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• W2146820210 abstract "The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles. The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles. Although lipid metabolism and homeostasis have been a research focus for decades, the increasing obesity and diabetes epidemic illustrates that there is an urgent need to pin down the molecular mechanisms regulating this complex network. Furthermore, the worldwide increase in coronary heart disease caused by arteriosclerotic plaques is associated with hypertriglyceridemia (1.Nordestgaard B.G. Benn M. Schnohr P. Tybjaerg-Hansen A. Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women.JAMA. 2007; 298: 299-308Crossref PubMed Scopus (1607) Google Scholar, 2.Bansal S. Buring J.E. Rifai N. Mora S. Sacks F.M. Ridker P.M. Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women.JAMA. 2007; 298: 309-316Crossref PubMed Scopus (1234) Google Scholar), which is also a symptom of type 2 diabetes and the metabolic syndrome. Diabetes and associated insulin resistance result in an impairment of the insulin-mediated suppression of very low density lipoprotein (VLDL) secretion in the liver and thereby in an altered lipoprotein pattern in the plasma (3.Choi S.H. Ginsberg H.N. Increased very low density lipoprotein (VLDL) secretion, hepatic steatosis, and insulin resistance.Trends Endocrinol. Metab. 2011; 22: 353-363Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). VLDL precursors are assembled in the endoplasmic reticulum (ER) and their maturation occurs in the postER compartments (4.Sundaram M. Yao Z. Recent progress in understanding protein and lipid factors affecting hepatic VLDL assembly and secretion.Nutr. Metab. (Lond.). 2010; 7: 35Crossref PubMed Scopus (118) Google Scholar). However, how this maturation is achieved is only partially understood (4.Sundaram M. Yao Z. Recent progress in understanding protein and lipid factors affecting hepatic VLDL assembly and secretion.Nutr. Metab. (Lond.). 2010; 7: 35Crossref PubMed Scopus (118) Google Scholar). The Golgi apparatus, as a postER compartment, is responsible for the processing and sorting of proteins, however, the contribution of specific Golgi compartments and their associated proteins for certain trafficking pathways, such as lipoprotein metabolism, is less well understood (4.Sundaram M. Yao Z. Recent progress in understanding protein and lipid factors affecting hepatic VLDL assembly and secretion.Nutr. Metab. (Lond.). 2010; 7: 35Crossref PubMed Scopus (118) Google Scholar). To elucidate the regulatory networks that finely orchestrate these processes, further studies are needed. ADP-ribosylation factor-related protein 1 (ARFRP1) [reviewed in (5.Hesse D. Jaschke A. Chung B. Schurmann A. Trans-Golgi proteins participate in the control of lipid droplet and chylomicron formation.Biosci. Rep. 2013; 33: 1-9Crossref PubMed Scopus (30) Google Scholar)] is a member of the Ras super-family of small guanosine triphosphatases (GTPases) that act as molecular switches (6.Colicelli J. Human RAS superfamily proteins and related GTPases.Sci. STKE. 2004; 2004: RE13Crossref PubMed Scopus (576) Google Scholar, 7.Wennerberg K. Rossman K.L. Der C.J. The Ras superfamily at a glance.J. Cell Sci. 2005; 118: 843-846Crossref PubMed Scopus (1009) Google Scholar). In the GTP-bound active state, ARFRP1 is located at the trans-Golgi (8.Zahn C. Hommel A. Lu L. Hong W. Walther D.J. Florian S. Joost H.G. Schürmann A. Knockout of Arfrp1 leads to disruption of ARF-like1 (ARL1) targeting to the trans-Golgi in mouse embryos and HeLa cells.Mol. Membr. Biol. 2006; 23: 475-485Crossref PubMed Scopus (39) Google Scholar) where it was shown to be involved in the targeting of certain proteins to the plasma membrane (9.Zahn C. Jaschke A. Weiske J. Hommel A. Hesse D. Augustin R. Lu L. Hong W. Florian S. Scheepers A. et al.ADP-ribosylation factor-like GTPase ARFRP1 is required for trans-Golgi to plasma membrane trafficking of E-cadherin.J. Biol. Chem. 2008; 283: 27179-27188Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 10.Hesse D. Hommel A. Jaschke A. Moser M. Bernhardt U. Zahn C. Kluge R. Wittschen P. Gruber A.D. Al-Hasani H. et al.Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1.Biochem. Biophys. Res. Commun. 2010; 394: 896-903Crossref PubMed Scopus (17) Google Scholar) and to intracellular storage vesicles (10.Hesse D. Hommel A. Jaschke A. Moser M. Bernhardt U. Zahn C. Kluge R. Wittschen P. Gruber A.D. Al-Hasani H. et al.Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1.Biochem. Biophys. Res. Commun. 2010; 394: 896-903Crossref PubMed Scopus (17) Google Scholar) as well as secreted proteins (11.Hesse D. Jaschke A. Kanzleiter T. Witte N. Augustin R. Hommel A. Puschel G.P. Petzke K.J. Joost H.G. Schupp M. et al.GTPase ARFRP1 is essential for normal hepatic glycogen storage and insulin-like growth factor 1 secretion.Mol. Cell. Biol. 2012; 32: 4363-4374Crossref PubMed Scopus (19) Google Scholar). The hepatocyte-specific deletion of Arfrp1 results in a disturbed glucose metabolism caused by a reduced plasma membrane localization of the glucose transporter GLUT2 and a marked growth retardation partially due to a lower secretion of the insulin-like growth factor IGF1 from hepatocytes (11.Hesse D. Jaschke A. Kanzleiter T. Witte N. Augustin R. Hommel A. Puschel G.P. Petzke K.J. Joost H.G. Schupp M. et al.GTPase ARFRP1 is essential for normal hepatic glycogen storage and insulin-like growth factor 1 secretion.Mol. Cell. Biol. 2012; 32: 4363-4374Crossref PubMed Scopus (19) Google Scholar). A lipodystrophic phenotype was initiated by a fat cell-specific deletion of Arfrp1. Diminished lipid droplet fusion and elevated lipolysis were responsible for smaller lipid droplets in brown adipose tissue (12.Hommel A. Hesse D. Volker W. Jaschke A. Moser M. Engel T. Bluher M. Zahn C. Chadt A. Ruschke K. et al.The ARF-like GTPase ARFRP1 is essential for lipid droplet growth and is involved in the regulation of lipolysis.Mol. Cell. Biol. 2010; 30: 1231-1242Crossref PubMed Scopus (42) Google Scholar). Furthermore, in a recent study it was shown that in the intestine ARFRP1 is involved in the formation and lipidation of lipid-carrying chylomicrons (13.Jaschke A. Chung B. Hesse D. Kluge R. Zahn C. Moser M. Petzke K.J. Brigelius-Flohe R. Puchkov D. Koepsell H. et al.The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium.Hum. Mol. Genet. 2012; 21: 3128-3142Crossref PubMed Scopus (25) Google Scholar). Besides its fundamental role in whole body glucose homeostasis, the liver is further essential for the distribution of lipids in the body via synthesis and secretion of VLDLs. Because the organization of VLDLs is very similar to that of chylomicrons, the aim of this study was to clarify the impact of the GTPase ARFRP1 and the trans-Golgi for lipoprotein assembly and release in the liver. Liver-specific Arfrp1-knockout (Arfrp1liv−/−) mice were generated as described (11.Hesse D. Jaschke A. Kanzleiter T. Witte N. Augustin R. Hommel A. Puschel G.P. Petzke K.J. Joost H.G. Schupp M. et al.GTPase ARFRP1 is essential for normal hepatic glycogen storage and insulin-like growth factor 1 secretion.Mol. Cell. Biol. 2012; 32: 4363-4374Crossref PubMed Scopus (19) Google Scholar) and compared with their control littermates (Arfrp1flox/flox). All experiments were performed at the age of 5 weeks because of an incomplete suppression of Arfrp1 expression at later time points. The animals were housed in a controlled environment (20 ± 2°C, 12/12 h light/dark cycle) and had free access to water and standard diet (V153x, Ssniff, Soest, Germany). For fasting conditions, diet was deprived overnight. All