Angiopoietin-like 4 promotes intracellular degradation of lipoprotein lipase in adipocytes
2016; Elsevier BV; Volume: 57; Issue: 9 Linguagem: Inglês
10.1194/jlr.m067363
ISSN1539-7262
AutoresWieneke Dijk, Anne P. Beigneux, Mikael Larsson, André Bensadoun, Stephen G. Young, Sander Kersten,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoLPL hydrolyzes triglycerides in triglyceride-rich lipoproteins along the capillaries of heart, skeletal muscle, and adipose tissue. The activity of LPL is repressed by angiopoietin-like 4 (ANGPTL4) but the underlying mechanisms have not been fully elucidated. Our objective was to study the cellular location and mechanism for LPL inhibition by ANGPTL4. We performed studies in transfected cells, ex vivo studies, and in vivo studies with Angptl4−/− mice. Cotransfection of CHO pgsA-745 cells with ANGPTL4 and LPL reduced intracellular LPL protein levels, suggesting that ANGPTL4 promotes LPL degradation. This conclusion was supported by studies of primary adipocytes and adipose tissue explants from wild-type and Angptl4−/− mice. Absence of ANGPTL4 resulted in accumulation of the mature-glycosylated form of LPL and increased secretion of LPL. Blocking endoplasmic reticulum (ER)-Golgi transport abolished differences in LPL abundance between wild-type and Angptl4−/− adipocytes, suggesting that ANGPTL4 acts upon LPL after LPL processing in the ER. Finally, physiological changes in adipose tissue ANGPTL4 expression during fasting and cold resulted in inverse changes in the amount of mature-glycosylated LPL in wild-type mice, but not Angptl4−/− mice. We conclude that ANGPTL4 promotes loss of intracellular LPL by stimulating LPL degradation after LPL processing in the ER. LPL hydrolyzes triglycerides in triglyceride-rich lipoproteins along the capillaries of heart, skeletal muscle, and adipose tissue. The activity of LPL is repressed by angiopoietin-like 4 (ANGPTL4) but the underlying mechanisms have not been fully elucidated. Our objective was to study the cellular location and mechanism for LPL inhibition by ANGPTL4. We performed studies in transfected cells, ex vivo studies, and in vivo studies with Angptl4−/− mice. Cotransfection of CHO pgsA-745 cells with ANGPTL4 and LPL reduced intracellular LPL protein levels, suggesting that ANGPTL4 promotes LPL degradation. This conclusion was supported by studies of primary adipocytes and adipose tissue explants from wild-type and Angptl4−/− mice. Absence of ANGPTL4 resulted in accumulation of the mature-glycosylated form of LPL and increased secretion of LPL. Blocking endoplasmic reticulum (ER)-Golgi transport abolished differences in LPL abundance between wild-type and Angptl4−/− adipocytes, suggesting that ANGPTL4 acts upon LPL after LPL processing in the ER. Finally, physiological changes in adipose tissue ANGPTL4 expression during fasting and cold resulted in inverse changes in the amount of mature-glycosylated LPL in wild-type mice, but not Angptl4−/− mice. We conclude that ANGPTL4 promotes loss of intracellular LPL by stimulating LPL degradation after LPL processing in the ER. ERRATUMJournal of Lipid ResearchVol. 