Proc. Natl. Acad. Sci. USAVol. 88, pp. 2254-2258, March 1991Medical Sciences

Transport of lipoprotein lipase across endothelial cells(triglyceride/fatty acids/adipose tissue/proteoglycans/glycosaminoglycans)

UDAY SAXENA, MICHAEL G. KLEIN, AND IRA J. GOLDBERG*Department of Medicine and Specialized Center of Research in Arteriosclerosis, Columbia University, College of Physicians and Surgeons,New York, NY 10032

Communicated by Richard J. Havel, December 26, 1990 (received for review March 8, 1990)

ABSTRACT Lipoprotein lipase (LPL), synthesized inmuscle and fat, hydrolyzes plasma triglycerides primarilywhile bound to luminal endothelial cell surfaces. To obtaininformation about the movement of LPL from the basal to theluminal endothelial cell surface, we studied the transport ofpurified bovine milk LPL across bovine aortic endothelial cellmonolayers. 125I-labeled LPL ('25I-LPL) added to the basalsurface of the monolayers was detected on the apical side of thecells in two compartments: (i) in the medium of the upperchamber, and (ii) bound to the apical cell surface. The amountof 125I-LPL on the cell surface, but not in the medium, reachedsaturation with time and LPL dose. Catalytically active LPLwas transported to the apical surface but very little LPLactivity appeared in the medium. Heparinase treatment of thebasal cell surface and addition of dextran sulfate (0.15 IAM) tothe lower chamber decreased the amount of 125I-LPL appear-ing on the apical surface. Similarly, the presence of increasingmolar ratios of oleic acid/bovine serum albumin at the basalsurface decreased the transport of active LPL across themonolayer. Thus, a saturable transport system, which requiresheparan sulfate proteoglycans and is inhibited by high concen-trations of free fatty acids on the basal side of the cells, appearsto exist for passage of enzymatically active LPL across endo-thelial cells. We postulate that regulation of LPL transport tothe endothelial luminal surface modulates the physiologicallyactive pool of LPL in vivo.

Lipoprotein lipase (LPL) bound to endothelial cell-surfaceheparan sulfate proteoglycans hydrolyzes triglycerides inplasma lipoproteins, especially chylomicrons and very lowdensity lipoproteins (1, 2). LPL is not synthesized by thevascular endothelial cells but is produced by underlyingadipocytes and myocytes (3-5). The physiologic actions ofLPL are mediated primarily by the pool ofLPL that is locatedat the luminal surface of the endothelium. Therefore, after itssynthesis and secretion, LPL must be transported from thebasal to the luminal side of the endothelial cell. How LPL isrouted from its initial site of synthesis to the endothelialsurface is not understood.Molecules can be transported across endothelial cells by

nonspecific and specific mechanisms (6, 7). The nonspecificprocesses include uptake by fluid phase and adsorptivepathways, which lead to transcytosis. Plasmalemmal vesi-cles, formed from the plasma membrane, shuttle betweenendothelial cell luminal and basal surfaces, discharging theircontents to the plasma or interstitial fluid compartments.Albumin transport across endothelial cells is an example ofsuch a process (8, 9). Smaller molecules like free fatty acids(FFAs) are thought to transfer across endothelial cells vialateral diffusion in cell membranes (10). Some movement canalso occur through gaps in the intercellular tight junctions,which form a barrier between the plasma and interstitial

compartments (6). Specific transport may require initialinteraction of a molecule with high-affinity receptors presenton the cell surface (11, 12). Insulin, for example, is bound toits receptor, internalized by endothelial cells, and then trans-ported from the apical to the basal endothelial cell surface inendocytic vesicles. LPL must be transported across endo-thelial cells in the opposite direction, from the basal to theapical surface of the cells.LPL transport was studied by using endothelial cell mono-

layers grown on a permeable polycarbonate filter system,which separates culture dishes into two compartments. Na-tive LPL and 251I-labeled LPL (125I-LPL) were added to thelower chamber and the appearance of radioactivity and LPLhydrolytic activity in the upper-chamber medium and on theapical cell surface were measured. In addition, the role ofproteoglycans in the transport of LPL was explored.

