Christian Jung

Induced release of membrane vesicles from rat adipocytes containing glycosylphosphatidylinositol-anchored microdomain and lipid droplet signalling proteins

Synthesis and degradation of lipids in mammalian adipocytes are tightly and coordinatedly regulated by insulin, fatty acids, reactive oxygen species and drugs. Conversely, the lipogenic or lipolytic state of adipocytes is communicated to other tissues by the secretion of soluble adipocytokines. Here we report that insulin, palmitate, H(2)O(2) and the antidiabetic sulfonylurea drug glimepiride induce the release of the typical lipid droplet (LD) protein, perilipin-A, as well as typical plasma membrane microdomain (DIGs) proteins, such as caveolin-1 and the glycosylphosphatidylinositol (GPI)-anchored proteins, Gce1 and CD73 from rat adipocytes. According to biochemical and morphological criteria these LD and GPI-proteins are embedded within two different types of phospholipid-containing membrane vesicles, collectively called adiposomes. Adiposome release was not found to be causally related to cell lysis or apoptosis. The interaction of Gce1 and CD73 with the adiposomes apparently depends on their intact GPI anchor. Pull-down of caveolin-1, perilipin-A and CD73 together with phospholipids (via binding to annexin-V) as well as mutually of caveolin-1 with CD73 or perilipin-A (via coimmunoprecipitation) argues for their colocalization within the same adiposome vesicle. Taken together, certain lipogenic and anti-lipolytic agents induce the specific release of a subset of LD and DIGs proteins, including certain GPI-proteins, in adiposomes from primary rat adipocytes. Given the (c)AMP-degrading activities of Gce1 and CD73 and LD-forming function of perilipin-A and caveolin-1, the physiological relevance of the release of adiposomes from adipocytes may rely on the intercellular transfer of lipogenic and anti-lipolytic information.

Intestinal cholesterol absorption: identification of different binding proteins for cholesterol and cholesterol absorption inhibitors in the enterocyte brush border membrane.

Absorption of cholesterol from the intestine is a central part of body cholesterol homeostasis. The molecular mechanisms of intestinal cholesterol absorption and the proteins mediating membrane transport are not known. We therefore aimed to identify the proteins involved in intestinal cholesterol absorption across the luminal brush border membrane of small intestinal enterocytes. By photoaffinity labeling using photoreactive derivatives of cholesterol and 2-azetidinone cholesterol absorption inhibitors, an 80-kDa and a 145-kDa integral membrane protein were identified as specific binding proteins for cholesterol and cholesterol absorption inhibitors, respectively, in the brush border membrane of small intestinal enterocytes. The 80-kDa cholesterol-binding protein did not interact with cholesterol absorption inhibitors and vice versa; cholesterol or plant sterols did not interfere with the 145-kDa molecular target for cholesterol absorption inhibitors. Both proteins showed an identical tissue distribution and were exclusively found at the anatomical sites of cholesterol absorption-duodenum, jejunum and ileum. Neither stomach, cecum, colon, rectum, kidney, liver nor fat tissue expressed the 80- or 145-kDa binding proteins for cholesterol and cholesterol absorption inhibitors. Both proteins are different from the hitherto described candidate proteins for the intestinal cholesterol transporter,-SR-BI, ABC G5/ABC G8 or ABC A1. Our data strongly suggest that intestinal cholesterol absorption is not facilitated by a single transporter protein but occurs by a complex machinery. Two specific binding proteins for cholesterol (80 kDa) and cholesterol absorption inhibitors (145 kDa) of the enterocyte brush border membrane are probable protein constituents of the mechanism responsible for the intestinal absorption of cholesterol.

Transfer of the glycosylphosphatidylinositol‐anchored 5′‐nucleotidase CD73 from adiposomes into rat adipocytes stimulates lipid synthesis

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)AMP‐degrading GPI‐proteins, among them the 5‐nucleotidase CD73. Here the role of adiposomes in GPI‐protein‐mediated information transfer was studied.

Translocation of Glycosylphosphatidylinositol-Anchored Proteins from Plasma Membrane Microdomains to Lipid Droplets in Rat Adipocytes Is Induced by Palmitate, H2O2, and the Sulfonylurea Drug Glimepiride

Inhibition of lipolysis by palmitate, H2O2, and the antidiabetic sulfonylurea drug, glimepiride, in rat adipocytes has been shown previously to rely on the concerted degradation of cAMP by the glycosylphosphatidylinositol (GPI)-anchored phosphodiesterase Gce1 and 5′-nucleotidase CD73, which both gain access to the lipid droplets (LDs). The present report demonstrates the translocation of Gce1 and CD73, harboring the intact GPI anchor, from detergent-insoluble glycolipid-enriched plasma membrane domains (DIGs) to the LDs in response to palmitate, H2O2, and glimepiride by analysis of their steady-state distribution using photoaffinity labeling and activity determination as well as of their redistribution after pulse or equilibrium metabolic labeling. We were surprised to find that palmitate, H2O2, and glimepiride induced the activation of the GPI-specific phospholipase C (GPI-PLC) at DIGs of rat adipocytes, leading to anchorless Gce1 and CD73. Inhibition of the GPI-PLC or the presence of nonhydrolyzable substrate analogs of Gce1 and CD73 interfered with the palmitate-, H2O2-, and glimepiride-induced 1) lipolytic cleavage of Gce1 and CD73, 2) translocation of their GPI-anchored versions from DIGs to LDs, 3) up-regulation of cAMP degradation, and 4) inhibition of lipolysis. These data suggest a novel insulin-independent antilipolytic mechanism in rat adipocytes, which relies on the palmitate-, H2O2-, and glimepiride-induced and GPI-PLC-dependent translocation of (c)AMP-degrading GPI-anchored proteins from the adipocyte plasma membrane to LDs. The findings may shed new light on the biogenesis and degradation of LDs in response to physiological and pharmacological stimuli.

Hydrogen peroxide-induced translocation of glycolipid-anchored (c)AMP-hydrolases to lipid droplets mediates inhibition of lipolysis in rat adipocytes

The insulin‐independent inhibition of lipolysis by palmitate, the anti‐diabetic sulphonylurea glimepiride and H2O2 in rat adipocytes involves stimulation of the glycosylphosphatidylinositol (GPI)‐specific phospholipase‐C (GPI‐PLC) and subsequent translocation of the GPI‐anchored membrane ectoproteins (GPI‐proteins), Gce1 and cluster of differentiation antigen (CD73), from specialized plasma membrane microdomains (DIGs) to cytosolic lipid droplets (LDs). This results in cAMP degradation at the LD surface and failure to activate hormone‐sensitive lipase. Reactive oxygen species (ROS) may trigger this sequence of events in response to palmitate and glimepiride.

Induced translocation of glycosylphosphatidylinositol-anchored proteins from lipid droplets to adiposomes in rat adipocytes

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.