57Issue 10PreviewANGPTL4 lowers the amount of LPL on the adipocyte cell surface. A: Western blot of cell lysates of gWAT explants from Angptl4−/− and wild-type mice incubated in the absence or presence of heparin (50 IU/ml) for 3 h. Western blots were probed with antibodies against LPL and HSP90 (as loading control). B: Western blot of cell lysates of adipocytes that had been differentiated from stromal vascular fractions of WAT from Angptl4−/− and wild-type mice and incubated in the absence or presence of heparin (10 IU/ml) for 20 min. Full-Text PDF Open Access Circulating triglyceride-rich lipoproteins, such as chylomicrons and VLDLs, supply tissues with lipid nutrients for storage or oxidation. Hydrolysis of circulating triglycerides is mediated by LPL, a glycoprotein found in multiple tissues, including adipose tissue, brain, heart, and skeletal muscle (1.Kersten S. Physiological regulation of lipoprotein lipase.Biochim. Biophys. Acta. 2014; 1841: 919-933Crossref PubMed Scopus (342) Google Scholar, 2.Goldberg I.J. Eckel R.H. Abumrad N. Regulation of fatty acid uptake into tissues: lipoprotein lipase- and CD36-mediated pathways.J. Lipid Res. 2009; 50 (Suppl): S86-S90Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 3.Wang H. Eckel R.H. Lipoprotein lipase in the brain and nervous system.Annu. Rev. Nutr. 2012; 32: 147-160Crossref PubMed Scopus (63) Google Scholar, 4.Young S.G. Zechner R. Biochemistry and pathophysiology of intravascular and intracellular lipolysis.Genes Dev. 2013; 27: 459-484Crossref PubMed Scopus (243) Google Scholar). LPL is produced by parenchymal cells and then transported to the luminal surface of capillaries by an endothelial cell protein, glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) (5.Davies B.S.J. Beigneux A.P. Barnes R.H. Tu Y. Gin P. Weinstein M.M. Nobumori C. Nyrén R. Goldberg I. Olivecrona G. et al.GPIHBP1 is responsible for the entry of lipoprotein lipase into capillaries.Cell Metab. 2010; 12: 42-52Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). A critical step in the maturation of LPL is asparagine-linked glycosylation (6.Ben-Zeev O. Doolittle M.H. Davis R.C. Elovson J. Schotz M.C. Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment.J. Biol. Chem. 1992; 267: 6219-6227Abstract Full Text PDF PubMed Google Scholar, 7.Simsolo R.B. Ong J.M. Kern P.A. Characterization of lipoprotein lipase activity, secretion, and degradation at different sites of post-translational processing in primary cultures of rat adipocytes.J. Lipid Res. 1992; 33: 1777-1784Abstract Full Text PDF PubMed Google Scholar, 8.Semenkovich C.F. Luo C.C. Nakanishi M.K. Chen S.H. Smith L.C. Chan L. In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion.J. Biol. Chem. 1990; 265: 5429-5433Abstract Full Text PDF PubMed Google Scholar). LPL is glycosylated within the endoplasmic reticulum (ER) via cotranslational transfer of oligosaccharide chains high in mannose residues (6.Ben-Zeev O. Doolittle M.H. Davis R.C. Elovson J. Schotz M.C. Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment.J. Biol. Chem. 1992; 267: 6219-6227Abstract Full Text PDF PubMed Google Scholar, 7.Simsolo R.B. Ong J.M. Kern P.A. Characterization of lipoprotein lipase activity, secretion, and degradation at different sites of post-translational processing in primary cultures of rat adipocytes.J. Lipid Res. 1992; 33: 1777-1784Abstract Full Text PDF PubMed Google Scholar, 8.Semenkovich C.F. Luo C.C. Nakanishi M.K. Chen S.H. Smith L.C. Chan L. In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion.J. Biol. Chem. 1990; 265: 5429-5433Abstract Full Text PDF PubMed Google Scholar, 9.Ben-Zeev O. Stahnke G. Liu G. Davis R.C. Doolittle M.H. Lipoprotein lipase and hepatic lipase: the role of asparagine-linked glycosylation in the expression of a functional enzyme.J. Lipid Res. 1994; 35: 1511-1523Abstract Full Text PDF PubMed Google Scholar). After being translocated to the Golgi apparatus, the high-mannose oligosaccharides are trimmed and replaced by more complex oligosaccharides (6.Ben-Zeev O. Doolittle M.H. Davis R.C. Elovson J. Schotz M.C. Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment.J. Biol. Chem. 1992; 267: 6219-6227Abstract Full Text PDF PubMed Google Scholar, 7.Simsolo R.B. Ong J.M. Kern P.A. Characterization of lipoprotein lipase activity, secretion, and degradation at different sites of post-translational processing in primary cultures of rat adipocytes.J. Lipid Res. 1992; 33: 1777-1784Abstract Full Text PDF PubMed Google Scholar, 8.Semenkovich C.F. Luo C.C. Nakanishi M.K. Chen S.H. Smith L.C. Chan L. In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion.J. Biol. Chem. 1990; 265: 5429-5433Abstract Full Text PDF PubMed Google Scholar, 9.Ben-Zeev O. Stahnke G. Liu G. Davis R.C. Doolittle M.H. Lipoprotein lipase and hepatic lipase: the role of asparagine-linked glycosylation in the expression of a functional enzyme.J. Lipid Res. 1994; 35: 1511-1523Abstract Full Text PDF PubMed Google Scholar). Endoglycosidase H (EndoH) cleaves high-mannose oligosaccharides from proteins, but not complex oligosaccharides (6.Ben-Zeev O. Doolittle M.H. Davis R.C. Elovson J. Schotz M.C. Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment.J. Biol. Chem. 1992; 267: 6219-6227Abstract Full Text PDF PubMed Google Scholar, 10.Davis R.C. Ben-Zeev O. Martin D. Doolittle M.H. Combined lipase deficiency in the mouse: evidence of impaired lipase processing and secretion.J. Biol. Chem. 1990; 265: 17960-17966Abstract Full Text PDF PubMed Google Scholar), making it possible to distinguish LPL within the ER and Golgi compartments. The catalytic activity of LPL along the capillary endothelium is considered rate-limiting for the lipolytic processing of plasma triglycerides and for the subsequent uptake of fatty acids by surrounding tissues (1.Kersten S. Physiological regulation of lipoprotein lipase.Biochim. Biophys. Acta. 2014; 1841: 919-933Crossref PubMed Scopus (342) Google Scholar, 4.Young S.G. Zechner R. Biochemistry and pathophysiology of intravascular and intracellular lipolysis.Genes Dev. 2013; 27: 459-484Crossref PubMed Scopus (243) Google Scholar, 11.Bartelt A. Bruns O.T. Reimer R. Hohenberg H. Ittrich H. Peldschus K. Kaul M.G. Tromsdorf U.I. Weller H. Waurisch C. et al.Brown adipose tissue activity controls triglyceride clearance.Nat. Med. 2011; 17: 200-205Crossref PubMed Scopus (1151) Google Scholar). To ensure that supply of fatty acids to tissues matches metabolic demand, LPL activity is under tight regulatory control. Although it is possible to identify small fluctuations in Lpl mRNA levels, LPL activity appears to be regulated mainly at the posttranslational level (1.Kersten S. Physiological regulation of lipoprotein lipase.Biochim. Biophys. Acta. 2014; 1841: 919-933Crossref PubMed Scopus (342) Google Scholar, 4.Young S.G. Zechner R. Biochemistry and pathophysiology of intravascular and intracellular lipolysis.Genes Dev. 2013; 27: 459-484Crossref PubMed Scopus (243) Google Scholar). An important physiological regulator of LPL is angiopoietin-like 4 (ANGPTL4). ANGPTL4 potently inhibits LPL activity in multiple tissues and regulates LPL activity during a variety of physiological conditions, including fasting, cold, and exercise (12.Catoire M. Alex S. Paraskevopulos N. Mattijssen F. Evers-van Gogh I. Schaart G. Jeppesen J. Kneppers A. Mensink M. Voshol P.J. et al.Fatty acid-inducible ANGPTL4 governs lipid metabolic response to exercise.Proc. Natl. Acad. Sci. USA. 2014; 111: E1043-E1052Crossref PubMed Scopus (97) Google Scholar, 13.Kroupa O. Vorrsjö E. Stienstra R. Mattijssen F. Nilsson S.K. Sukonina V. Kersten S. Olivecrona G. Olivecrona T. Linking nutritional regulation of Angptl4, Gpihbp1, and Lmf1 to lipoprotein lipase activity in rodent adipose tissue.BMC Physiol. 