MATERIALS AND METHODSBovine Milk LPL: Purification and Radioiodination. LPL

was purified from fresh unpasteurized milk, radioiodinated,and stored at -70TC as described (13). At the time of storage,LPL preparations had a specific activity of approximately20-30 mmol ofFFA per hour per mg ofprotein. Labeled LPLhad a specific radioactivity activity of -500 dpm per ng ofprotein and >90%o of the counts were precipitated by 10%otrichloroacetic acid. Heat-inactivated LPL, prepared byheating LPL for 1 hr at 520C, was used in some experiments.

Endothelial Cell Monolayers. Primary cultures of bovineaortic endothelial cells were established as described forporcine aortic endothelial cells (13). Cells were plated onto25-mm polycarbonate filters (pore diameter, 3.0 gm; Nucle-pore), according to the method of Shasby et al. (14). Eachgelatin- and fibronectin-coated filter was seeded with 8-10 x105 cells in 1.5 ml of Dulbecco's modified Eagle's medium(DMEM) containing 10% bovine calf serum, antibiotics (100units of penicillin per ml and 100 gg of streptomycin per ml;Hazelton Research Products, Lenexa, KS), and glutamine(1%). The media in the upper chambers (1.5 ml) and lowerchambers (2.6 ml), separated by the filter, were replacedevery other day. Experiments were conducted 5-6 days afterseeding the endothelial cells. For 12 hr prior to each exper-iment, the cells were maintained in DMEM without calfserum to reduce the number of surface proteoglycan bindingsites occupied by serum proteins.The barrier function of the endothelial cell monolayer was

examined by several methods. After a 1-hr incubation at370C, there was a 3.5-fold greater amount of 125I-LPL in themedium of the upper chamber when filters that did notcontain any cells were used compared to the filters containingthe monolayers; a 10-fold greater amount of 1251-LPL was

Abbreviations: LPL, lipoprotein lipase; BSA, bovine serum albu-min; FFA, free fatty acid.*To whom reprint requests should be addressed at: Department ofMedicine, Columbia University, College of Physicians and Sur-geons, 630 West 168 Street, New-York, NY 10032.

2254

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Nadl. Acad. Sci. USA 88 (1991) 2255

found in the upper chamber if the filters were not coated withcollagen and fibronectin. In pilot experiments, the seedingdensity of the cells was increased 5-fold to =4 million cellsper well. This did not change the transport of '25I-LPL intothe medium, compared to the filters on which the usualnumber of cells was seeded, demonstrating that increasingthe number of cells seeded did not increase the barrierfunction of the monolayer. Transport rates of [14C]albuminfrom the basal to the apical side of the monolayers were<1.5% per hr, a rate similar to that reported by Stoll andSpector (15). At the conclusion of each LPL transportexperiment, the monolayers were stained with 2% toluideneblue to verify the uniformity of the monolayer.

Transport Studies. On the day of the experiment, culturemedia from both chambers were aspirated and the cells werewashed three times with DMEM containing 3% bovine serumalbumin (DMEM-BSA). All further steps were carried outwith DMEM-BSA. After washing, LPL (radioiodinated orunlabeled) was added to the lower chamber, and then thechambers were incubated at 370C for up to 1 hr. At the endof the incubation period, the chambers were transferred tothe cold room (40C) and washed three times with coldDMEM-BSA. LPL associated with the apical cell surfacewas then released by the addition ofDMEM-BSA containingheparin (100 units/ml; Organon) at 40C for 10 min to the upperand lower chambers. 1"I-LPL radioactivity present in thethree compartments-(i) medium from the upper chamber,(ii) apical cell surface, and (iii) basal cell surface-wasmeasured. In other experiments, LPL enzymatic activity inthese three compartments was assayed by using a highspecific activity substrate emulsion (16). To confirm thatactive LPL was present on the apical surface of endothelialcells and to avoid inhibition ofthe assay by heparin, LPL wasalso directly assayed while bound to the cells (13).

