ApoA-IV modulates the secretory trafficking of apob. and the size of triglyceride-rich lipoproteins

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1 ApoA-IV modultes the secretory trfficking of pob nd the size of triglyceride-rich lipoproteins Richrd B. Weinerg 1,2 Jmes W. Gllgher 3,4, Meliss A. Fritius 3, nd Gregory S. Shelness 3,5 Deprtments of Internl Medicine 1, Physiology & Phrmcology 2, nd Pthology 3 Wke Forest School of Medicine, Winston-Slem, North Crolin Current ddress 4 : Deprtment of Biology, Lincoln University, Lincoln University, PA To who correspondence should e ddressed: E-mil: gshelnes@wkehelth.edu; Tel.: ; Fx: Running Title: ApoA-IV nd secretion of pob-contining lipoproteins Arevitions: po, polipoprotein; BSA, ovine serum lumin; Dox, doxycycline, ER, endoplsmic reticulum; FBS, fetl ovine serum; MTP, microsoml triglyceride trnsfer protein; PL, phospholipid; RT-PCR, rel time PCR; SDS-PAGE, sodium dodecyl sulfte-polycrylmide gel electrophoresis; Tet, tetrcycline; TG, triglyceride; VLDL, very low density lipoprotein

2 ABSTRACT Although the evidence linking poa-iv expression nd triglyceride (TG)-rich lipoprotein ssemly nd secretion is compelling, the intrcellulr mechnisms y which poa-iv could modulte these processes remin poorly understood. We therefore exmined the functionl impct of poa-iv expression on endogenous pob, TG, nd VLDL secretion in stly trnsfected McA-RH7777 rt heptom cells. Expression of poa-iv modified with the ER retention signl, KDEL (poa-iv-kdel) drmticlly decresed oth the rte nd efficiency of endogenous pob secretion, suggesting presecretory interction etween poa-iv-kdel nd pob or pob-contining lipoproteins. Expression of ntive poa-iv using either constitutive or tetrcycline-inducile promoter, delyed the initil rte of pob secretion nd reduced the finl secretion efficiency y ~40%. However, wheres poa-iv-kdel reduced TG secretion y 75%, expression of ntive poa-iv cused 20-35% increse in TG secretion, ccompnied y ~55% increse in VLDL-ssocited pob, n increse in the TG:phospholipid rtio of secreted d<1.006 lipoproteins, nd 10.1 nm increse in pek VLDL 1 prticle dimeter. Ntive poa-iv expression hd negligile impct on expression of the MTP gene. These dt suggest tht y intercting with pob in the secretory pthwy, poa-iv lters the trfficking kinetics of pobcontining TG-rich lipoproteins through cellulr lipidtion comprtments, which, in turn, enhnces prticle expnsion nd increses TG secretion. Supplementry key words: lipoprotein ssemly; triglyceride; lipid trfficking; very low density lipoprotein; polipoprotein B; polipoprotein A-IV 2

3 INTRODUCTION The growth, reproduction, nd survivl of ll multicellulr orgnisms re dependent upon the efficient sorption, trnsport, nd storge of exogenous lipids (1). The ssemly nd secretion of triglyceride (TG)-rich lipoproteins y the intestine nd liver is centrl to these criticl metolic processes (2-4). TG-rich lipoprotein ssemly is thought to occur in two sequentil steps, in which the endoplsmic reticulum (ER)-loclized cofctor, microsoml triglyceride trnsfer protein (MTP), plys n essentil role (5, 6). In the first step, MTP directs the co-trnsltionl lipidtion of polipoprotein (po) B in the ER with phospholipid nd TG to form precursor prticles with dimeters of ~10-20 nm (7-10). In the second step, MTP promotes the movement of lipid from the cytosol into the ER to form luminl lipid droplets, which then fuse with pob precursor prticles in the ER nd/or post-er comprtments (11-16). In the liver this results in the secretion of VLDL prticles with dimeters rnging from nm; however, in the intestine pob-contining lipoproteins expnd into chylomicrons, with dimeters in excess of one micron (1, 4, 17). The ility of intestinl enterocytes to generte such lrge lipoproteins is unrelted to APOBEC1-medited editing of pob100 mrna to yield pob48 (18). Therefore, other intrcellulr fctors must ply role in the expnsion of chylomicrons prticles. ApoA-IV my e one such fctor. ApoA-IV, the lrgest memer of the exchngele polipoprotein fmily (19), is synthesized y intestinl enterocytes during lipid sorption nd secreted into mesenteric lymph on the surfce of nscent chylomicrons (20, 21). Although rod spectrum of physiologic functions hs een proposed for poa-iv in the three nd hlf decdes since its first description (22), sustntil ody of evidence supports role in chylomicron ssemly: 1) poa-iv synthesis in intestinl enterocytes increses up to 5-fold during sorption of long-chin ftty cids (23, 24), which requires chylomicron ssemly, ut not during sorption of short-chin ftty cids, which do not (25); 2) intestinl poa-iv 3

