title: Association of (c)AMP-degrading glycosylphosphatidylinositol-anchored proteins with lipid droplets is induced by palmitate, H2O2 and the sulfonylurea drug, glimepiride, in rat adipocytes.
abstract: Inhibition of lipolysis in rat adipocytes by palmitate, H2O2 and the antidiabetic sulfonylurea drug, glimepiride, has been demonstrated to rely on the upregulated conversion of cAMP to adenosine by enzymes associated with lipid droplets (LD) rather than on cAMP degradation by the insulin-stimulated microsomal phosphodiesterase 3B (Müller, G., Wied, S., Over, S., and Frick, W. (2008) Biochemistry 47, 1259-1273). Here these two enzymes were identified as the glycosylphosphatidylinositol (GPI)-anchored phosphodiesterase, Gce1, and the 5'-nucleotidase, CD73, on basis of the following findings: (i) Photoaffinity labeling with 8-N3-[32P]cAMP and [14C]5'-FSBA of LD from palmitate-, glucose oxidase- and glimepiride-treated, but not insulin-treated and basal, adipocytes led to the identification of 54-kDA cAMP- and 62-kDa AMP-binding proteins. (ii) The amphiphilic proteins were converted into hydrophilic versions and released from the LD by chemical or enzymic treatments specifically cleaving GPI anchors, but resistant toward carbonate extraction. (iii) The cAMP-to-adenosine conversion activity was depleted from the LD by adsorption to (c)AMP-Sepharose. (iv) cAMP-binding to LD was increased upon challenge of the adipocytes with palmitate, glimepiride or glucose oxidase and abrogated by phospholipase C digestion. (v) The 62-kDa AMP-binding protein was labeled with typical GPI anchor constituents and reacted with anti-CD73 antibodies. (vi) Inhibition of the bacterial phosphatidylinitosol-specific phospholipase C or GPI anchor biosynthesis blocked both agent-dependent upregulation and subsequent loss of cAMP-to-adenosine conversion associated with LD and inhibition of lipolysis. (vii) Gce1 and CD73 can be reconstituted into and exchanged between LD in vitro. These data suggest a novel insulin-independent antilipolytic mechanism engaged by palmitate, glimepiride and H2O2 in adipocytes which involves the upregulated expression of a GPI-anchored PDE and 5'-nucleotidase at LD. Their concerted action may ensure degradation of cAMP and inactivation of hormone-sensitive lipase in the vicinity of LD.
authors.affiliation: Sanofi-Aventis Pharma, Therapeutic Department Metabolism, 65926 Frankfurt am Main, Germany. Guenter.Mueller@sanofi-aventis.com
authors.forename: Günter
authors.initials: G
authors.lastname: Müller
authors.forename: Sabine
authors.initials: S
authors.lastname: Over
authors.forename: Susanne
authors.initials: S
authors.lastname: Wied
authors.forename: Wendelin
authors.initials: W
authors.lastname: Frick
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008081:Liposomes / Q000378:metabolism*; D008297:Male; D008954:Models, Biological; D019308:Palmitic Acid / Q000494:pharmacology*; D051381:Rats; D013453:Sulfonylurea Compounds / Q000494:pharmacology*
chemical_list: D002352:Carrier Proteins; D017261:Glycosylphosphatidylinositols; D008081:Liposomes; D013453:Sulfonylurea Compounds; D019308:Palmitic Acid; C057619:glimepiride; D006861:Hydrogen Peroxide; D000242:Cyclic AMP; D015720:5'-Nucleotidase