Cellular LPL Measurements. 1251-LPL associated with thecell (representing internalized LPL) was measured afterremoving surface-associated LPL with heparin, lifting thecell monolayers with 0.025% trypsin (370C for 5 min), cen-trifuging at 2500 x g for 10 min, and washing the cell pelletswith DMEM-BSA. To analyze the cell-associated 125I-LPL,the cell pellets were lysed in buffer containing 10 mMTris-HCl (pH 7.5), 1% 3-[(3-cholamidopropyl)dimethylam-monio]-1-propanesulfonate, and 1% octyl glucoside. The cellextract was sonicated for 20 sec with a Branson sonifier. Theextracts were then centrifuged at 2500 x g for 10 min and thesupernatants were precipitated with 10%o trichloroacetic acid.The precipitates were then analyzed by SDS/PAGE using a7.5% gel.

RESULTSConcentration and Time Dependence of LPL Transport

Across Monolayers. To determine whether LPL was trans-ported across endothelial cells from the basal to the apicalsurface, increasing concentrations of radioiodinated LPL(0.5-120 ug) were added to the lower chamber and the cellswere incubated for 1 hr at 37°C. With increasing amounts ofLPL (Fig. LA), the amount of 1251-LPL bound to the apicalsurface increased with the addition ofup to 60 ,ug of 125I-LPL,and then remained unchanged as more LPL was added (up to120 ,ug). In contrast, there was a linear increase in the amountof 115I-LPL in the medium from the upper chamber. Most(90% + 2%) of the 125I-LPL found in the medium and thatreleased by heparin from the apical surface was precipitableby 10% trichloroacetic acid. These results demonstrate that125I-LPL is transported across endothelial cells without muchdegradation.The time course of LPL transport was studied by using 20

pug of 1251-LPL added to the lower chamber. The amount of125I-LPL found on the apical surface (released by heparin)

A 800-'

600-j

fi' 400

i 200

n.Lv-0 40 80 120

LPL added (IJg)

B0C

-Ia.-C

0

C

D

151

301

:j200 L

-j

10

%£

0 30 60 90 120Time (minutes)

0.-i

0O

4

100

80

60

40

-20

. n

*10 aC-J0.8I-a

6 U

4 CD

2 Q

0 4

0 20 40 60Time (minutes)

0 20 40rime (minutes)

60

FIG. 1. Time and dose dependence of LPL transport across endo-thelial cell monolayers. (A) Effect of addition of increasing amounts ofLPL. 125I-LPL in DMEM-BSA was added to the lower chamber (basalsurface) of endothelial cell monolayers and the cells were incubated at37TC for 1 hr. Amounts of 15I-LPL in the upper-chamber medium (0)and released from the apical surface by heparin (A) are shown. Valuespresented are the means of three separate observations ± SEM. (B)Time course of transport of 1"I-LPL across endothelial cell monolay-ers. 125I-LPL (20 pAg) in DMEM-BSA was added to the lower chamberof endothelial cell monolayers. The cells were incubated at 37"C and125I-LPL present in the upper-chamber medium (e) and on the apicalcell surface (A) was measured at the indicated times. Each data pointrepresents the average oftriplicate dishes. (C) Time course oftransportofLPL activity. Unlabeled LPL (10jLg) in DMEM-BSA was added tothe lowerchamberand the experiment was then performed as describedfor 125I-LPL (see B), except that triglyceride hydrolytic activity ofLPLin the upper-chamber medium (e) and on the apical surface (A) wasassayed by using a high specific activity substrate emulsion (16). Theresults are the averages of duplicate determinations and are presentedas FFA generated per ml ofmedium per hr. (D) Direct measurement ofLPL activity on the apical surface of endothelial cells. In a separateexperiment, bovine LPL(lOpug) in DMEM-BSA was added to the lowerchamber and at the times indicated the media in the lower and upperchambers were removed, the cells were washed, and 1.5 ml ofDMEM-BSA containing 100 Al of substrate emulsion was added to the upperchamber and LPL activity was determined as described. Data shownare averages of assays from duplicate dishes for each time point.

increased for 20 min, remained relatively constant until 60min, and then decreased (Fig. 1B). A linear increase of

Medical Sciences: Saxena et al.

