Upregulated phospholipase D activity toward glycosylphosphatidylinositol-anchored proteins in micelle-like serum complexes in metabolically deranged rats and humans

Abstract

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 that are lower in insulin-resistant/obese rats compared with 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 that were generated by full-length GPI-AP in the 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 toward 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 toward 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.

Interaction of Full-Length Glycosylphosphatidylinositol-Anchored Proteins with Serum Proteins and Their Translocation to Cells In Vitro Depend on the (Pre-)Diabetic State in Rats and Humans

“…Strikingly, GPLD1 activity measurement by chip-based sensing enabled the differentiation of lean Wistar, lean normoinsulinemic normoglycemic ZF, and lean mildly hyperinsulinemic normoglycemic ZDF rats, which are characterized by similar blood glucose in conjunction with no (Wistar, ZF) or only mild elevation of plasma insulin level (ZDF) ( Table 1). These data confirmed the results of a previous study that had been obtained with different animals belonging to the same six categories of genotype and nutritional state [30]. The presence of the Ca 2+ -chelating agent Pha reduced the serum-induced specific phase shift by more than 90% ( Figure S1d), which is compatible with the known Ca 2+ -dependence of GPLD1 [37,38] and the specificity of the assay.…”

“…Chemical synthesis of PIG41, protein determination, preparation of primary rat adipocytes, assay for AChE enzymic activity, preparation of α-toxin from the culture supernatant of Clostridium septicum and bAChE from bovine erythrocytes, coupling of α-toxin to the gold chip surface of long-chain 3D CM-dextran sam ® 5 chips using conventional EDC/NHS-based protocol in a SamX instrument (SAW/Nanotemper, Bonn/Munich, Germany), and SAW measurement, instrumentation and evaluation were performed as has been described in detail previously [28][29][30]50]. Before injection of serum samples into CM-dextran chips, 0.1 vol.…”

“…Full-length GPI-APs, such as bAChE from bovine erythrocytes, reconstituted together with (lyso)phospholipids and cholesterol into micelle-like complexes at an "optimized" constituent ratio, were demonstrated to represent preferred substrates for degradation by serum GPI-PLD activity [30]. So far, in mammalian serum this activity has been attributed to GPLD1 exclusively [51].…”

“…Recently, full-length GPI-APs were detected in the incubation medium of isolated rat adipocyte plasma membranes and cultured rat adipocytes in vitro, as well as in serum of metabolically deranged rats and humans [28] or old rats [29]. Importantly, they were found to be embedded together with (lyso)phospholipids and cholesterol in micelle-like complexes, called micelle-like GPI-AP complexes, rather than in lipid-free aggregates, vesicles, or lipoproteins [30].…”

“…One protective mechanism may rely on the cleavage of the GPI anchor by GPI-specific phospholipase D (GPI-PLD) activity, which in mammalian serum is constituted solely by GPI-PLD1 (GPLD1), according to our current knowledge [36][37][38]. Recent studies revealed that the serum concentration of full-length GPI-APs is correlated to serum GPLD1 activity and blood glucose/plasma insulin levels in diabetic rats and human patients [28,30]. This was interpreted as counterregulation between the release of full-length GPI-APs into the circulation due to metabolic stress and their lipolytic degradation.…”