4 synthesis is simultneously inhiited when chylomicron secretion is locked y the surfctnt PL-81 (26); 3) within the enterocyte, poa-iv co-loclizes with pob in pre-golgi secretory trnsport vesicles (27); 4) the dynmic interfcil properties of poa-iv re idelly suited to stilizing expnding lipid interfces (28, 29); 5) plsm poa-iv levels rise promptly fter ftty mel (30), nd re depressed in intestinl disorders in which TG sorption is impired (31); 6) poa-iv expression in the intestinl cell line, IPEC-1, increses ulk TG trnsport y enling formtion nd secretion of lrger TG-rich lipoproteins (32, 33). Although the evidence linking poa-iv expression nd chylomicron ssemly nd secretion is compelling, the intrcellulr mechnisms y which poa-iv might impct these processes remin poorly understood. For poa-iv to fcilitte the expnsion of TG-rich lipoproteins it must interct either directly with pob or with the surfce of pob-contining lipoproteins within the secretory pthwy. In previous studies, we co-trnsfected poa-iv, pob, nd MTP into COS cells nd found tht poa-iv modified with the C-terminl ER retention signl, KDEL (po A-IV- KDEL), inhiited the secretion of pob constructs lrger thn pob25, suggesting the existence of protein-protein interction etween poa-iv nd specific sequences in the N-terminl region of the pob molecule in the erly stges of TG-rich lipoprotein ssemly (34). Herein, we hve extended these investigtions to exmine the effect of poa-iv on endogenous pob trfficking kinetics nd the physicl properties of secreted pob-contining TG-rich lipoproteins. To do so, we stly trnsfected poa-iv nd poa-iv-kdel constructs into McA-RH7777 rt heptom cells, which, unlike rodent liver, lck endogenous expression of poa-iv, nd thus provide null ckground (35), Our dt suggest tht poa-iv lters the secretory trfficking of pob nd/or pob-contining lipoproteins through cellulr lipidtion comprtments, which in turn, enhnces prticle expnsion nd, ultimtely, TG secretion. 4

5 EXPERIMENTAL PROCEDURES Plsmids Humn poa-iv nd poa-iv-kdel expression plsmids were descried previously (34). Plsmids contining the tetrcycline regultor (ptet-on) nd the tetrcycline response element (ptre2hyg) were otined from Clontech Lortories, Inc. (Mountin View, CA). Humn poa-iv cdna ws generted y polymerse chin rection using linerized poa-iv plsmid DNA s templte nd 5 nd 3 flnking primers contining engineered BglII nd MluI restriction enzyme sites, respectively. Following BglII nd MluI digestion, the cdna ws ligted to BmHI nd MluI-digested ptre2hyg. The integrity of the plsmid ws verified y sequence nlysis. Metolic rdioleling of trnsfected McA-RH7777 lines Stle nd inducile poa-iv expressing McA-RH7777 cell lines were generted s descried previously (36). Unless otherwise indicted, McA-RH7777 cells were grown in 100 mm dishes contining DMEM (Medi Tech, Mnsss, VA) with 10% FBS (growth medium). In some experiments with the inducile cells, 1 µg/ml doxycycline (Dox) ws dded to the medi. Two h prior to experiments, medi ws replced with growth medium supplemented with 0.8 mm oleic cid complexed to 1.5% ftty cid-free BSA (oth from Sigm-Aldridge, St. Louis, MO). Trnsfected cells were metoliclly rdioleled y incution for the indicted times with 100 µci/ml [ 35 S]Met nd Cys (EsyTg Express Protein Leling Mix; Perkin Elmer Life Sciences, Wlthm, MA) in Met- nd Cys-deficient DMEM (Gico-Life Technologies), lso supplemented with 10% FBS nd 0.8 mm oleic cid. Pulse-chse studies were performed in 60 mm dishes. After 10 min pulse with [ 35 S]Met/Cys s descried ove, cells were wshed nd incuted with olete-contining growth medium, contining n dditionl 2.5 mm Met nd 1 mm Cys, for the indicted times. Cell lyste nd medi smple were prepred s descried previously (34, 37) nd sujected to immunoprecipittion with rit nti-poa-iv or got nti-humn pob 5

6 (Acdemy Biomedicl, Houston, TX). Immune complexes were frctionted y SDS-PAGE nd dried gels were exposed to BioMx MS film cked with BioMx TrnsScreen-LE intensifying screen (Estmn Kodk, Rochester, NY) t 70 C; nd intensities were quntified using Moleculr Dynmics 445 SI phosphorimger or Fujifilm BAS5000 phosphorimger. These dt were used to clculte pob secretion efficiency, intrcellulr retention, nd degrdtion. Quntifiction of intrcellulr nd secreted lipids from trnsfected McA-RH7777 cells Trnsfected cells were plted in 150 mm dishes t 50% confluence. Twenty-four hours lter cells were incuted in growth medium supplemented with 0.8 mm olete for 2 h. Medi ws then replced with growth medium contining 0.8 mm olete nd 10 µci [ 3 H]olete nd the cells were incuted for n dditionl 18 h. Medi ws then removed, concentrted in Centricon YM-50 concentrtor (Millipore, Billeric, MA) nd sujected to Bligh-Dyer extrction (36, 38). Extrcted lipids were frctioned y thin lyer chromtogrphy using heptne-ether-cetic cid [90:30:1]; nds were visulized y incution in iodine vpor nd TG nd phospholipid (PL) contining frctions were cut from the plte nd quntified y liquid scintilltion counting (36). Protein concentrtion of cell extrcts ws determined y the icinchoninic cid method (39) using the Pierce BCA Protein Assy Regent kit (Thermo Scientific, Rockford, IL).Rockford, IL) Isoltion nd chrcteriztion of VLDL Medi from trnsfected cells ws djusted to volume of 3 ml with phosphte uffered sline nd centrifuged for 14 h t 100,000 rpm in TLA100.3 rotor using TL-100 tletop ultrcentrifuge (Beckmn Coulter, Bre, CA). The d<1.006 g/ml top (1 ml) nd d>1.006 g/ml ottom (2 ml) frctions were collected y tue slicing, nd pob ws isolted y immunoprecipittion nd nlyzed y 4-20% SDS-PAGE nd fluorogrphy. Lipoproteins in the VLDL 1 size rnge were isolted y cumultive rte density grdient ultrcentrifugtion, nd the 6

