Müller G, Wied S, Crecelius A, Kessler A, Eckel J
Hoechst AG, Hoechst Marion Roussel, Frankfurt am Main, Germany.
Endocrinology. 1997 Aug;138(8):3459-75. doi: 10.1210/endo.138.8.5308.
Polar headgroups of free glycosyl-phosphatidylinositol (GPI) lipids or protein-bound GPI membrane anchors have been shown to exhibit insulin-mimetic activity in different cell types. However, elucidation of the molecular mode of action of these phospho-inositolglycan (PIG) molecules has been hampered by 1) lack of knowledge of their exact structure; 2) variable action profiles; and 3) rather modest effects. In the present study, these problems were circumvented by preparation of PIG-peptides (PIG-P) in sufficient quantity by sequential proteolytic (V8 protease) and lipolytic (phosphatidylinositol-specific phospholipase C) cleavage of the GPI-anchored plasma membrane protein, Gce1p, from the yeast Saccharomyces cerevisiae. The structure of the resulting PIG-P, NH2-Tyr-Cys-Asn-ethanolamine-PO4-6(Man1-2)Man1-2Man1-+ ++6Man1-4GlcNH(2)1-6myo-inositol-1,2-cyclicPO4, was revealed by amino acid analysis and Dionex exchange chromatography of fragments generated enzymatically or chemically from the neutral glycan core and is in accordance with the known consensus structures of yeast GPI anchors. PIG-P stimulated glucose transport and lipogenesis in normal, desensitized and receptor-depleted isolated rat adipocytes, increased glycerol-3-phosphate acyltransferase activity and translocation of the glucose transporter isoform 4, and inhibited isoproterenol-induced lipolysis and protein kinase A activation in adipocytes. Furthermore, PIG-P was found to stimulate glucose transport in isolated rat cardiomyocytes and glycogenesis and glycogen synthase in isolated rat diaphragms. The concentration-dependent effects of the PIG-P reached 70-90% of the maximal insulin activity with EC50-values of 0.5-5 microM. Chemical or enzymic cleavages within the glycan or peptide portion of the PIG-P led to decrease or loss of activity. The data demonstrate that PIG-P exhibits a potent insulin-mimetic activity which covers a broad spectrum of metabolic insulin actions on glucose transport and metabolism.
游离糖基磷脂酰肌醇（GPI）脂质或与蛋白质结合的GPI膜锚定物的极性头部基团已被证明在不同细胞类型中表现出胰岛素模拟活性。然而，这些磷酸肌醇聚糖（PIG）分子的分子作用模式的阐明受到以下因素的阻碍：1）对其确切结构缺乏了解；2）作用谱可变；3）效应相当有限。在本研究中，通过对酿酒酵母的GPI锚定质膜蛋白Gce1p进行连续的蛋白水解（V8蛋白酶）和脂解（磷脂酰肌醇特异性磷脂酶C）切割，制备了足够量的PIG肽（PIG-P），从而规避了这些问题。通过对从中性聚糖核心酶促或化学产生的片段进行氨基酸分析和戴安离子交换色谱，揭示了所得PIG-P的结构，即NH2-Tyr-Cys-Asn-乙醇胺-PO4-6(Man1-2)Man1-2Man1-+ ++6Man1-4GlcNH(2)1-6肌醇-1,2-环磷酸，这与酵母GPI锚定物的已知共有结构一致。PIG-P刺激正常、脱敏和受体缺失的分离大鼠脂肪细胞中的葡萄糖转运和脂肪生成增加甘油-3-磷酸酰基转移酶活性和葡萄糖转运蛋白异构体4的转位，并抑制异丙肾上腺素诱导的脂肪分解和脂肪细胞中的蛋白激酶A激活。此外，发现PIG-P刺激分离的大鼠心肌细胞中的葡萄糖转运以及分离的大鼠膈肌中的糖原生成和糖原合酶。PIG-P的浓度依赖性效应达到最大胰岛素活性的70-90%，EC50值为0.5-5 microM。PIG-P的聚糖或肽部分内的化学或酶促切割导致活性降低或丧失。数据表明，PIG-P表现出强大的胰岛素模拟活性，涵盖了胰岛素对葡萄糖转运和代谢的广泛代谢作用。
