Association of (c)AMP-Degrading Glycosylphosphatidylinositol-Anchored Proteins with Lipid Droplets Is Induced by Palmitate, H2O2 and the Sulfonylurea Drug, Glimepiride, in Rat Ad

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• 作者：G&uuml ; nter M&uuml ; ller ; Sabine Over ; Susanne Wied ; Wendelin Frick
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：February 5, 2008
• 年：2008
• 卷：47
• 期：5
• 页码：1274 - 1287
• 全文大小：877K
• 年卷期：v.47,no.5(February 5, 2008)
• ISSN：1520-4995

文摘

Inhibition of lipolysis in rat adipocytes by palmitate, H₂O₂ and the antidiabetic sulfonylureadrug, glimepiride, has been demonstrated to rely on the upregulated conversion of cAMP to adenosine byenzymes associated with lipid droplets (LD) rather than on cAMP degradation by the insulin-stimulatedmicrosomal 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)-anchoredphosphodiesterase, Gce1, and the 5'-nucleotidase, CD73, on basis of the following findings: (i) Photoaffinitylabeling 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 andreleased from the LD by chemical or enzymic treatments specifically cleaving GPI anchors, but resistanttoward carbonate extraction. (iii) The cAMP-to-adenosine conversion activity was depleted from the LDby adsorption to (c)AMP-Sepharose. (iv) cAMP-binding to LD was increased upon challenge of theadipocytes 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 withanti-CD73 antibodies. (vi) Inhibition of the bacterial phosphatidylinitosol-specific phospholipase C orGPI 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 bereconstituted into and exchanged between LD in vitro. These data suggest a novel insulin-independentantilipolytic mechanism engaged by palmitate, glimepiride and H₂O₂ in adipocytes which involves theupregulated expression of a GPI-anchored PDE and 5'-nucleotidase at LD. Their concerted action mayensure degradation of cAMP and inactivation of hormone-sensitive lipase in the vicinity of LD.