7 prticle size distriution ws nlyzed using Zetsizer Nno-S model ZEN1600 dynmic lser light-scttering instrument (Mlvern Instruments, Worcestershire, UK) t 633 nm (36). Gene expression in inducile McA-RH7777 cells Inducile poa-iv expressing McA-RH7777 cells were grown in 100 mm dishes contining DMEM with 10% FBS with nd without 1 µg/ml doxycycline (Dox). After 48 h cellulr RNA ws extrcted using TRIzol (Invitrogen). Totl RNA ws converted into cdna using Omniscript RT kits (Qigen, Vlenci, CA). RT-PCR ws performed on 7500 Fst Rel Time PCR System (Applied Biosystems, Foster City, CA). A typicl PCR rection (20 µl) contined 10 µl 2 Fst SYBR Green Mster Mix (Applied Biosystems), 1 µl ech of 5 µm forwrd nd reverse primers, nd 1:10 dilution of cdna. Copy numers were normlized to GAPDH The following primers were used: poa-iv, forwrd TTC CTG AAG GCT GCG GTG CTG, reverse CTG CTG AGT GAC ATC CGT CTT CTG; microsoml triglyceride trnsfer protein (MTP), forwrd CCT ACC AGG CCC AAC AAG AC, reverse CGC TCA ATT TTG CAT GTA TCC. Immunolot nlyses were performed with nti-mouse MTP monoclonl ntiody (BD Biosciences) nd rit ntihumn poa-iv, s descried previously (34, 40). 7

8 RESULTS Stle trnsfection of McA-RH7777 cells with poa-iv nd poa-iv-kdel lters endogenous pob secretion McA-RH7777 cells were stly trnsfected with poa-iv, po A-IV-KDEL, or control neomycin resistnce plsmid (Neo), stimulted with BSA-olete, nd rdioleled with [ 35 S]Met nd Cys for 4 h. Cell lyste nd medi frctions were sujected to immunoprecipittion with nti-poa-iv or nti-pob ntiody nd nlyzed y SDS-PAGE nd fluorogrphy. As shown in Fig. 1A, in cells trnsfected with poa-iv, oth the cell (C) nd medi (M) frctions displyed nd with the expected electrophoretic moility of ~48 kd (compre Lnes 1-4 to Lnes 5-8). In contrst, in cells trnsfected with poa-iv-kdel, the poa-iv nd ws present in cell lystes (rrowhed, Fig. 1B) ut not in the medi frctions, demonstrting, s expected, tht the KDEL ER retention-recycling signl drmticlly reduced poa-iv secretion. To ssess the impct of poa-iv-kdel on endogenous pob secretion, cell nd medi smples from Neo nd poa-iv KDEL-trnsfected cells were exmined for pob100 content. As shown in Fig. 1C, poa-iv- KDEL expression resulted in ~75% reduction in pob in medi compred to Neo cells (compre Lnes 2, 4, nd 6, with Lnes 8, 10, nd 12), suggesting n intrcellulr interction etween poa-iv-kdel nd either pob or pob-contining lipoproteins (34). To quntittively ssess the impct of poa-iv nd poa-iv-kdel expression on pob secretion kinetics, cells were sujected to 10 min pulse with [ 35 S]Met nd Cys followed y min chse with medi contining cold mino cids. As nticipted, poa-iv-kdel expression reduced oth the initil rte of pob secretion into the culture cell medi nd the finl secretion efficiency reltive to Neo controls y ~80%. Interestingly, ntive poa-iv lso reduced the initil rte nd finl efficiency of pob secretion to level intermedite etween the Neo nd poa-iv-kdel trnsfected cells (Fig. 1D). Recovery of pob from the cell lystes indicted tht in the poa-iv trnsfected cells there ws trend towrds greter intrcellulr 8

9 pob retention t 60 minutes (p=0.07); however, y 120 minutes very little of the initil pulseleled pob remined within the cells under ll three trnsfection conditions (Fig. 1E). As expected, pob underwent rpid degrdtion t ll times in the poa-iv-kdel trnsfected cells, wheres pob degrdtion ws slower, ut similr in ntive poa-iv cells nd Neo trnsfected cells (Fig. 1F). These dt estlish tht poa-iv expression lters the rte nd efficiency of endogenous pob secretion. Stle trnsfection of McA-RH7777 cells with poa-iv nd poa-iv-kdel lters lipid secretion We next exmined the effect of poa-iv nd poa-iv-kdel on lipid secretion. In ccord with its strong inhiitory effect on pob secretion, poa-iv-kdel expression cused drmtic decrese in the secretion of TG (Fig. 2A) nd PL (Fig. 2B), ssocited with reciprocl increse in cell-ssocited TG (Fig. 2D). In contrst, poa-iv incresed TG secretion (Fig. 2A), despite the decrese in pob secretion depicted in Figs.1C nd D, ut with little chnge in intrcellulr TG (Fig. 2D). The constnt intrcellulr TG levels could e consequence of homeosttic lnce etween secretion nd cellulr TG synthesis. ApoA-IV-induced stimultion in TG secretion ws lso ccompnied y ~40% increse in the medi TG:PL rtio reltive to Neo controls (Fig. 2C), consistent with n increse in the size of secreted pob-contining lipoproteins. Dox-induced poa-iv expression in McA-RH7777 cells lters pob nd lipid secretion To ssure tht the oserved differences in pob nd lipid secretion were not cused y clonl vrition mong stly trnsfected cell lines, cell line ws creted tht expressed poa-iv under the control of tetrcycline (Tet) responsive promoter. Upon incution in medi contining doxycycline (Dox), these cells displyed drmtic increse in poa-iv undnce in cell lystes nd medi (Fig. 3A, compre Lnes 1-6 to Lnes 7-12). To ssess how poa-iv 9