animal experiments were approved by the ethics committee of the State Agency of Environment, Health, and Consumer Protection (State of Brandenburg, Germany). For the quantification of ApoC3 in plasma and liver samples (25 μg) a rat/mouse ELISA was used and applied according to the manufacturer's instructions (Apc3, Abnova, Germany). Nonesterified free fatty acid, triglyceride, and cholesterol content of plasma was determined as described (14.Schulz N. Himmelbauer H. Rath M. van Weeghel M. Houten S. Kulik W. Suhre K. Scherneck S. Vogel H. Kluge R. et al.Role of medium- and short-chain L-3-hydroxyacyl-CoA dehydrogenase in the regulation of body weight and thermogenesis.Endocrinology. 2011; 152: 4641-4651Crossref PubMed Scopus (32) Google Scholar). Plasma cholesterol was determined by enzymatic colorimetric assay (Cholesterol liquicolor; Human, Wiesbaden, Germany). For the quantitative determination of triglyceride content from murine livers (15.Lubura M. Hesse D. Neumann N. Scherneck S. Wiedmer P. Schurmann A. Non-invasive quantification of white and brown adipose tissues and liver fat content by computed tomography in mice.PLoS ONE. 2012; 7: e37026Crossref PubMed Scopus (57) Google Scholar), livers were homogenized in 10 mM sodium dihydrogen phosphate, 1 mM EDTA, and 1% polyoxyethylene(10)tridecyl ether; incubated for 5 min at 37°C; and the triglycerides in the supernatant were detected with a commercial kit (RandoxTR-210, Crumlin, UK). To obtain lipoprotein fractions for fast-protein liquid chromatography (FPLC) analysis, each individual plasma sample was loaded on a filtration chromatography column onto a Superose 6 10/300 GL column (GE Healthcare), which separates lipoproteins according to their size, and cholesterol and triglyceride concentrations were continuously measured in the effluent using an enzymatic assay at an optical density of 492 nm as described (16.Molica F. Burger F. Thomas A. Staub C. Tailleux A. Staels B. Pelli G. Zimmer A. Cravatt B. Matter C.M. et al.Endogenous cannabinoid receptor CB1 activation promotes vascular smooth-muscle cell proliferation and neointima formation.J. Lipid Res. 2013; 54: 1360-1368Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 17.Brunham L.R. Kruit J.K. Iqbal J. Fievet C. Timmins J.M. Pape T.D. Coburn B.A. Bissada N. Staels B. Groen A.K. et al.Intestinal ABCA1 directly contributes to HDL biogenesis in vivo.J. Clin. Invest. 2006; 116: 1052-1062Crossref PubMed Scopus (419) Google Scholar, 18.Peters J.M. Hennuyer N. Staels B. Fruchart J.C. Fievet C. Gonzalez F.J. Auwerx J. Alterations in lipoprotein metabolism in peroxisome proliferator-activated receptor alpha-deficient mice.J. Biol. Chem. 1997; 272: 27307-27312Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). This system allows separation of the three major lipoprotein classes; VLDLs, LDLs, and HDLs. Cholesterol or triglyceride concentrations were determined in the eluted fractions. Accumulated data were analyzed by the Millenium 20/0 program (Waters). The area under each peak is proportional to the lipid concentration in the respective lipoprotein fraction. Results are expressed as lipid concentration in VLDLs, LDLs, and HDLs as previously described (16.Molica F. Burger F. Thomas A. Staub C. Tailleux A. Staels B. Pelli G. Zimmer A. Cravatt B. Matter C.M. et al.Endogenous cannabinoid receptor CB1 activation promotes vascular smooth-muscle cell proliferation and neointima formation.J. Lipid Res. 2013; 54: 1360-1368Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Moreover, data are also presented as cholesterol- and triglyceride-mean profiles obtained with each individual plasma sample of each group. To determine maximum hepatic VLDL secretion, basal blood samples were collected from the tail vein after fasting overnight. Triton WR-1339 (15% in saline, 0.5 g/kg) was injected