2012; 12: 13Crossref PubMed Scopus (63) Google Scholar, 14.Dijk W. Heine M. Vergnes L. Boon M.R. Schaart G. Hesselink M.K. Reue K. van Marken Lichtenbelt W.D. Olivecrona G. Rensen P.C. et al.ANGPTL4 mediates shuttling of lipid fuel to brown adipose tissue during sustained cold exposure.eLife. 10.7554/eLife.08428. 2015; Google Scholar). For example, changes in the expression of ANGPTL4 (originally called fasting-induced adipose factor) allow for swift changes in adipose tissue LPL activity during fasting (13.Kroupa O. Vorrsjö E. Stienstra R. Mattijssen F. Nilsson S.K. Sukonina V. Kersten S. Olivecrona G. Olivecrona T. Linking nutritional regulation of Angptl4, Gpihbp1, and Lmf1 to lipoprotein lipase activity in rodent adipose tissue.BMC Physiol. 2012; 12: 13Crossref PubMed Scopus (63) Google Scholar, 15.Kersten S. Mandard S. Tan N.S. Escher P. Metzger D. Chambon P. Gonzalez F.J. Desvergne B. Wahli W. Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene.J. Biol. Chem. 2000; 275: 28488-28493Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). Expression of ANGPTL4 is induced by fatty acids via PPARs as part of a feedback mechanism aimed at preventing lipid overload within cells (16.Georgiadi A. Lichtenstein L. Degenhardt T. Boekschoten M.V. van Bilsen M. Desvergne B. Müller M. Kersten S. Induction of cardiac Angptl4 by dietary fatty acids is mediated by peroxisome proliferator-activated receptor beta/delta and protects against fatty acid-induced oxidative stress.Circ. Res. 2010; 106: 1712-1721Crossref PubMed Scopus (109) Google Scholar, 17.Dijk W. Kersten S. Regulation of lipoprotein lipase by Angptl4.Trends Endocrinol. Metab. 2014; 25: 146-155Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Genetic studies have strongly supported a role for ANGPTL4 in determining plasma triglyceride levels in humans and have linked inactivating variants in the ANGPTL4 gene to a reduced risk of coronary heart disease (18.Romeo S. Pennacchio L.A. Fu Y. Boerwinkle E. Tybjaerg-Hansen A. Hobbs H.H. Cohen J.C. Population-based resequencing of ANGPTL4 uncovers variations that reduce triglycerides and increase HDL.Nat. Genet. 2007; 39: 513-516Crossref PubMed Scopus (425) Google Scholar, 19.Dewey F.E. Gusarova V. O'Dushlaine C. Gottesman O. Trejos J. Hunt C. Van Hout C.V. Habegger L. Buckler D. Lai K-M. V. et al.Inactivating variants in ANGPTL4 and risk of coronary artery disease.N. Engl. J. Med. 2016; 374: 1123-1133Crossref PubMed Scopus (310) Google Scholar, 20.Myocardial Infarction Genetics and CARDIoGRAM Exome Consortia Investigators Coding variation in ANGPTL4, LPL, and SVEP1 and the risk of coronary disease.N. Engl. J. Med. 2016; 374: 1134-1144Crossref PubMed Scopus (340) Google Scholar). However, cross-sectional studies have not revealed a clear correlation between the plasma levels of ANGPTL4 and triglycerides (21.Robciuc M.R. Naukkarinen J. Ortega-Alonso A. Tyynismaa H. Raivio T. Rissanen A. Kaprio J. Ehnholm C. Jauhiainen M. Pietiläinen K.H. Serum angiopoietin-like 4 protein levels and expression in adipose tissue are inversely correlated with obesity in monozygotic twins.J. Lipid Res. 2011; 52: 1575-1582Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 22.Smart-Halajko M.C. Robciuc M.R. Cooper J.A. Jauhiainen M. Kumari M. Kivimaki M. Khaw K-T. Boekholdt S.M. Wareham N.J. Gaunt T.R. et al.The relationship between plasma angiopoietin-like protein 4 levels, angiopoietin-like protein 4 genotype, and coronary heart disease risk.