10 expression ffects the initil kinetics of pob secretion, pulse chse experiments were performed in cells induced for 48 h either without or with Dox in the tissue culture medi. As shown in Fig. 3B, Dox-induced poa-iv expression reduced the initil pob secretion rte y 44%, reltive to the Dox (-) control, in good greement with dt otined using the constitutive cell lines (Fig. 1D). To ssess the impct of poa-iv expression on pob prticle chrcteristics, cells were rdioleled with [ 35 S]Met nd Cys for 4 hours in the sence nd presence of Dox, nd medi were sujected to density grdient centrifugtion t d=1.006 g/ml. As shown in Figs. 4A nd 4B, the percentge of pob in the d<1.006 top frction incresed from 55% to 84% with Dox-induced poa-iv expression. As ws lso seen in constitutive poa-iv expression experiments (Fig. 2), Dox-induced poa-iv expression incresed medi TG (Fig. 4C) nd the medi TG:PL rtio (pnel E), gin suggesting tht poa-iv expression fcilitted the secretion of lrger, more TG-rich lipoproteins. Dox-induced poa-iv expression in McA-RH7777 cells increses VLDL prticle size As the previous results demonstrted tht poa-iv expression incresed TG secretion in ssocition with n incresed percentge of pob tht forms VLDL, we directly mesured the effect of poa-iv expression on VLDL prticle dimeter. When n inducile poa-iv cell line ws incuted with Dox for 48 hours, intrcellulr poa-iv incresed drmticlly (Fig. 5A). Medi from these Dox-treted cell ws then sujected to density grdient centrifugtion to otin the VLDL 1 frction, nd its prticle size distriution ws determined y dynmic light scttering (36). Addition of Dox cused rightwrd shift in VLDL 1 prticle distriution (Fig. 5B), cused y n increse in pek dimeter from 59.4 to 69.5 nm (Fig. 5C), nd n increse in the intensity-weighted men ("Z-verged") dimeter from to nm. Together these dt suggest tht Dox-induced poa-iv expression in McA-RH7777 cells fcilittes second step prticle lipidtion, therey resulting in the secretion of lrger, more TG-enriched lipoproteins. 10

11 Dox-induced poa-iv expression in McA-RH7777 cells does not lter MTP gene expression As it ws previously reported tht the effects of poa-iv on TG trnsport in IPEC1 cells ws ssocited with n increse in endogenous MTP expression (41), we ssessed whether the chnges in lipid nd lipoprotein secretion nd prticle chrcteristics oserved in our Doxinducile McA-RH7777 cells could lso e ttriutle in whole or prt to similr upregultion of MTP. To ssess the effect of poa-iv expression on MTP, cells were incuted for 48 h either without or with Dox nd poa-iv nd MTP gene expression ws mesured y quntittive RT-PCR. As shown in Fig. 6A, s expected, incution with Dox incresed poa-iv gene expression ~5-fold; however, in the sme cells MTP gene expression displyed nonsignificnt increse of 25% (Fig 6B). To confirm this outcome, extrcts from oth -Dox nd +Dox treted cells were sujected to immunolot nlysis. While poa-iv ws upregulted y Dox, no detectle chnge in MTP protein undnce ws oserved (Fig. 4C). 11

12 DISCUSSION Previous studies in porcine neontl intestinl cells (IPEC-1) hve demonstrted direct impct of poa-iv on the iogenesis of TG-rich lipoproteins (32, 33), specificlly tht expression of ntive nd C-terminl modified humn poa-iv constructs increses trnscellulr TG trnsport y enling secretion of lrger TG-rich lipoproteins, while decresing secretion of pob. As our previous study in COS cells found evidence for direct presecretory protein-protein interction etween poa-iv nd pob (34), we sked whether poa-iv could fcilitte lipoprotein expnsion y ltering the trfficking kinetics of pob. To exmine this issue, we used McA- RH7777 cells s model, ecuse they possesses the requisite cellulr mchinery (i.e., constitutive expression of pob nd MTP) to ssemle nd secrete TG-rich lipoproteins in the VLDL size rnge (42). Furthermore while rodent heptocytes express endogenous poa-iv nd its expression is strongly induced y incresed liver ft content (43-45), McA-RH7777 cells lck poa-iv expression, nd thus provide n essentilly null ckground (35). Using this system we oserved tht lthough trnsfection of poa-iv-kdel nd ntive poa-iv oth reduced the secretory efficiency of pob reltive to control cells, poa-iv-kdel mrkedly inhiited cellulr TG secretion, wheres ntive poa-iv incresed TG secretion into medi, the ssocition of pob with d<1.006 g/ml lipoproteins, nd the dimeter of VLDL 1 prticles. Considered together, these dt suggest tht poa-iv my ct s chperone tht modultes the trfficking of pob through the secretory pthwy, therey prolonging the residence time of nscent pobcontining prticles in cellulr comprtments where lipidtion occurs (13, 14, 16, 46), enhncing TG loding nd, ultimtely, incresing TG secretion. Although these dt corroorte the previous studies of Lu et l., who found tht constitutive nd Dox-induced over-expression of poa-iv in IPEC-1 cells fcilittes trnscellulr TG trnsport y incresing lipoprotein size (32, 33), they re seemingly t odds with work of Yo et l. (35). These investigtors oserved tht constitutive expression of rt poa-iv under control 12