II.i-- ----

IIIIII

I'i

2256 Medical Sciences: Saxena et al.

radioactivity occurred in the upper-chamber medium for upto 120 min. A similar time-dependent transport of LPLenzymatic activity released from the apical surface by hep-arin was found; LPL activity increased for 20 min and thendecreased slightly over the next 40 min (Fig. 1C). In contrast,there was very little activity present in the medium at all timesbetween 5 and 60 min. These data measuring enzyme activitysuggest that only enzyme bound to the apical surface isenzymatically active. Radioiodinated LPL present in themedium in the prior experiments may represent transport ofenzymatically inactive LPL molecules. Alternatively, LPL inthe medium may have lost its catalytic activity during theexperiments.Measurements ofLPL activity released from the apical cell

surface by heparin might have underestimated the actualamount of transported LPL due to instability of the enzymeor interference with triglyceride hydrolysis in the assay byheparin. LPL activity assayed by addition of emulsion di-rectly to the upper-chamber medium is shown in Fig. 1D. Atime course of LPL transport similar to that in Fig. 1C wasobtained; LPL activity on the apical surface peaked at 20min. A comparison of the activity of LPL transported to theapical surface with that of the original preparation wasperformed. Using the data obtained in Fig. 1B and the LPLactivity in Fig. 1D, at 20 min the specific activity of trans-ported LPL was 2.1 mmol of FFA mg-1'hr-1. This specificactivity was greater than the specific activity of 1.6 mmol ofFFA-mg-1hr-1 of the same preparation of purified LPLincubated in buffer for 20 min at 370C. In contrast, at 20 minthe specific activity of LPL in the upper-chamber mediumwas markedly reduced (0.05 mmol ofFFA mg-1-hr-1). Thus,LPL on the apical cell surface appeared to be protectedagainst inactivation when compared to LPL in the medium orcontrol incubated LPL.To further examine the hypothesis that enzymatically

inactive LPL may be preferentially transported directly to themedium, LPL was deliberately heat inactivated. Althoughthis enzyme was enzymatically inactive, SDS/PAGE analy-sis showed that the heat-inactivated LPL was the same sizeas unheated LPL. Use of the heated enzyme produced a2.8-fold increase in the amount ofLPL present in the mediumcompared to unheated LPL. By contrast, compared to con-trol LPL, heat inactivation of LPL reduced the amountbound to the apical surface by 40%. These data suggest thatinactive LPL may be preferentially transported to the me-diumRequirement of Basal Heparan Sulfate Proteoglycans for

LPL Transport. To test whether specific transport of LPLacross the cells requires initial binding of LPL to heparansulfate proteoglycans on the basal cell surface, the basal sideof the monolayers was treated with heparinase. In anotherexperiment, LPL binding to proteoglycans was reduced bythe addition of dextran sulfate to the lower chamber. Theheparinase treatment decreased the amount of 125I-LPLbound to the basal cell surface by >80o relative to controlcells (not treated by heparinase) (Fig. 2). The amount of125I-LPL transported to the apical cell surface decreased by62%. Heparinase pretreatment did not alter the binding ofLPL to the apical cell surface, suggesting that heparan sulfatebinding sites on that surface were not affected by the pre-treatment. With dextran sulfate addition to the lower cham-ber, there was an 88% decrease in the amount of 125I-LPLbound to the basal cell surface compared to control incuba-tion (no dextran sulfate) and a corresponding decrease in theamount of 125I-LPL found on the a ical surface (95% de-crease). In contrast, the amount of 5I-LPL transported tothe medium in the upper chamber did not change with dextransulfate addition and decreased only 11% with heparinasetreatment. These results suggest that the binding of LPL toheparan sulfate proteoglycans on the basal cell surface is

-. on

20-_

.1

If

-Henari'nase Dextrar, -:"IfratL

FIG. 2. Effect of heparinase pretreatment and dextran sulfate onthe transport of LPL. The basal surface of endothelial cell mono-layers was treated with 0.1 unit of heparinase (ICN) for 2 hr at 370Cprior to adding 125I-LPL (8 ,ug). In a separate experiment, 125I-LPL(8 ,ug) was added to the bottom chamber together with dextran sulfate(0.15 1uM). The cells were then incubated at 3TC for 1 hr. 125I-LPLin the upper-chamber medium (n) and 125I-LPL released from theapical (a) and basal (a) cell surfaces after addition of heparin weredetermined. Results are expressed as percent ofa control incubation.

required for the transport of LPL to the apical cell surface,whereas the transport into the upper-chamber medium isminimally affected by the removal of heparan sulfate bindingsites.