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 2277-2282Crossref PubMed Scopus (60) Google Scholar, 23.Robciuc M.R. Tahvanainen E. Jauhiainen M. Ehnholm C. Quantitation of serum angiopoietin-like proteins 3 and 4 in a Finnish population sample.J. Lipid Res. 2010; 51: 824-831Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 24.Mehta N. Qamar A. Qu L. Qasim A.N. Mehta N.N. Reilly M.P. Rader D.J. Differential association of plasma angiopoietin-like proteins 3 and 4 with lipid and metabolic traits.Arterioscler. Thromb. Vasc. Biol. 2014; 34: 1057-1063Crossref PubMed Scopus (61) Google Scholar). These observations suggest that the plasma pool of ANGPTL4 may not be primarily responsible for regulating plasma triglyceride levels and that the inhibitory effects of ANGPTL4 on LPL activity may not occur exclusively on the surface of capillaries. Indeed, Robciuc et al. (25.Robciuc M.R. Skrobuk P. Anisimov A. Olkkonen V.M. Alitalo K. Eckel R.H. Koistinen H.A. Jauhiainen M. Ehnholm C. Angiopoietin-like 4 mediates PPAR delta effect on lipoprotein lipase-dependent fatty acid uptake but not on beta-oxidation in myotubes.PLoS One. 2012; 7: e46212Crossref PubMed Scopus (46) Google Scholar) proposed that ANGPTL4 could inhibit LPL activity not only at the cell surface, but also intracellularly, partly based on microscopy studies showing colocalization of LPL and ANGPTL4 within cells. At the same time, recent studies have raised the possibility that ANGPTL4 regulation of LPL might occur within the subendothelial spaces rather than along the capillary lumen (26.Davies B.S.J. Goulbourne C.N. Barnes R.H. Turlo K.A. Gin P. Vaughan S. Vaux D.J. Bensadoun A. Beigneux A.P. Fong L.G. et al.Assessing mechanisms of GPIHBP1 and lipoprotein lipase movement across endothelial cells.J. Lipid Res. 2012; 53: 2690-2697Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 27.Chi X. Shetty S.K. Shows H.W. Hjelmaas A.J. Malcolm E.K. Davies B.S.J. Angiopoietin-like 4 modifies the interactions between lipoprotein lipase and its endothelial cell transporter GPIHBP1.J. Biol. Chem. 2015; 290: 11865-11877Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 28.Nilsson S.K. Anderson F. Ericsson M. Larsson M. Makoveichuk E. Lookene A. Heeren J. Olivecrona G. Triacylglycerol-rich lipoproteins protect lipoprotein lipase from inactivation by ANGPTL3 and ANGPTL4.Biochim. Biophys. Acta. 2012; 1821: 1370-1378Crossref PubMed Scopus (32) Google Scholar). Studies of 3T3-L1 adipocytes suggested that inhibition of LPL activity by ANGPTL4 begins only after these proteins arrive at the cell surface (29.Makoveichuk E. Vorrsjö E. Olivecrona T. Olivecrona G. Inactivation of lipoprotein lipase in 3T3-L1 adipocytes by angiopoietin-like protein 4 requires that both proteins have reached the cell surface.Biochem. Biophys. Res. Commun. 2013; 441: 941-946Crossref PubMed Scopus (21) Google Scholar). However, the extent to which the cultured cell studies are relevant to LPL activity in adipose tissue in vivo is uncertain. Accordingly, our objective in the current studies was to investigate the cellular location and mechanism for LPL inhibition by ANGPTL4 in adipose tissue. To address this objective, we used a combination of cell culture studies, and ex vivo and in vivo studies of adipocytes and adipose tissue from wild-type and Angptl4−/− mice. All animal experiments were performed in accordance with Directive 2010/63/EU from the European Union. All animal studies were reviewed and approved by the Animal Ethics Committee of Wageningen University. CHO pgsA-745 cells were cultured as described previously (30.Voss C.V. Davies B.S.J. Tat S. Gin P. Fong L.G. Pelletier