intraperitoneally to block lipoprotein lipase and blood was sampled 1, 2, 4, and 6 h after application. After centrifugation, total triglyceride content was determined using a serum triglyceride determination kit (TR0100, Sigma, USA). To investigate VLDL composition, mice were fasted overnight and blood samples were subjected to fractionation (350,000 g, 4 h, 16°C; OptiPrep, Axis-Shield, UK) in order to separate different lipoprotein fractions. Subsequently, triglyceride and cholesterol content of fractions were determined and apolipoprotein distribution was assessed by Western blotting. Primary hepatocytes were isolated from 12-week-old mice and Arfrp1 expression was suppressed by siRNA as described (11.Hesse D. Jaschke A. Kanzleiter T. Witte N. Augustin R. Hommel A. Puschel G.P. Petzke K.J. Joost H.G. Schupp M. et al.GTPase ARFRP1 is essential for normal hepatic glycogen storage and insulin-like growth factor 1 secretion.Mol. Cell. Biol. 2012; 32: 4363-4374Crossref PubMed Scopus (19) Google Scholar). To determine ApoB48/100 synthesis and degradation in primary hepatocytes, intracellular pools of cysteine and methionine were depleted by incubation with Cys-free and Met-free DMEM for 30 min prior to labeling with [35S] Promix (100 μCi/500 μl) for 2 h (19.Chen Z. Fitzgerald R.L. Li G. Davidson N.O. Schonfeld G. Hepatic secretion of apoB-100 is impaired in hypobetalipoproteinemic mice with an apoB-38.9-specifying allele.J. Lipid Res. 2004; 45: 155-163Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). For chase experiments cells were consecutively incubated with normal DMEM for 3 h. Immunoprecipitation of ApoB48/100 from cell lysates and cultured media/supernatant was carried out, separated on a 6% gel which was dried prior to visualization on a phosphor imager system (Storm 820, Molecular Dynamics). Band intensity was quantified by Image Quant 5.2 (Molecular Dynamics). To determine the rate of de novo triglyceride synthesis, primary hepatocytes were transfected with scrambled siRNA or siArfrp1 and then incubated with [14C]-labeled glucose (1 μCi/ml) overnight (20.Henkel J. Frede K. Schanze N. Vogel H. Schurmann A. Spruss A. Bergheim I. Puschel G.P. Stimulation of fat accumulation in hepatocytes by PGE(2)-dependent repression of hepatic lipolysis, beta-oxidation and VLDL-synthesis.Lab. Invest. 2012; 92: 1597-1606Crossref PubMed Scopus (41) Google Scholar). Triglycerides from cells were isolated by chloroform-methanol extraction and the addition of CaCl2 to prevent contamination of the aqueous phase with lipids. Lipid extracts were dried and redissolved in chloroform prior to counting in a scintillation counter (LS6000LL; Beckman Coulter, Krefeld, Germany). Livers of fasted Arfrp1liv−/− and control mice were minced in homogenization buffer [0.25 M sucrose, 1 mM EDTA, 20 mM HEPES KOH (pH 7.4), and protease inhibitor], homogenized by a potter, and filtered through gauze (70 μm). After removing the nuclei by centrifuging at 1,500 g for 10 min at 4°C, the homogenate was separated by Optiprep density gradient centrifugation according to the manufacturer's instructions (Axis-Shield, UK). The gradient was collected in fractions of 1 ml at the end of the centrifugation. Aliquots were used to determine triglyceride concentration in fractions (Serum Triglyceride Determination kit, Sigma, Germany). Proteins in the fractions were concentrated using trichloroacetic acid to be analyzed by Western blotting (13.Jaschke A. Chung B. Hesse D. Kluge R. Zahn C. Moser M. Petzke K.J. Brigelius-Flohe R. Puchkov D. Koepsell H. et al.The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium.Hum. Mol. Genet. 2012; 21: 3128-3142Crossref PubMed Scopus (25) Google Scholar). The activity of microsomal triglyceride transfer protein (MTP) was determined as described in Jaschke et al. (13.Jaschke A. Chung B. Hesse D. Kluge R. Zahn C. Moser M. Petzke K.J. Brigelius-Flohe R. Puchkov D. Koepsell H. et al.The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium.Hum. Mol. Genet. 