Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), which are anchored at the surface of mammalian cultured and tissue cells through a carboxy-terminal GPI glycolipid, are susceptible to release into incubation medium and (rat and human) blood, respectively, in response to metabolic stress and ageing. Those GPI-APs with the complete GPI still attached form micelle-like complexes together with (lyso)phospholipids and cholesterol and are prone to degradation by serum GPI-specific phospholipase D (GPLD1), as well as translocation to the surface of acceptor cells in vitro. In this study, the interaction of GPI-APs with GPLD1 or other serum proteins derived from metabolically deranged rat and humans and their translocation were measured by microfluidic chip-and surface acoustic wave-based sensing of micelle-like complexes reconstituted with model GPI-APs. The effect of GPI-AP translocation on the integrity of the acceptor cell surface was studied as lactate dehydrogenase release. For both rats and humans, the dependence of serum GPLD1 activity on the hyperglycemic/hyperinsulinemic state was found to be primarily based on upregulation of the interaction of GPLD1 with micelle-like GPI-AP complexes, rather than on its amount. In addition to GPLD1, other serum proteins were found to interact with the GPI phosphoinositolglycan of full-length GPI-APs. Upon incubation of rat adipocytes with full-length GPI-APs, their translocation from the micelle-like complexes (and also with lower efficacy from reconstituted high-density lipoproteins and liposomes) to acceptor cells was observed, accompanied by upregulation of their lysis. Both GPI-AP translocation and adipocyte lysis became reduced in the presence of serum proteins, including (inhibited) GPLD1. The reduction was higher with serum from hyperglycemic/hyperinsulinemic rats and diabetic humans compared to healthy ones. These findings suggest that the deleterious effects of full-length GPI-APs following spontaneous release into the circulation of metabolically deranged rats and humans are counterbalanced by upregulated interaction of their GPI anchor with GPLD1 and other serum proteins. Thereby, translocation of GPI-APs to blood and tissue cells and their lysis are prevented. The identification of GPI-APs and serum proteins interacting within micelle-like complexes may facilitate the prediction and stratification of diseases that are associated with impaired cell-surface anchorage of GPI-APs, such as obesity and diabetes.