13 of the constitutive CMV promoter in McA-RH7777 cells hd no ovious effect on the size of d<1.006 g/ml lipoprotein prticles s ssessed y electron microscopy (35). However, these studies used serum-free medi nd much lower concentrtion of olete (0.1 mm), which my hve resulted in su-optiml stimultion of pob nd VLDL secretion, therey oscuring n effect of poa-iv. Another importnt cvet in compring these studies is tht rt poa-iv is much more hydrophoic thn humn po A-IV (29) nd lcks one of four EQQQ repets tht re unique feture of the humn poa-iv C-terminus (47). As discussed elow, this deletion occurs in region tht my medite the interction etween poa-iv nd pob. Given the criticl role of MTP in the ssemly nd secretion of TG-rich lipoproteins (5) it is pertinent to consider whether the impct of poa-iv on TG secretion oserved in the present studies ws medited y n effect on MTP gene expression. Using the IPEC-1 intestinl cell model, Yo et l. (40) oserved tht Dox-induced expression of swine poa-iv incresed MTP gene expression y ~80%; in previous study this ws ssocited with 2-fold increse in TG secretion (33). However, Dox-induced expression of truncted "pig-like" humn poa-iv (lcking the distinctive C-terminl repeted EQQQ motif) incresed MTP gene expression y ~50%, ut in the previous study this ws ssocited with 25-fold increse in TG secretion (33). In the present study we oserved tht Dox-induced expression of humn poa-iv cused only 25% increse in MTP mrna undnce nd no detectle chnge in protein levels (Fig 6), with n ssocited 1.4-fold increse in TG secretion. Differences in the cell models nd the structures of the trnsfected poa-iv proteins preclude direct comprison mong these findings. Nonetheless, we elieve tht the non-significnt induction of MTP gene expression my not e the sole fctor determining the impct of poa-iv on TG-rich lipoprotein ssemly in our heptocyte model. 13

14 We previously noted tht the inhiitory effect of poa-iv-kdel on pob secretion in COS cells ppers etween pob21-25 nd is independent of pob lipidtion (34). Furthermore, IPEC-1 cell trnsfection studies found tht deletion of residues 345 to 357 in the poa-iv C-terminus increses trnscellulr TG trnsport 25-fold, ut tht dditionl trunction y only 11 residues completely ltes the effect (33). These dt re consistent with protein-protein interction etween specific domins in poa-iv nd pob, nd suggest tht poa-iv my ct s secretory pthwy chperone for pob. In this regrd, poa-iv displys slower secretion kinetics thn pob (34, 35), nd thus its ssocition with pob could retrd export of pobcontining TG-rich prticles from the ER nd/or slow their pssge through post-er lipidtion comprtments. ApoA-IV could lso serve s ridge etween pob nd other ER chperone proteins tht modulte pob secretion, such s ERp72, GRP94, clreticulin, nd BiP (48, 49). While these dt elucidte how poa-iv could modulte lipoprotein ssemly nd lipid sorption t the cellulr level, the puzzling issue hs remined tht no ovious phenotypes were oserved in either poa-iv knockout (50) or humn po A-IV trnsgenic mice (51). One possiility is tht in these studies lipid sorption ws determined y plsm TG ppernce curves fter single lipid olus. Given tht the intestine possesses lrge reserve cpcity for lipid sorption (52, 53), it is possile tht these nimls were not given ig enough lipid chllenge to discern n effect of poa-iv deletion or over-expression on dietry ft sorption efficiency. In this regrd, we recently reported tht lthough ft lnce nd lymph duct cnnultion studies in wild type nd poa-iv knock-out mice found no effect of poa-iv on totl dietry ft sorption, lipid trnsport studies using everted gut scs reveled tht poa-iv fcilittes TG trnsport in the proximl gut (54). These dt estlish tht the ility of poa-iv to fcilitte TG sorption t the cellulr level is in fct discernle t the su-orgn level, ut is msked in the intct niml y the functionl redundncy of the smll owel. Thus, the impct of poa-iv gene expression on intestinl lipid sorption in vivo my e mnifest only under 14