C. Mottler C.D. Bensadoun A. Beigneux A.P. Young S.G. Mutations in lipoprotein lipase that block binding to the endothelial cell transporter GPIHBP1.Proc. Natl. Acad. Sci. USA. 2011; 108: 7980-7984Crossref PubMed Scopus (50) Google Scholar, 31.Beigneux A.P. Davies B.S.J. Tat S. Chen J. Gin P. Voss C.V. Weinstein M.M. Bensadoun A. Pullinger C.R. Fong L.G. et al.Assessing the role of the glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) three-finger domain in binding lipoprotein lipase.J. Biol. Chem. 2011; 286: 19735-19743Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). CHO pgsA-745 cells were electroporated with an expression vector for Flag-tagged human ANGPTL4 (hANGPTL4) (either the full-length protein hANGPTL4[1–406] or the N-terminal domain of hANGPTL4[1–160]) or a vector for V5-tagged human LPL (or empty vector). After the cell transfections, the cells were mixed and coplated in the same well of a 24-well plate. After 24 h, the cell culture medium was collected for a LPL activity assay. After 48 h, the cell culture medium was collected and cell lysates were prepared for SDS-PAGE and Western blotting. ANGPTL4 and LPL expression vectors were also cotransfected into CHO pgsA-745 cells. For those studies, the cells were co-electroporated with a vector for Flag-tagged hANGPTL4 (either hANGPTL4[1–406] or hANGPTL4[1–160]) and a vector for V5-tagged human LPL (hLPL) (or empty vector). After 24 h, the cell culture medium was collected for a LPL activity assay. After 48 h, the cell culture medium was collected and cell lysates were prepared for SDS-PAGE and Western blotting. Inguinal or gonadal white adipose tissue (gWAT) was removed from Angptl4−/− and wild-type mice and placed in DMEM (Lonza) supplemented with 1% penicillin/streptomycin (P/S) and 1% BSA (Sigma-Aldrich, Houten, The Netherlands). Fat pads from 2–3 mice were pooled, minced with scissors, and digested for 1 h at 37°C in collagenase-containing medium [DMEM with 3.2 mM CaCl2, 1.5 mg/ml collagenase type II (C6885; Sigma-Aldrich), 10% FCS, 0.5% BSA, and 15 mM HEPES]. Next, the cell suspension was filtered through a 100 μm cell strainer (Falcon) and centrifuged at 1,600 rpm for 10 min. The supernatant fluid was removed, and the pellet containing the stromal vascular fraction was resuspended in erythrocyte lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) and incubated for 2–3 min at room temperature. After neutralization, cells were centrifuged at 1,200 rpm for 5 min. Pelleted cells were resuspended in DMEM containing 10% FCS and 1% P/S and plated. After reaching confluency, cells were differentiated according to standard protocol for 3T3-L1 cells with addition of 1 μM rosiglitazone (32.Alex S. Lange K. Amolo T. Grinstead J.S. Haakonsson A.K. Szalowska E. Koppen A. Mudde K. Haenen D. Al-Lahham S. et al.Short-chain fatty acids stimulate angiopoietin-like 4 synthesis in human colon adenocarcinoma cells by activating peroxisome proliferator-activated receptor γ.Mol. Cell. Biol. 2013; 33: 1303-1316Crossref PubMed Scopus (177) Google Scholar). Tissue samples from ANGPTL4 knockout mice (Angptl4−/−), wild-type mice, and Angptl4-Tg mice from previously published studies were used for analyses of LPL glycosylation and mass (13.Kroupa O. Vorrsjö E. Stienstra R. Mattijssen F. Nilsson S.K. Sukonina V. Kersten S. Olivecrona G. Olivecrona T. Linking nutritional regulation of Angptl4, Gpihbp1, and Lmf1 to lipoprotein lipase activity in rodent adipose tissue.BMC Physiol. 