Peak M, Rochford J J, Borthwick A C, Yeaman S J, Agius L
Department of Medicine, The Medical School, University of Newcastle upon Tyne, UK.
Diabetologia. 1998 Jan;41(1):16-25. doi: 10.1007/s001250050861.
In hepatocytes glycogen storage is stimulated by insulin and this effect of insulin is counteracted by epidermal growth factor (EGF). The mechanism by which insulin stimulates glycogen synthesis in liver is unknown. We investigated the involvement of candidate protein kinases in insulin signalling in hepatocytes. Both insulin and EGF activated extracellular regulated kinase 2 (ERK-2), p70rsk and protein kinase B (PKB) and inactivated glycogen synthase kinase-3 (GSK-3). Whereas EGF caused a greater activation of ERK-2 than insulin, the converse was true for PKB. The stimulation by insulin of ERK-2 was blocked by a mitogen-activated protein (MEK) inhibitor (PD 98059) and of p70rsk by rapamycin. However, these inhibitors, separately or in combination, did not block the stimulation of glycogen synthesis by insulin, indicating that activation of these kinases is not essential for the stimulation of glycogen synthesis by insulin. Mono Q fractionation of hepatocyte extracts resolved a single myelin basic protein (MBP) kinase peak from extracts of EGF-treated cells (peak 1, eluting at 200 mmol/l NaCl) and two peaks from insulin-treated cells (peak 1 eluting at 200 mmol/l NaCl and peak 2 eluting at 400 mmol/l NaCl). In the combined presence of insulin and EGF, activation of peak 2 was abolished. In situ MBP kinase assays and immunoblotting established that peak 1 coincides with ERK-2 and peak 2 is not an activated form of ERK-1 or ERK-2. It is concluded that PKB, which is activated to a greater extent by insulin than EGF, and peak 2, which is activated by insulin and counteracted by EGF, are possible candidates in mediating the stimulation of glycogen synthesis by insulin.
在肝细胞中，胰岛素可刺激糖原储存，而表皮生长因子（EGF）可抵消胰岛素的这种作用。胰岛素刺激肝脏中糖原合成的机制尚不清楚。我们研究了候选蛋白激酶在肝细胞胰岛素信号传导中的作用。胰岛素和EGF均可激活细胞外调节激酶2（ERK-2）、p70rsk和蛋白激酶B（PKB），并使糖原合酶激酶-3（GSK-3）失活。虽然EGF对ERK-2的激活作用比胰岛素更强，但PKB的情况则相反。胰岛素对ERK-2的刺激可被丝裂原活化蛋白（MEK）抑制剂（PD 98059）阻断，对p70rsk的刺激可被雷帕霉素阻断。然而，这些抑制剂单独或联合使用均不能阻断胰岛素对糖原合成的刺激，这表明这些激酶的激活对于胰岛素刺激糖原合成并非必不可少。对肝细胞提取物进行单Q分级分离，从EGF处理细胞的提取物中分离出一个单一的髓鞘碱性蛋白（MBP）激酶峰（峰1，在200 mmol/L NaCl处洗脱），从胰岛素处理细胞的提取物中分离出两个峰（峰1在200 mmol/L NaCl处洗脱，峰2在400 mmol/L NaCl处洗脱）。在胰岛素和EGF共同存在的情况下，峰2的激活被消除。原位MBP激酶测定和免疫印迹表明，峰1与ERK-2一致，峰2不是ERK-1或ERK-2的活化形式。结论是，胰岛素比EGF更能激活的PKB以及被胰岛素激活并被EGF抵消的峰2，可能是介导胰岛素刺激糖原合成的候选因子。