15 circumstnces where intestinl lipid trnsport cpcity is impired, such s the neontl period (55). For exmple, estrogen relted receptor-α knockout mice disply decresed intestinl poa-iv levels nd impired ft sorption in pups, ut not in dults (56). In summry, these dt estlish tht oth constitutive nd regulted expression of humn poa-iv in McA-RH7777 rt heptom cells lters the secretory trfficking of pob nd fcilittes TG trnsport y incresing the size nd core TG content of lipoproteins. When considered in the light of previous work on the structure nd function of poa-iv nd pob, we propose tht y functioning s secretory chperone for pob, poa-iv increses the residence time of nscent pob-contining lipoproteins in intrcellulr comprtments where second step lipidtion occurs, therey llowing more time for them to undergo core expnsion efore secretion. In the liver, incresed poa-iv expression induced y heptic ft ccumultion could ply role in mediting more efficient TG export (57). In the intestine, such intrcellulr ctions of poa-iv, under certin specific physiologicl conditions, could fcilitte more efficient sorption of dietry lipids. 15

16 Acknowledgments This work ws supported y Ntionl Institutes of Helth grnts HL49373 (G.S.S.) nd HL30897 (R.B.W.). J.W.G ws supported y predoctorl fellowship from the Americn Hert Assocition, Mid-Atlntic Affilite 16

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19 29. Weinerg, R. B., V. R. Cook, J. A. DeLozier, nd G. S. Shelness Dynmic interfcil properties of humn polipoprotein A-IV nd B17 t the ir/wter nd oil/wter interfce. J.Lipid Res. 41: Green, P. H., R. M. Glickmn, J. W. Riley, nd E. Quinet Humn polipoprotein A-IV. Intestinl origin nd distriution in plsm. J.Clin.Invest. 65: Kog, S., Y. Miyt, A. Funkoshi, nd H. Iyshi Plsm polipoprotein A-IV levels decrese in ptients with chronic pncretitis nd mlsorption syndrome. Digestion 32: Lu, S., Y. Yo, S. Meng, D. Cheng, nd D. D. Blck Overexpression of polipoprotein A-IV enhnces lipid trnsport in neworn swine epitheli cells. J.Biol.Chem. 277: Lu, S., Y. Yo, X. Y. Cheng, S. Mitchell, S. Y. Leng, S. M. Meng, J. W. Gllgher, G. S. Shelness, G. S. Morris, J. Mhn, S. Frse, C. M. Mnsch, R. B. Weinerg, nd D. D. Blck Overexpression of polipoprotein A-IV enhnces lipid secretion in IPEC-1 cells y incresing chylomicron size. J. Biol. Chem. 281: Gllgher, J. W., R. B. Weinerg, nd G. S. Shelness poa-iv tgged with the ER retention signl KDEL perturs the intrcellulr trfficking nd secretion of pob. J. Lipid Res. 45: Yo, Z., S. J. Luer, D. A. Snn, nd S. Fzio ApoA-IV is secreted on discrete HDL prticles y the rt heptom cell line McA-RH7777 trnsfected with ApoA-IV cdna. Arterioscler.Throm. 13: Blde, A. M., M. A. Fritius, L. Hou, R. B. Weinerg, nd G. S. Shelness Biogenesis of polipoprotein A-V nd impct its on VLDL triglyceride secretion. J. Lipid Res. 52: Sellers, J. A., nd G. S. Shelness Lipoprotein ssemly cpcity of the mmmry tumor-derived cell line C127 is due to the expression of functionl microsoml triglyceride trnsfer protein. J.Lipid Res. 42: Sellers, J. A., L. Hou, H. Athr, M. M. Hussin, nd G. S. Shelness A Drosophil microsoml triglyceride trnsfer protein homolog promotes the ssemly nd secretion of humn polipoprotein B - Implictions for humn nd insect lipid trnsport nd metolism. J.Biol.Chem. 278: Smith, P. K., R. I. Krohn, G. T. Hermnson, A. K. Mlli, F. H. Grtner, M. D. Provenzno, E. K. Fujimoto, N. M. Goeke, B. J. Olson, nd K. D. C Protein ssy using icinchoninic cid. Anl. Biochem. 150: Cheng, D., P. S. McArthur, S. Rong, J. S. Prks, nd G. S. Shelness Alterntive splicing ttenutes trnsgenic expression directed y the ApoE promoter-enhncer sed expression vector, pliv11. J. Lipid Res. 51: Yo, Y., S. Lu, Y. Hung, C. C. Beemn-Blck, R. Lu, X. Pn, M. M. Hussin, nd D. D. Blck Regultion of microsoml triglyceride trnsfer protein y polipoprotein A-IV in 19