2012; 21: 3128-3142Crossref PubMed Scopus (25) Google Scholar). Liver tissues were homogenized and 100 μg of protein were used for the assay. The methods were performed as described (11.Hesse D. Jaschke A. Kanzleiter T. Witte N. Augustin R. Hommel A. Puschel G.P. Petzke K.J. Joost H.G. Schupp M. et al.GTPase ARFRP1 is essential for normal hepatic glycogen storage and insulin-like growth factor 1 secretion.Mol. Cell. Biol. 2012; 32: 4363-4374Crossref PubMed Scopus (19) Google Scholar). The following gene expression assays were used: Eef2 (Mm01171434_g1), Apob (Mm01545159_m1), Dgat2 (Mm00499530_m1), and Fasn (Mm00662319_m1) (all from Applied Biosystems, Darmstadt, Germany). For protein detection the following primary antibodies were used: PLIN2 [Western blot (WB), 1:1,000; immunohistochemistry (IHC), 1:500 (Progen Biotechnik, Heidelberg, Germany)]; ApoA1 [WB, 1:2,000 (Abcam, Cambridge, UK)]; ApoB48/100 [WB, 1:1,000; IHC, 1:500 (Meridian Life Science, Memphis, TN)]; ApoC3 [IHC, 1:100 (Abcam)]; carboxylesterase 1/esterase-x [WB, 1:1,000 (Abcam)]; MTP [WB, 1:1,000 (Abcam)]; calnexin [WB, 1:2,000 (Abcam)]; syntaxin-6 [WB, 1:2,000 (BD Bioscience, Heidelberg, Germany)]; p115 [WB, 1:2,000 (BD Bioscience)]; and ARFRP1 (11.Hesse D. Jaschke A. Kanzleiter T. Witte N. Augustin R. Hommel A. Puschel G.P. Petzke K.J. Joost H.G. Schupp M. et al.GTPase ARFRP1 is essential for normal hepatic glycogen storage and insulin-like growth factor 1 secretion.Mol. Cell. Biol. 2012; 32: 4363-4374Crossref PubMed Scopus (19) Google Scholar). Secondary antibodies were labeled with horse-radish peroxidase or with immunofluorescent dyes for visualization on a confocal microscope (Leica-TCS-SP2; Microsystems, Heidelberg, Germany). Furthermore, ApoA1 and ApoB48/100 were measured by immunonephelometric assays using specific mouse antibodies developed in rabbits as previously described (21.Brunham L.R. Singaraja R.R. Duong M. Timmins J.M. Fievet C. Bissada N. Kang M.H. Samra A. Fruchart J.C. McManus B. et al.Tissue-specific roles of ABCA1 influence susceptibility to atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 548-554Crossref PubMed Scopus (89) Google Scholar). To visualize stored triglycerides in liver sections, Oil-Red O staining was utilized. Therefore, microscopic slides were air dried, fixed in 4% formaldehyde, and washed in 60% isopropyl alcohol. Staining was performed for 10 min in Oil-Red O solution (0.3% Oil-Red O in 60% isopropyl alcohol) and thereafter slides were rinsed in 60% isopropyl alcohol before nuclei were counterstained with hematoxylin. The morphometric measurement of lipid droplets was performed using a Keyence BZ-9000 microscope for picture acquisition and the corresponding software for quantification (Neu-Isenburg, Germany). Statistical differences were determined by nonparametric Mann-Whitney-Wilcoxon test with significance levels set at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). Data are presented as means ± standard error of the mean (SEM). For statistical analysis and for graphical presentation GraphPad Prism (5.0; GraphPad Software, San Diego, CA) was used. VLDLs are the main cargo of triglycerides released by the liver under fasting conditions. To investigate the synthesis and release of VLDLs from the liver, mice expressing or lacking ARFRP1 in the liver were exposed to an overnight fasting period and plasma triglycerides and apolipoproteins were investigated. The amount of free fatty acids in the plasma of Arfrp1liv−/− and control mice was not different (Fig. 1A), however, plasma triglycerides of Arfrp1liv−/− mice were significantly lower compared with the control animals (Fig. 1A). This difference in plasma triglycerides was mainly due to a lower amount of triglycerides in the VLDL fraction of the plasma of Arfrp1liv−/− mice [density < 1.006 g/ml in Optiprep (22.Graham J.M. Higgins J.A. Gillott T. Taylor T. Wilkinson J. Ford T. Billington D. A novel method for the rapid separation of plasma lipoproteins using self-generating gradients of iodixanol.Atherosclerosis. 