Data Supplement for "Upregulated Phospholipase D Activity Towards Glycosylphosphatidylinositol-Anchored Proteins in Micelle-Like Serum Complexes in Metabolically Deranged Rats and Humans"

Data Supplement for Publication

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.