20 neworn swine intestinl epithelil cells. Am. J. Physiol. Gstrointest. Liver Physiol. 300: G357- G Borén, J., S. Rusteus, nd S.-O. Olofsson Studies on the ssemly of polipoprotein B-100- nd B-48-contining very low density lipoproteins in McA-RH7777 cells. J.Biol.Chem. 269: Lngner, C. A., E. H. Birkenmeier, O. Ben-Zeev, M. C. Schotz, H. O. Sweet, M. T. Dvisson, nd J. I. Gordon The ftty liver dystrophy (fld) muttion. A new mutnt mouse with developmentl normlity in triglyceride metolism nd ssocited tissue-specific defects in lipoprotein lipse nd heptic lipse ctivities. J. Biol. Chem. 264: Horton, J. D., N. A. Shh, J. A. Wrrington, N. N. Anderson, S. W. Prk, M. S. Brown, nd J. L. Goldstein Comined nlysis of oligonucleotide microrry dt from trnsgenic nd knockout mice identifies direct SREBP trget genes. Proc.Ntl.Acd.Sci.USA 100: Inui, Y., Y. Keno, K. Fukud, T. Igur, T. Mkmur, K. Tokung, Kwt S., nd Y. Mtsuzw Modultion of polipoprotein gene expression in ftty liver of oese rts: enhnced APOA-IV, ut no APOB expression y high sucrose diet. Int J Oes Relt Met Disord. 21: Gusrov, V., J. Seo, M. L. Sullivn, S. C. Wtkins, J. L. Brodsky, nd E. A. Fisher Golgi-ssocited Mturtion of Very Low Density Lipoproteins Involves Conformtionl Chnges in Apolipoprotein B, ut Is Not Dependent on Apolipoprotein E. J. Biol. Chem. 282: Weinerg, R. B Identifiction of functionl domins in the plsm polipoproteins y nlysis of inter-species sequence vriility. J. Lipid Res. 35: Zhng, J. Y., nd H. Herscovitz Nscent lipidted polipoprotein B is trnsported to the Golgi s n incompletely folded intermedite s proed y its ssocition with network of endoplsmic reticulum moleculr chperones, GRP94, ERp72, BiP, clreticulin, nd cyclophilin B. J.Biol.Chem. 278: Linnik, K. M., nd H. Herscovitz Multiple Moleculr Chperones Interct with Apolipoprotein B during Its Mturtion. J. Biol. Chem. 273: Weinstock, P. H., C. L. Bisgier, T. Hyek, K. Alto-Setl, E. Sehyek, L. Wu, P. Sheiffele, M. Merkel, A. D. Essenurg, nd J. L. Breslow Decresed HDL cholesterol levels ut norml lipid sorption, growth, nd feeding ehvior in polipoprotein A-IV knockout mice. J. Lipid Res. 38: Alto-Setl, K., C. L. Bisgier, A. Ho, K. A. Kieft, M. G. Trer, H. J. Kyden, R. Rmkrishnn, A. Wlsh, A. D. Essenurg, nd J. L. Breslow Intestinl expression of humn polipoprotein A-IV in trnsgenic mice fils to influence dietry lipid sorption or feeding ehvior. J. Clin. Invest. 93: Crey, M. C., D. M. Smll, nd C. M. Bliss Lipid Digestion nd Asorption. Annu. Rev. Physiol. 45:

21 53. Field, F. J., nd S. N. Mthur Intestinl lipoprotein synthesis nd secretion. Prog.Lipid Res. 34: Simon, T., V. R. Cook, A. Ro, nd R. B. Weinerg Impct of murine intestinl polipoprotein A-IV expression on regionl lipid sorption, gene expression, nd growth. J. Lipid Res. 52: Levy, E., nd D. Ménrd Developmentl spects of lipid nd lipoprotein synthesis nd secretion in humn gut. Microsc. Res. Tech. 49: Crrier, J. C., G. V. Delois, C. Chmpigny, E. Levy, nd V. Giguère Estrogenrelted receptor lph (ERRlph) is trnscriptionl regultor of polipoprotein A-IV nd controls lipid hndling in the intestine. J.Biol.Chem. 279: Horton, J. D., H. Shimno, R. L. Hmilton, M. S. Brown, nd J. L. Goldstein Disruption of LDL receptor gene in trnsgenic SREBP-1 mice unmsks hyperlipidemi resulting from production of lipid-rich VLDL. J.Clin.Invest.. 103: Chung, S., A. K. Gere, J. Seo, G. S. Shelness, nd J. S. Prks A novel role for ABCA1-generted lrge pre-et migrting nscent HDL in the regultion of heptic VLDL triglyceride secretion. J. Lipid Res. 51:

22 FIGURE LEGENDS Figure 1. Effect of poa-iv nd poa-iv-kdel on endogenous pob secretion in McA- RH7777 cells. Pnels A nd B: Duplicte dishes of McA-RH7777 cells stly trnsfected with poa-iv (pnel A, lnes 5-8), poa-iv-kdel (pnel B, lnes 5-8) or control neomycin (Neo) vector (pnels A nd B, lnes 1-4) were rdioleled with [ 35 S]Met nd Cys for 4 h nd equl liquots of cell lystes (C) nd medi (M) were immunoprecipitted with nti-poa-iv ntiody followed y SDS-PAGE nd fluorogrphy. Arrowheds in pnel B show position of the poa-iv- KDEL nd. Pnel C: Neo nd poav-kdel cells were leled s descried ove nd cell lyste nd medi smples were immunoprecipitted with nti-pob ntiody nd nlyzed s ove. Pnels D nd E: Neo, poa-iv, nd poa-iv-kdel cells were pulse rdioleled for 10 min with [ 35 S]Met nd Cys nd then chsed for the indicted times with medi contining cold mino cids. At the end of ech chse period, leled pob100 protein in cell lystes nd medi ws quntified y immunoprecipittion, SDS-PAGE, nd phosphorimger nlysis to determine the percentge of newly synthesized pob tht ws secreted into the medi (D), retined within the cells (E), nd degrded (F). Dt re mens ± SE; n = 3. Where error rs re not visile the SE is less thn the size of the symol. Time points with different letters re (D): p=0.001 t 60 min, p=0.001 t 90 min, nd p=0.003 t 120 min; (F): p=0.044 t 60 min, p=0.055 t 90 min, nd p=0.014 t 120 min y ANOVA. Figure 2. Lipid secretion is ltered y expression of poa-iv nd poa-iv-kdel in McA- RH7777 cells. McA-RH7777 cells stly trnsfected with control neomycin vector (Neo), poa- IV (AIV) or poa-iv-kdel (AIV-KDEL) were incuted with medi contining 10% serum, 0.8 mm olete, nd 10µCi [ 3 H]olete for 18 h. Lipids were isolted from medi nd cell lystes y solvent extrction, seprted y TLC, nd quntitted y liquid scintilltion counting. Lipid dt re normlized to cell protein. Pnel A, medi TG; Pnel B, medi PL; Pnel C, medi TG:PL 22