1996; 124: 125-135Abstract Full Text PDF PubMed Scopus (77) Google Scholar)] (Fig. 1B) visible by the quantification of triglycerides after fractionation by ultracentrifugation (Fig. 1B, upper panel) as well as by FPLC (Fig. 1B, lower panel). The VLDL fraction of Arfrp1liv−/− mice contained fewer triglycerides (Arfrp1flox/flox, 31.1 ± 3.7 mg/dl; Arfrp1liv−/−, 19.6 ± 2.3 mg/dl; P < 0.05), whereas more triglycerides were found in the LDL fraction (density 1.01–1.030 g/ml; Arfrp1flox/flox, 11.8 ± 1.0 mg/dl; Arfrp1liv−/−, 17.1 ± 1.3 mg/dl; P < 0.05). This observation indicated that the lack of ARFRP1 alters lipid distribution in the different plasma lipoprotein fractions. Accordingly, fractions 16–30 (density > 1.030 g/ml) from Arfrp1liv−/− plasma that are supposed to contain HDLs are a mixture of smaller VLDLs plus true HDLs that exhibit not only lower triglycerides (Fig. 1B) but also lower cholesterol concentrations (Fig. 1D; Arfrp1flox/flox, 63.1.1 ± 2.3 mg/dl; Arfrp1liv−/−, 50.9 ± 2.6 mg/dl; P < 0.01). This resulted in a tendency of total plasma cholesterol to be reduced in Arfrp1liv−/− mice (Fig. 1C). In addition, ApoB48/100 content in the HDL-containing plasma fractions of Arfrp1liv−/− mice was slightly elevated (Fig. 1E). A reduced plasma triglyceride accumulation in Arfrp1liv−/− mice was accentuated by the application of the lipoprotein lipase inhibitor Triton WR-1339 after fasting, which allows determination of triglyceride release by the liver. In control mice, a strong increase of plasma triglycerides indicated an accumulation of VLDL particles in the plasma (Fig. 2A). In comparison, only a slight increase in triglycerides was observed in the plasma of Arfrp1liv−/− mice. As a consequence, hepatic triglycerides revealed elevated lipid levels in Arfrp1liv−/− mice compared with control livers (Fig. 2B). ApoB48/100, as the main structural lipoprotein in VLDLs, was elevated in the plasma of Arfrp1liv−/− mice as determined by WB analysis and immunonephelometric assay (Fig. 3A, upper and middle panel). In addition, plasma fractionation after ultracentrifugation and consecutive WB analysis of the VLDL fraction indicated an accumulation of ApoB48/100 in VLDL particles (Fig. 3A, lower panel). Furthermore, immunohistochemical analyses of liver sections revealed an intracellular accumulation of ApoB48/100 in Arfrp1liv−/− livers (Fig. 3B). A similar increase of ApoB100 was determined in the VLDL fraction from Arfrp1liv−/− mice after Triton WR-1335 treatment (Fig. 3C). In order to test whether ARFRP1 influences ApoB48/100 synthesis, we measured mRNA of Apob by quantitative real-time PCR without detecting differences between the genotypes (Fig. 3D). For studying the ApoB48/100 protein synthesis and clearance, we isolated hepatocytes from 12-week-old C57BL/6 mice, suppressed Arfrp1 expression by transfecting siRNA, and performed a pulse-chase experiment. The amount of ApoB48/100 immunoprecipitated from the lysates and from the supernatant was unaltered after depletion of Arfrp1 2 h after treatment with 35S-labeled amino acids (Fig. 3E, upper panel). The stability of ApoB48/100 was unaffected in siArfrp1 hepatocytes compared with control transfected cells (Fig. 3E, lower panel). Besides ApoB48/100, other apolipoproteins such as ApoC3, which with 40% of protein mass in secreted VLDLs represents a major apoprotein in these lipoproteins (23.Talmud P.J. Hawe E. Martin S. Olivier M. Miller G.J. Rubin E.M. Pennacchio L.A. Humphries S.E. Relative contribution of variation within the APOC3/A4/A5 gene cluster in determining plasma triglycerides.Hum. Mol. Genet. 