Inactivation of lipoprotein lipase in 3T3-L1 adipocytes by angiopoietin-like protein 4 requires that both proteins have reached the cell surface.
Lipoprotein lipase (LPL) and angiopoietin-like protein 4 (Angptl4) were studied in 3T3-L1 adipocytes. Transfections of the adipocytes with Angptl4 esiRNA caused reduction of the expression of Angptl4 to about one fourth of that in cells treated with vehicle only. This resulted in higher levels of LPL activity both on cell surfaces (heparin-releasable) and in the medium, while LPL activity within the cells remained unaffected. This demonstrated that even though both proteins are made in the same cell, Angptl4 does not inactivate LPL during intracellular transport. Most of the Angptl4 protein was present as covalent dimers and tetramers on cell surfaces, while within the cells there were only monomers. LPL gradually lost activity when incubated in medium, but there was no marked difference between conditioned medium from normal cells (rich in Angptl4) and medium after knockdown of Angptl4. Hence Angptl4 did not markedly accelerate inactivation of LPL in the medium. Experiments with combinations of different cells and media indicated that inactivation of LPL occurred on the surfaces of cells producing Angptl4.
Department of Medical Biosciences, Physiological Chemistry, Umeå University, SE-901 87 Umeå, Sweden.
Elena
E
Makoveichuk
Evelina
E
Vorrsjö
Thomas
T
Olivecrona
Gunilla
G
Olivecrona
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D000075803:Angiopoietin-Like Protein 4; D042682:Angiopoietins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D016895:Culture Media, Serum-Free / Q000378:metabolism; D055785:Gene Knockdown Techniques; D008071:Lipoprotein Lipase / Q000378:metabolism*; D051379:Mice; D055503:Protein Multimerization; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
D000075803:Angiopoietin-Like Protein 4; D042682:Angiopoietins; C407088:Angptl4 protein, mouse; D016895:Culture Media, Serum-Free; D034741:RNA, Small Interfering; D008071:Lipoprotein Lipase
10mM phosphate, 0.15M NaCl, pH 7.4; 3-Isobutyl-1-methylxantine; 3T3-L1; Adipocytes; Angiopoietin-like protein 4; Angptl4; BSA; DEX; DMEM; Dulbecco’s modified Eagle’s medium; HI-FCS; Heparin-releasable; LPD; LPL; Lipoprotein lipase; MIX; PBS; RNAi; angiopoietin-like protein 4; bovine serum albumin; dexamethasone; endoribonuclease-prepared siRNA pools comprised of a heterogeneous mixture of siRNAs that all target the same mRNA sequence; esiRNA; heat-inactivated fetal calf serum; lipoprotein lipase; lipoprotein-deficient