Internalization of LPL During Transport. To examinewhether LPL bound to the basal cells surface was internal-ized under conditions in which LPL was transported to theapical cell surface, 125I-LPL was bound to the basal surfacefor 2 hr at 40C and the monolayers were then incubated for 20min at either 40C or 370C and cellular 125I-LPL was measured.1251-LPL on the apical cell surface was 41% lower at 4°C thanat 37rC and there was a >80%o decrease in the amount ofcell-associated radioactivity at 40C. When 1251-LPL and dex-tran sulfate were added together to the lower chamber, a64%decrease in the amount ofLPL radioactivity associated withthe cells relative to control cells was observed. However,when dextran sulfate was added to the upper chamber ofmonolayers in which 1251-LPL was present in the lowerchamber, there was no decrease (and in some experimentsthere was a small increase) in the cell-associated radioactiv-ity. Analysis of the cell-associated radioactivity (Fig. 3)showed that the mobility of 1251-LPL associated with the cellswas identical to that of control 1251-LPL. Because internal-ization occurs under the same conditions that demonstratetransport of 125I-LPL to the apical surface, it suggests thatinternalization is required for proteoglycan-mediated LPLtransport.A second question is whether LPL that moves to the

upper-chamber medium requires intracellular pathways ortransfers via paracellular mechanisms. If LPL in the upperchamber is decreased with treatments that affect cellular

9

69

1 2

-46-3.

FIG. 3. SDS/polyacrylamide gel analysis of control and inter-nalized 1251-LPL. Internalized LPL from endothelial cells (lane 2)and 125I-LPL incubated under similar conditions in the absence ofcells (lane 1) were applied to a 7.5% polyacrylamide gel. The gel wasrun at 20 mA for 60 min, fixed, and exposed to Kodak X-Omat filmfor 2 weeks. Approximately 2000 cpm was applied to each lane.

G-1

Proc. Natl. Acad. Sci. USA 88 (1991)

Proc. Natl. Acad. Sci. USA 88 (1991) 2257

functions, this would support a role for an intracellularpathway of LPL transport to the medium. Incubation at 40Creduced the amount ofLPL found in the upper chamber after1 hr by -5O%. When cells were treated with colchicine orsodium cyanide, however, an increase rather than a decreasein LPL was found in the upper chamber. This occurreddespite a decrease in the amount of internalized 1251-LPL(53% decrease with colchicine and 45% decrease with sodiumcyanide), also observed at 40C, and suggested that thesemetabolic inhibitors may have disrupted the integrity of theendothelial monolayers (17). Thus, whether LPL movementto the upper-chamber medium is via cellular or paracellularmechanisms cannot be concluded from these experiments.

Inhibition of LPL Transport by Increased FFA Concentra-tions. We have previously shown that FFAs can dissociateLPL bound to endothelial cell surfaces (13, 18) and hypoth-esized that addition of FFA should decrease the transport ofLPL to the apical endothelial surface. To examine the effectof FFAs on LPL transport, increasing molar ratios of oleicacid/BSA were added to the lower chamber together withunlabeled LPL. The addition of oleic acid markedly de-creased the amount of LPL enzymatic activity that wastransported to the apical cell surface (Fig. 4A). No LPLactivity was measurable in the medium (data not shown),consistent with the results shown in Fig. 1C.To specifically assess the effects of fatty acids on the

transport ofLPL protein that was bound to the basal surfaceof the cells, 1251-LPL was allowed to bind to the basal surfacefor 2 hr at 40C. The medium containing the unbound"-5I-LPLwas removed from the lower chamber and replaced withmedium containing increased molar ratios ofoleic acid/BSA.The cells were incubated for 1 hr at 370C. With increasing

0

C.)