2002; 11: 3039-3046Crossref PubMed Scopus (341) Google Scholar), are synthesized by the liver and attached to VLDLs prior to secretion. In the plasma of fasted Arfrp1liv−/− mice, ApoC3, that acts as an inhibitor of plasma lipoprotein lipase (24.Jong M.C. Rensen P.C. Dahlmans V.E. van der Boom H. van Berkel T.J. Havekes L.M. Apolipoprotein C–III deficiency accelerates triglyceride hydrolysis by lipoprotein lipase in wild-type and apoE knockout mice.J. Lipid Res. 2001; 42: 1578-1585Abstract Full Text Full Text PDF PubMed Google Scholar), was significantly less abundant than in Arfrp1flox/flox mice (Fig. 3F, upper panel). This difference was most obvious in the VLDL-containing plasma part after fractionation by ultracentrifugation (Fig. 3F, middle panel). However, ApoC3 appeared to accumulate in liver extracts of Arfrp1liv−/− mice because the levels were significantly higher in total liver extracts of these mice (Fig. 3F, lower panel). Nevertheless, the mRNA of Apoc3 was unaltered (data not shown). IHC analysis of livers indicated an intracellular accumulation of ApoC3 in Arfrp1liv−/− mice which was colocalized with ApoB48/100 (Fig. 3B). Because ARFRP1 is known to act at the trans-Golgi (8.Zahn C. Hommel A. Lu L. Hong W. Walther D.J. Florian S. Joost H.G. Schürmann A. Knockout of Arfrp1 leads to disruption of ARF-like1 (ARL1) targeting to the trans-Golgi in mouse embryos and HeLa cells.Mol. Membr. Biol. 2006; 23: 475-485Crossref PubMed Scopus (39) Google Scholar), we next isolated intracellular fractions (ER and Golgi compartments) from liver homogenates of Arfrp1flox/flox and Arfrp1liv−/− mice by gradient centrifugation and detected the apoprotein and triglyceride concentrations. As expected, ARFRP1 associated with the trans-Golgi fractions that were syntaxin-6 positive (Fig. 4A). In the ER (positive for calnexin), levels of ApoB48 were not different between the genotypes. However, in the Golgi fractions (trans- as well as cis/median-Golgi) of Arfrp1liv−/− livers, we detected more ApoB48 than in the corresponding fractions from Arfrp1flox/flox livers (Fig. 4B). Comparing ApoB48 levels of all fractions from Arfrp1flox/flox and Arfrp1liv−/− livers, it appears that the latter contain more ApoB48 (supplementary Fig. III). Measurement of the triglycerides in the intracellular fractions showed only moderate differences. In samples of Arfrp1liv−/− liver the triglyceride concentrations were higher in the cis/median-Golgi (positive for p115) but lower in the ER fractions, whereas the trans-Golgi fractions exhibited similarly low levels in both genotypes (Fig. 4C). ApoA1, as the major protein component of HDLs, is synthesized in the liver and the intestine (25.Januzzi J.L. Azrolan N. O'Connell A. Aalto-Setala K. Breslow J.L. Characterization of the mouse apolipoprotein Apoa-1/Apoc-3 gene locus: genomic, mRNA, and protein sequences with comparisons to other species.Genomics. 1992; 14: 1081-1088Crossref PubMed Scopus (36) Google Scholar). The amount of ApoA1 in the plasma of Arfrp1liv−/− mice was reduced as determined by WB and immunonephelometric assay (Fig. 5, upper and middle panels). However, Apoa1 mRNA was unaltered between genotypes (data not shown), whereas total ApoA1 was markedly reduced in Arfrp1liv−/− liver lysates (Fig. 5, lower panel) The isolation of triglycerides from the livers of fasted mice revealed a higher triglyceride concentration in Arfrp1liv−/− mice (Fig. 6B), which was also obvious after Oil-Red O staining of liver sections (Fig. 6A). This elevated lipid storage in Arfrp1liv−/− livers was accompanied by an increased hepatic expression of PLIN2 and localization of PLIN2 to the lipid droplets" @default.
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