2012; 12: 13Crossref PubMed Scopus (63) Google Scholar, 14.Dijk W. Heine M. Vergnes L. Boon M.R. Schaart G. Hesselink M.K. Reue K. van Marken Lichtenbelt W.D. Olivecrona G. Rensen P.C. et al.ANGPTL4 mediates shuttling of lipid fuel to brown adipose tissue during sustained cold exposure.eLife. 10.7554/eLife.08428. 2015; Google Scholar, 33.Köster A. Chao Y.B. Mosior M. Ford A. Gonzalez-DeWhitt P.A. Hale J.E. Li D. Qiu Y. Fraser C.C. Yang D.D. et al.Transgenic angiopoietin-like (angptl)4 overexpression and targeted disruption of angptl4 and angptl3: regulation of triglyceride metabolism.Endocrinology. 2005; 146: 4943-4950Crossref PubMed Scopus (349) Google Scholar, 34.Lichtenstein L. Mattijssen F. De Wit N.J. Georgiadi A. Hooiveld G.J. Van Der Meer R. He Y. Qi L. Köster A. Tamsma J.T. et al.Angptl4 protects against severe proinflammatory effects of saturated fat by inhibiting fatty acid uptake into mesenteric lymph node macrophages.Cell Metab. 2010; 12: 580-592Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 35.Mandard S. Zandbergen F. Van Straten E. Wahli W. Kuipers F. Müller M. Kersten S. The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity.J. Biol. Chem. 2006; 281: 934-944Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Angptl4−/− mice, wild-type mice, and Angptl4-Tg mice were on a C56BL/6J background for >10 generations. Wild-type and Angptl4-Tg mice were littermates. In Angptl4−/− mice, part of the Angptl4 gene was deleted by homologous recombination in embryonic stem cells, resulting in a nonfunctional ANGPTL4 protein (33.Köster A. Chao Y.B. Mosior M. Ford A. Gonzalez-DeWhitt P.A. Hale J.E. Li D. Qiu Y. Fraser C.C. Yang D.D. et al.Transgenic angiopoietin-like (angptl)4 overexpression and targeted disruption of angptl4 and angptl3: regulation of triglyceride metabolism.Endocrinology. 2005; 146: 4943-4950Crossref PubMed Scopus (349) Google Scholar, 34.Lichtenstein L. Mattijssen F. De Wit N.J. Georgiadi A. Hooiveld G.J. Van Der Meer R. He Y. Qi L. Köster A. Tamsma J.T. et al.Angptl4 protects against severe proinflammatory effects of saturated fat by inhibiting fatty acid uptake into mesenteric lymph node macrophages.Cell Metab. 2010; 12: 580-592Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Angptl4-Tg mice overexpress the Angptl4 gene in various tissues under the endogenous promoter (35.Mandard S. Zandbergen F. Van Straten E. Wahli W. Kuipers F. Müller M. Kersten S. The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity.J. Biol. Chem. 2006; 281: 934-944Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Brown adipose tissue samples from Angptl4−/− and wild-type mice exposed to cold or thermoneutral temperature for 10 days were from a study by Dijk et al. (14.Dijk W. Heine M. Vergnes L. Boon M.R. Schaart G. Hesselink M.K. Reue K. van Marken Lichtenbelt W.D. Olivecrona G. Rensen P.C. et al.ANGPTL4 mediates shuttling of lipid fuel to brown adipose tissue during sustained cold exposure.eLife. 10.7554/eLife.08428. 2015; Google Scholar). gWAT samples from fed, fasted, and refed Angptl4−/− and wild-type mice were from a study described by Kroupa et al. (13.Kroupa O. Vorrsjö E. Stienstra R. Mattijssen F. Nilsson S.K. Sukonina V. Kersten S. Olivecrona G. Olivecrona T. Linking nutritional regulation of Angptl4, Gpihbp1, and Lmf1 to lipoprotein lipase activity in rodent adipose tissue.BMC Physiol. 