23 rtio; Pnel D, cell TG. Dt re mens ± SD (n=3). Brs leled with different letters re p<0.05 y ANOVA. Figure 3. Inducile expression of poa-iv decreses the rte of endogenous pob100 secretion. Pnel A: Triplicte dishes of McA-RH7777 cells stly trnsfected with Tet-inducile poa-iv were incuted without (-) or with (+) 1 µg/ml doxycycline (Dox) for 48 hrs. Cells were then rdioleled with [ 35 S]Met nd Cys for 4 h, followed y immunoprecipittion of cell lystes (C) nd medi (M) with nti-poa-iv ntiody, SDS-PAGE nd fluorogrphy. Pnel B: Triplicte dishes of McA-RH7777 cells were incuted without or with Dox, s indicted, nd then sujected to 10 min pulse with [ 35 S]Met nd Cys followed y 0, 30 nd 60 min chse with medi contining cold mino cids. Dt re men ± SD (n=3); where error rs re not visile the SD is less thn the size of the symol. Time points with different letters re p=0.013 t 30 min nd p=0.034 t 60 min y ANOVA. Figure 4. Expression of poa-iv in McA-RH7777 cells increses the percentge of pob secreted s VLDL nd increses ulk TG secretion. Pnel A: Triplicte dishes of Tetinducile poa-iv McA-RH7777 cells were incuted without (-) or with (+) 1 µg/ml doxycycline (Dox) for 48 h. Cells were then rdioleled with [ 35 S]Met nd Cys for 6 h nd the medi ws sujected to density grdient centrifugtion to otin VLDL d<1.006 g/ml top (T) nd d>1.006 g/ml ottom (B) frctions. ApoB ws immunoprecipitted nd nlyzed y SDS-PAGE nd fluorogrphy. Pnel B: Bnd intensities were quntified y phosphorimger nlysis nd the percentge of totl pob in the top d<1.25 g/ml ws plotted. Pnels C, D, nd E: Cells treted with nd without Dox were incuted with [ 3 H]olete for 18 h nd rdioleled lipids were nlyzed s descried in Fig. 2. Dt re mens ± SD (n=3). Brs leled with different letters re p<0.05 y ANOVA. 23

24 Figure 5. Effect of poa-iv expression on VLDL size distriution. Tet-inducile poa-iv McA-RH7777 cells were incuted without (-) or with (+)1 µg/ml doxycycline (Dox) for 48 h nd then treted with DMEM contining 20% FBS nd 0.4 mm olete complexed to 0.75% BSA for 4 h. Pnel A: cell lystes (50 µg) were nlyzed y 12.5% SDS-PAGE followed y immunolot nlysis with nti-poa-iv ntiody; Pnel B: Medi were hrvested nd sujected to cumultive rte flottion ultrcentrifugtion, s descried (12, 36, 58) nd the size distriutions in the VLDL 1 frctions were determined using dynmic lser light scttering; Pnel C: pek VLDL 1 prticle dimeters (men ± SE; n=3, *p<0.001 y unpired t-test). Figure 6. Impct of Dox-induced expression of poa-iv on MTP mrna nd protein undnce. Tet-Inducile poa-iv McA-RH7777 cells were incuted in the sence (-) or presence (+) of 1 µg/ml doxycycline (Dox) for 48 h nd poa-iv (A) nd MTP (B) gene expression ws mesured y RT-PCR, normlized to GAPDH, nd shown s the fold increse in the presence of Dox. Dt re mens ± SD, n=3, *p<0.001 y unpired t-test. C: Immunolot nlysis of trnsfected humn poa-iv nd endogenous rt MTP. 24

25 Fig. 1 Neo A poaiv C M C M C M C M poaiv 1 B Neo poaiv-kdel C M C M C M C M poaiv 1 C Neo poaiv-kdel C M C M C M C M C M C M (% initil) c c c E Time (min) Time (min) F (% initil) pob Degrdtion pob Secretion D 7 (% initil) 2 Cell Associted pob 1 Time (min) 25 pob

26 Fig. 2 Medi TG (dpm/mg cell protein) Medi TG:PL Rtio A C c c Medi PL (dpm/mg cell protein) Cell TG (dpm/mg cell protein) B D 26

27 Fig 3 A -Dox +Dox C M C M C M C M C M C M poaiv B pob Secretion (% initil) -Dox +Dox Time (min) 27

28 Medi PL (dpm/mg cell protein) D Medi TG B C E 28 (dpm/mg cell protein) A Medi TG:PL Rtio % d < g/ml Fig. 4 -Dox +Dox T B T B T B T B T B T B pob100

29 Fig. 5 A -Dox +Dox poaiv B Volume (%) C Dimeter (nm) * -Dox +Dox 29

30 Fig. 6 A * B Reltive Expression C Dox +Dox poaiv MTP (Dox) 30