0

a.

o100

80'

60

40f

20

0

0

0)

c

-J

if)NY

'A

0.5:11:1 2:1 4:1

2:1

Oleic acid/BSA, mol

FIG. 4. Effect of oleic acid/BSA molar ratios on the transport ofLPL. (A) LPL activity. LPL (8 ug) was added together withincreasing molar ratios of oleic acid/BSA to the lower chamber andthe cells were incubated at 370C for 1 hr. After the incubation, LPLactivity on the apical surface was determined by using a radioactivetriglyceride emulsion as described. (B) '"I-LPL. 125I-LPL (8 ,ug) wasallowed to bind to the basal surface of the monolayer for 2 hr at 4TC.The medium in both chambers was changed, increasing molar ratiosof oleic acid/albumin were added to the lower chamber, and theendothelial cell monolayer was incubated for 1 hr at 370C. Radioac-tivity in the upper-chamber medium (0) and on the apical cell surface(*) was determined.

concentrations of fatty acids in the lower-chamber medium,less 125I-LPL was found on the apical surface (Fig. 4B).Therefore, higher concentrations ofoleic acid decreased totalLPL protein transport, as well as transport of catalyticallyactive LPL to the apical surface. In contrast to cell-surfaceLPL, the amount of '25I-LPL transported to the mediumincreased with addition of FFA. This increase of 1"I-LPL inthe medium may have been due to increased nonspecifictransport ofLPL by the cells or to an increase in transport ofLPL that was inactivated by high concentrations of FFA.

DISCUSSIONOur results demonstrate that LPL protein and LPL catalyticactivity are transported across endothelial cell monolayers.The transported LPL was found in two compartments: (i)LPL bound to the apical cell surface, which was specificallyassessed by its release with heparin, and (ii) LPL present inmedium in the upper chamber. Results presented in Fig. 1 Cand D showed that the LPL bound to the apical cell surfaceeffectively hydrolyzed a substrate triglyceride emulsion.Although 125I-LPL was found in the medium, very little activeLPL was found in this compartment. Data from other exper-iments showed that the LPL increases in these compartmentswere not parallel (Fig. 1 A and B). The medium LPLincreased linearly with addition of increasing concentrationsof LPL to the lower chamber, whereas the increase in theamount of LPL bound to the apical cell surface appeared toreach saturation.LPL transport to the apical surface and to the upper-

chamber medium appears to involve two distinct pathways.Several experiments suggest that apical surface LPL andLPL in the medium are not precursors of each other. Byblocking the binding of LPL to heparan sulfate proteogly-cans, we were able to markedly decrease only the LPL on theapical surface. We therefore believe that the apical surfaceLPL is not the precursor of most of the LPL in the medium.An alternative possibility, that LPL is first transported to theupper-chamber medium and then associates with heparansulfate proteoglycans on the apical surface, is also unlikelybecause the LPL increase on the apical cell surface occursprior to, not after, LPL appearing in the medium. In addition,if LPL was first bound to the basal surface (Fig. 4B) or waspresent in the lower chamber (Fig. lA), similar amounts ofLPL appeared on the apical surface despite marked differ-ences in the amount of LPL in the upper-chamber medium.Thus, our results are most consistent with two transportpathways: one that delivers LPL to the apical surface ofendothelial cells, and one through which inactive LPL travelsto the upper-chamber medium.

Is the pathway of LPL transport to the apical surfacedemonstrated in our studies operative in vivo? One issue tobe considered when responding to this question is whetherthe pathway allows LPL to retain its enzymatic activity. Asshown in Fig. 1D, LPL transported to the apical surface ofendothelial cells does not lose its enzymatic activity and,compared to LPL incubated in medium alone, actually has agreater specific activity. Moreover, the calculated specificactivity of apical surface LPL is likely to be an underestimateof the true specific activity of the enzyme. This is becauseLPL bound to solid supports is markedly less active thanLPL in solution (19).A second issue is whether the efficiency of LPL transport

is comparable to other receptor-mediated transcytosis path-ways. King and Johnson (11) reported that =8% of insulinadded to the apical surface of endothelial cells was trans-ported to the lower chamber within 1 hr. In our studies, LPLwas added to the lower-chamber medium, separated from thebasal surface of the cells by the polycarbonate filter. Thus,the concentration of LPL directly in contact with the cells