2012; 12: 13Crossref PubMed Scopus (63) Google Scholar). Hearts from fed and overnight-fasted Angptl4−/− and wild-type mice were collected and snap-frozen in liquid nitrogen. For "post-heparin" tissues, wild-type and Angptl4−/− mice were fed or fasted for 16 h. The mice were then anesthetized with isoflurane and injected with heparin (100 IU/kg in 0.9% NaCl) through the jugular vein. After 5 min, the mice were euthanized by cervical dislocation. gWAT was dissected, frozen immediately in liquid nitrogen, and stored at −80°C for further analyses. LPL activity levels in culture medium and cell lysates of transfected cells was assessed in duplicate with a [3H]triolein substrate (PerkinElmer) as previously described (36.Bengtsson-Olivecrona G. Olivecrona T. Assay of lipoprotein lipase and hepatic lipase.In Lipoprotein Analysis: A Practical Approach. R. E. Skinner and C. A. Converse, editors. Oxford University Press, Oxford, UK. 1992; : 169-185Google Scholar). gWAT was harvested from Angptl4−/− and wild-type mice and placed in DMEM supplemented with 1% P/S and 1% BSA. Fat pads were minced into small pieces, which were further divided to make small mounds of WAT (∼50–100 mg of tissue). WAT explants were placed into wells containing medium (DMEM with 1% P/S and 10% FCS) with or without heparin (50 IU/ml). Explants were incubated for different times (as indicated in the figure legends), after which the medium was harvested and explant weights were determined. Explants were immediately lysed to prepare protein extracts. Protein concentrations in explants were determined and equal amounts of protein were used for SDS-PAGE and Western blotting studies. Adipocytes that had been differentiated from the stromal vascular fractions of Angptl4−/− and wild-type mice were prepared as described earlier. Upon differentiation, adipocytes were lysed in NP40 lysis buffer [50 mM Tris-HCl (pH 8.0), 0.5% NP40, 150 mM NaCl, 5 mM MgCl2] supplemented with protease and phosphatase inhibitors (Roche). After centrifugation, the supernatant fluid (NP40S) was mixed with 2× Laemmli sample buffer and heated at 65°C for 15 min. The pellet was resuspended in PBS and 2× Laemmli sample buffer and heated at 95°C for 30 min (NP40P). NP40S and NP40P fractions were then loaded onto SDS-PAGE for further analyses by Western blotting. Fat pads, explants, and differentiated adipocytes were lysed in a mild RIPA-like lysis buffer [25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol; Thermo Scientific, Landsmeer, The Netherlands] containing protease and phosphatase inhibitors (Roche). Lysates were centrifuged 2–3 times at 13,000 rpm for 10 min to remove fat droplets. Protein lysates (10–30 μg protein per lane), medium (10–15 μl), or plasma (0.75 μl) samples were diluted in Laemmli sample buffer, cooked for 5 min at 95°C, and loaded onto precast TGX gels (Bio-Rad, Veenendaal, The Netherlands). For Native PAGE, protein lysates were diluted in a SDS-free and DTT-free sample buffer, directly loaded onto precast TGX gels, and run in a Tris-glycine running buffer without SDS. The separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane with a Transblot Turbo system (Bio-Rad). Membranes were probed with a goat anti-mouse LPL antibody (37.Weinstein M.M. Yin L. Beigneux A.P. Davies B.S.J. Gin P. Estrada K. Melford K. Bishop J.R. Esko J.D. Dallinga-Thie G.M. et al.Abnormal patterns of lipoprotein lipase release into the plasma in GPIHBP1-deficient mice.J. Biol. Chem. 2008; 283: 34511-34518Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), a rabbit anti-mouse HSP90 antibody (Cell Signaling Technology; #4874), a rabbit anti-mouse H2A antibody (Abcam; #ab18255), a rat anti-mouse ANGPTL4 antibody (Adipogen; #Kairos 142-2), or a rabbit anti-mouse ADIPOQ antibody (ThermoScientific, #PAI-054) at 1:5,000 (LPL), 1:2,000 (HSP90, ANGPTL4), or 1:1,000 (H2A, ADIPOQ) dilution. All incubations were performed in TBS (pH 7.5), 0.1% Tween-20, and 5% nonfat dry milk. Membran
Referência(s)