PLD_GAM_Suppl_Mat_revised.docx

Glycosylphosphatidylinositol-anchored proteins (GPI-AP) with the complete glycolipid anchor attached have previously been shown to be released from the outer plasma membrane leaflet of rat adipocytes in positive correlation to cell size and blood glucose/insulin levels of the donor rats. Furthermore, they are present in rat and human serum, however, at amounts which are lower in insulin-resistant/obese rats compared to normal ones. These findings prompted further evaluation of the potential of full-length GPI-AP for the prediction and stratification of metabolically deranged states. A comparison of the signatures of horizontal surface acoustic waves which were generated by full-length GPI-AP in course of their specific capture by and subsequent dissociation from a chip-based sensor between those from rat serum and those reconstituted into lipidic structures strongly argues for expression of full-length GPI-AP in serum in micelle-like complexes in concert with phospholipids, lysophospholipids and cholesterol. Both the reconstituted and the rat serum complexes were highly sensitive towards mechanical forces, such as vibration. Furthermore, full-length GPI-AP reconstituted into micelle-like complexes represented efficient substrates for cleavage by serum glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD). These findings raised the possibility that the upregulated release of full-length GPI-AP into micelle-like serum complexes from metabolically deranged cells is compensated by elevated GPI-PLD activity. In fact, serum GPI-PLD activity towards full-length GPI-AP in micelle-like complexes, but not in detergent micelles, was positively correlated to early states of insulin resistance and obesity in genetic and diet-induced rat models as well as to the body weight in humans. Moreover, the differences in the degradation of GPI-AP in micelle-like complexes were found to rely in part on the interaction of serum GPI-PLD with an activating serum factor. These data suggest, that serum GPI-PLD activity measured with GPI-AP in micelle-like complexes is indicative of enhanced release of full-length GPI-AP from relevant tissues into the circulation as a consequence of early metabolic derangement in rats and humans.

糖基磷脂酰肌醇锚定蛋白（Glycosylphosphatidylinositol-anchored proteins, GPI-AP）在完整结合糖脂锚定的状态下，此前已被证实可从大鼠脂肪细胞的质膜外小叶释放，且释放量与供体大鼠的细胞体积、血糖及胰岛素水平呈正相关。此类蛋白同时存在于大鼠与人类血清中，但在胰岛素抵抗/肥胖大鼠体内的含量低于正常对照个体。上述发现推动了针对全长GPI-AP用于预测和分层代谢紊乱状态的潜力开展深入评估。通过对比大鼠血清来源的全长GPI-AP与重组入脂质结构的全长GPI-AP，在被芯片传感器特异性捕获及后续解离过程中产生的水平表面声波特征，结果强烈支持血清中全长GPI-AP以胶束样复合物形式存在，并与磷脂、溶血磷脂及胆固醇共同组装。无论是重组获得的复合物还是大鼠血清来源的复合物，均对振动等机械力表现出高度敏感性。此外，重组入胶束样复合物的全长GPI-AP，可作为血清糖基磷脂酰肌醇特异性磷脂酶D（Glycosylphosphatidylinositol-specific phospholipase D, GPI-PLD）进行切割反应的高效底物。上述发现提出一种可能性：当代谢紊乱细胞向循环中释放的全长GPI-AP进入胶束样血清复合物的水平上调时，该过程可通过升高的GPI-PLD活性得到代偿。实际检测结果证实，以胶束样复合物（而非去垢剂胶束）中的全长GPI-AP为底物测得的血清GPI-PLD活性，与遗传型及饮食诱导的胰岛素抵抗和肥胖大鼠模型的早期病程呈正相关，同时与人类受试者的体重亦呈正相关。此外，血清中GPI-AP在胶束样复合物中的降解差异，部分源于血清GPI-PLD与一种激活型血清因子的相互作用。综上，本研究数据表明，以胶束样复合物中的GPI-AP为底物检测得到的血清GPI-PLD活性，可作为大鼠与人类早期代谢紊乱发生时，相关组织向循环系统释放全长GPI-AP水平升高的指示指标。