Medical Sciences: Saxena et aL

2258 Medical Sciences: Saxena et al.

was initially somewhat less than that in the chamber. If theamount of LPL on the apical surface is compared to thatadded to the lower chamber (Fig. 1), its transport appears tobe very low (<2%). However, when LPL was first bound tothe basal cell surface at 4'C (Fig. 4B) followed by a 1-hrincubation at 370C, the amount of LPL present on the apicalsurface was 15-22% of that originally on the basal surface.Therefore, once bound to the basal surface, LPL is efficientlytransported across endothelial cell monolayers.Our studies suggest a crucial role for heparan sulfate

proteoglycans in the movement of enzymatically active LPLacross endothelial cells. A requirement for the participationof basal cell-surface heparan sulfate proteoglycans in thetransport of LPL to the apical cell surface was tested bydigestion of glycosaminoglycans with heparinase and byaddition of dextran sulfate, which prevents association of theenzyme with heparan sulfate. Both interventions decreasedthe amount of LPL transported to the apical surface, sug-gesting that LPL is transported to the apical surface byreceptor-mediated processes in which heparan sulfate pro-teoglycans serve as receptors for LPL. Such a role forproteoglycans in cellular transport has not previously beendescribed. Cell-surface proteoglycans are integral mem-brane-bound proteins and are ideally suited to serve thisfunction (20). Furthermore, we have reported (21) that theinteraction of LPL with its endothelial cell-surface bindingsite is resistant to dissociation with acid pH, perhaps ex-plaining why LPL is not targeted to lysosomes. Using im-munohistochemistry, Blanchette-Mackie et al. (22) havedemonstrated LPL protein within endothelial cells that maybe in the process of transcytosis.The transport of inactive LPL into the medium may be via

a nonspecific fluid phase or adsorptive mechanisms, pro-cesses that are operative in vascular endothelial cells (7).Such mechanisms are sensitive to molecular shape, size, andcharge (6, 23). In this regard, nonspecific transport pathwaysmay preferentially transport inactive, monomeric LPL mol-ecules as opposed to dimeric active LPL (24, 25). Therefore,the LPL transport pathway leading to the medium may not beinvolved in movement of catalytically active LPL, but itmight allow for disposal of inactive enzyme from the inter-cellular space. The recent description of a mutant, inactive,non-heparin-binding LPL molecule found in the plasma of ahyperchylomicronemic patient (26) suggests that a non-proteoglycan-mediated pathway for transport of LPL func-tions in vivo.

Addition of oleic acid to the basal surface of endothelialcells decreased the transport of active LPL across endothelialcells. This reduction may be due to diminished binding ofLPL to the basal surface, since high concentrations of oleicacid decrease LPL binding to endothelial cells (13, 18). Thisdecrease in LPL transport to the luminal cell surface mayplay a role in regulation of LPL activity specifically inadipose tissue. Adipose tissue LPL activity is relatively highin fed animals and is reduced during fasting (2, 3). Althoughfeeding increases LPL activity, Semb and Olivecrona (27)demonstrated that LPL protein synthetic rate is the same inadipose tissue from fed and fasting animals. Ong and Kern(28) reported that human LPL mRNA and protein levels arenot different during fasting and feeding. Thus, alterations inLPL activity may be regulated by mechanisms other thanthose affecting LPL mRNA transcription and LPL proteinsynthesis. Paradoxically, Doolittle et al. (29) showed thatLPL mRNA and protein in rat adipose tissue increasedduring fasting, while LPL activity decreased. These investi-gators postulated that more LPL protein may be enzymati-

cally inactive in adipose from fasting compared to fed ani-mals.Adipocyte hormone-sensitive lipase is activated during

starvation, causing the release of FFA into the interstitialspace. If our in vitro results are relevant to the in vivosituation, the generation of FFA during starvation coulddecrease LPL transport to the endothelial luminal surface.This would lead to increased LPL protein in interstitial fluid,where it can be rapidly inactivated, and to decreased LPLactivity in adipose tissue. Thus, a change in LPL activitywithout a change in LPL mass could occur. Such a mecha-nism for regulation ofLPL via changes in LPL transport maybe a posttranslational process that, in part, modulates LPLactivity during feeding and fasting.

We thank Ester Rosenstark for assistance with preparation of themanuscript. This work was supported by Grant HL 21006(SCOR)from the National Heart, Lung, and Blood Institute. I.J.G. is therecipient of an Established Fellowship from the New York HeartAssociation.

