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Background and purpose: Adipocytes release membrane vesicles called adiposomes, which harbor the glycosylphosphatidylinositol-anchored proteins (GPI proteins), Gce1 and CD73, after induction with palmitate, H2O2 and the sulphonylurea drug glimepiride. The role of lipid droplets (LD) in trafficking of GPI proteins from detergent-insoluble, glycolipid-enriched, plasma membrane microdomains (DIGs) to adiposomes in rat adipocytes was studied. Experimental approach: Redistribution of Gce1 and CD73 was followed by pulse-chase and long-term labelling, western blot analysis and activity determinations with subcellular fractions and cell-free systems exposed to palmitate, H2O2 and glimepiride. Key results: In response to these signals, Gce1 and CD73 disappeared from DIGs, then transiently appeared in LD and finally were released into adiposomes from small, and, more efficiently, large adipocytes. From DIGs to LD, Gce1 and CD73 were accompanied by cholesterol. Cholesterol depletion from DIGs or LD caused accumulation at DIGs or accelerated loss from LD and release into adiposomes, respectively, of the GPI proteins. Blockade of translocation of Gce1, CD73, caveolin-1 and perilipin-A from DIGs to LD blocked LD biogenesis and long term-accumulation of LD interfered with induced release of the GPI proteins into adiposomes. GPI protein release was up-regulated upon long term-depletion of LD. Adiposomes were released by a DIGs-based cell-free system, but only in presence of LD. Conclusions: GPI proteins are translocated from DIGs to LD prior to their release into adiposomes, which is regulated by cholesterol, LD content and LD biogenesis. This detour may serve to transfer information about the LD content and to control lipolysis/esterification between large and small adipocytes via GPI protein-harbouring adiposomes.

Background and purpose:In addition to predominant localization at detergent-insoluble, glycolipid-enriched plasma membrane microdomains (DIGs), glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-proteins) have been found associated with lipid droplets (LDs) and adiposomes. Adiposomes are vesicles that are released from adipocytes in response to anti-lipolytic and lipogenic signals, such as H2O2, palmitate and the antidiabetic sulfonylurea drug, glimepiride, and harbour (c)AMPdegrading GPI-proteins, among them the 5-nucleotidase CD73. Here the role of adiposomes in GPI-protein-mediated information transfer was studied. Experimental approach: Adiposomes were incubated with isolated rat adipocytes under various conditions. Trafficking of CD73 and lipid synthesis were analysed. Key results: Upon blockade of GPI-protein trafficking, CD73 specifically associated with DIGs of small, and to a lower degree, large, adipocytes. On reversal of the blockade, CD73 appeared at cytosolic LD in time-adiposome concentration-and signal (H2O2 > glimepiride > palmitate)-dependent fashion. The salt-and carbonate-resistant association of CD73 with structurally intact DIGs and LD was dependent on its intact GPI anchor. Upon incubation with small and to a lower degree, large adipocytes, adiposomes increased lipid synthesis in the absence or presence of H2O2, glimepiride and palmitate and improved the sensitivity toward these signals. Upregulation of lipid synthesis by adiposomes was dependent on the translocation of CD73 with intact GPI anchors from DIGs to LD. Conclusions:The signal-induced transfer of GPI-anchored CD73 from adiposomes via DIGs to LD of adipocytes mediates paracrine upregulation of lipid synthesis within the adipose tissue.

Increased supply of fatty acids to muscle and liver is causally involved in the insulin resistance syndrome. Using a tissue microdialysis technique in Wistar and Zucker fatty (ZF) rats, we determined tissue glycerol levels as a marker of lipolysis in gastrocnemius muscle (gMT), subcutaneous adipose (SAT), and visceral adipose tissue (VAT) as well as the reduction of plasma free fatty acids, glycerol, and triglycerides caused by the antilipolysis-specific adenosine-A1 receptor agonist (ARA). In Wistar and ZF rats, ARA significantly lowered dialysate glycerol levels in SAT, VAT, and gMT. Whereas in SAT and VAT the decrease in dialysate glycerol indicated adipocytic antilipolysis, this decrease in gMT was not caused by a direct effect of ARA on intramyocellular lipolysis, as demonstrated by the lack of inhibition of the protein kinase A activity ratio in gMT. In addition, no differences of the fed-starved-refed dynamics of intramyocellular triglyceride levels compared with untreated controls were measured by in vivo 1 Hspectroscopy, excluding any adenylate cyclase-independent antilipolysis in muscle. Treatment with ARA resulted in pronounced reductions of plasma free fatty acids, glycerol, and triglycerides. Furthermore, in ZF rats, ARA treatment caused an immediate improvement of peripheral insulin sensitivity measured by the euglycemic-hyperinsulinemic glucose clamp technique.

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