state was decreased in P-32-labeled cells. PTG overexpression did not alter PP1 protein levels but increased PP1 activity 6-fold against phosphorylase in vitro. In contrast, there was no change in PP1 activity measured using myelin basic protein, suggesting that PTG overexpression specifically directed PP1 activity against glycogen-metabolizing enzymes. To investigate the metabolic consequences of altering PTG levels, glucose uptake and storage in 3T3-L1 adipocytes was measured. PTG overexpression did not affect 2-deoxy-glucose transport rates in basal and insulin-stimulated cells but dramatically enhanced glycogen synthesis rates under both conditions. Despite the large increases in cellular glucose flux upon PTG overexpression, basal and insulin-stimulated glucose incorporation into lipid were unchanged. Cumulatively, these data indicate that PTG overexpression in 3T3-L1 adipocytes discretely stimulates PP1 activity against glycogen synthase and phosphorylase, resulting in a marked and specific increase in glucose uptake and storage as glycogen.