1. Cheng, C. F., Oosta, G. M., Bensadoun, A. & Rosenberg,R. D. (1981) J. Biol. Chem. 256, 12893-128%.

2. Garfinkel, A. S. & Schotz, M. C. (1987) in Plasma Lipopro-teins, ed. Gotto, A. M., Jr. (Elsevier, New York), pp. 335-337.

3. Eckel, R. H. (1989) N. Engl. J. Med. 320, 1060-1068.4. Nilsson-Ehle, P., Garfinkel, A. S. & Schotz, M. C. (1980)

Annu. Rev. Biochem. 49, 667-693.5. Olivecrona, T. & Bengtsson-Olivecrona, G. (1987) in Lipopro-

tein Lipase, ed. Borensztajn, J. (Evener, Chicago), pp. 15-58.6. Simionescu, N. (1983) Phys. Res. 63, 1537-1579.7. Renkin, E. M. (1985) J. Appl. Physiol. 58, 315-325.8. Shasby, D. M. & Shasby, S. S. (1985) Circ. Res. 57, 903-908.9. Siflinger-Birmboim, A., Del Vecchio, P. J., Cooper, J. A. &

Malik, A. B. (1986) J. Appl. Physiol. 61, 2035-2039.10. Scow, R. 0. & Blanchette-Mackie, J. E. (1985) Prog. Lipid

Res. 24, 197-241.11. King, G. L. & Johnson, S. M. (1985) Science 227, 1583-1586.12. Hachiya, H. L., Halban, P. A. & King, G. L. (1988) Am. J.

Physiol. 255, C459-C464.13. Saxena, U., Witte, L. D. & Goldberg, I. J. (1989) J. Biol.

Chem. 264, 4349-4355.14. Shasby, D. M., Shasby, S. S., Sullivan, J. H. & Peach, M. J.

(1982) Circ. Res. 51, 657-661.15. Stoll, L. L. & Spector, A. A. (1987) J. Cell. Physiol. 133,

103-110.16. Eckel, R. H., Goldberg, I. J., Steiner, L. & Paterniti, J. R., Jr.

(1988) Diabetes 37, 610-615.17. Shasby, D. M., Shasby, S. S., Sullivan, J. M. & Peach, M. J.

(1982) Circ. Res. 51, 657-661.18. Saxena, U. & Goldberg, I. J. (1990) Biochim. Biophys. Acta

1043, 161-168.19. Posner, I., Wang, C. S. & McConathy, W. J. (1983) Arch.

Biochem. Biophys. 226, 306-316.20. Fransson, L. A. (1987) Trends Biol. Sci. 12, 406-411.21. Saxena, U., Klein, M. G. & Goldberg, I. J. (1990) J. Biol.

Chem. 265, 12880-12886.22. Blanchette-Mackie, E. J., Masuno, H., Dwyer, N. K., Olive-

crona, T. & Scow, R. 0. (1989) Am. J. Physiol. 256, E818-E828.

23. Schneeberger, E. E. (1983) Fed. Proc. Fed. Am. Soc. Exp.Biol. 42, 2419-2424.

24. DelVecchio, P. J., Siflinger-Birnboim, A., Shepard, J. M.,Bizios, R., Cooper, J. A. & Malik, A. B. (1987) Fed. Proc. Fed.Am. Soc. Exp. Biol. 46, 2511-2515.

25. Osborne, J. C., Jr., Bengtsson-Olivecrona, G., Lee, N. S. &Olivecrona, T. (1985) Biochemistry 24, 5606-5611.

26. Beg, 0. U., Meng, M. S., Skarlatos, S. I., Perviato, L., Brun-zell, J. D., Brewer, H. B., Jr., & Fojo, S. S. (1990) Proc. Natl.Acad. Sci. USA 87, 3474-3478.

27. Semb, H. & Olivecrona, T. (1989) Biochem. J. 267, 505-511.28. Ong, J. M. & Kern, P. A. (1989) J. Clin. Invest. 84, 305-311.29. Doolittle, M. H., Ben-Zeev, O., Elovson, J., Martin, D. &

Kirchgessner, T. G. (1990) J. Biol. Chem. 265, 4570-4577.

Proc. Natl. Acad. Sci. USA 88 (1991)