Biochimica et Biophysica Acta, 1095 ( 1991) 30 - 38 © 1991 Elsevier Science Publishers B.V. All rights reserved 0167-4889/91/$03.50 ADONIS 0167488991002591
36
BBAMCR 13024
HDL3-retroendocytosis in cultured small intestinal crypt cells: a novel mechanism of cholesterol efflux Gerhard Rogler, Gerhard Herold and Eduard F. Stange Department of Internal Medicine ii, University of UIra, Ulm (F.K G.)
(Receiveu 29 March 1991) (Revisedmanuscriptreceived8 July 1991) Key words: High density ]ipoprotein; Receptor; Retroendocytosis; Cholesterol; IEC-6; (Rat intestinal crypt cell)
The present study in IEC-6 crypt.derived rat epithelial cells describes a retroendocytotic pathway for HDL~. These intestinal cells exhibited specific binding of apoE free HDL~ with a maximal binding capacity of 2980 n g / m g cell protein and a K a of 36A / z g / m l . Specific binding was competed for by HDL 3 but not by I,DL. Apparent internalisation of HDL~ was low, degradation was negligible and intact particles were rescereted into the medium within 2 h. Electron microscopic studies showed binding and Internalisation of gold-labeled HDL 3 in coated pit regions and transport in endosomes distinct from lysosomes to lipid droplets. De nero cholesterol sTnthosis from [~4C]octanoate was enhanced nearly 2-fold by HDL 3 and the surplus of newly formed cholesterol was recovered in the medium. It was concluded that intact HDL 3 was bound specifically to intestinal cells and was resecreted through a process of retroendocytasis probably mediating effiux of cellular cholesterol. Introduction The small intestinal mucosa is actively involved both in the absorption and synthesis of cholesterol [1,2]. Enterocytes also display receptors for low [3] and high density lipoproteins [4-8] which were reported to mediate rapid uptake and subsequent degradation of these particles. Thus, the mucosal cell is exposed to cholesterol from a multitude of sources and current evidence suggests that these pools of cholesterol are compartmentalized intracellulady [9,10]. The export of intestinal cholesterol is similarly complex because it may occur via sloughing of ceils from the villus tip or by formation and lymphatic secretion of lipoproteins, particularly chylomicrons [1]. A recent study in the live rat found absorption and lymphatic secretion of bile derived cholesterol in the absence of triaeylglyeerois in the lumen, i.e., under circumstances of limited ehyiomicron iormation [11]. Since HDL partides were suggested to mediate reverse cholesterol
transport in other tissues and cells [12,13], we hypothesized a similar role for this iipoprotein fraction in the gut, The present work describes the HDL3,-pathway in
Correspondence:G. Hero|d, Departmentof Internal Medicine It, Universityof UIm, Robert Koch Sir. 8, 7900 UIm, F.R.G.
eooo 0o 5000
~ 40 20
~4000
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80
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~g HDLa / ml m e d i u m
Fig, 1, lEe-6 cells at day 5 of culture were incubatedat 4oC for 2 h in 2 ml of DMEM supplementedwith 5% of lipoprotvin-deficient (LPD) FC$ and iodinated HDL3 in concentrations up iv 100 p,g lipoprotein prvtein/mL Unspecific binding was measured using a 50-fold surplus of unlabeled lipoprotein and binding procedures were stopped by chilling on ice for 15 rain. Specific binding was calculated from the difference between total and unspecificbinding. Maximal binding and Kd were determined using Scatchard plotting as shown above, o, unspecificbinding; v, specificbinding;o, total binding,
3i cultured small intestinal cells and its putative role in mediating cholesterol efflux from the gut.
2000
Methods and Materials
1750
Cell culture
1500
Rat intestinal epithelial cells IEC-6 were obtained from American Type Culture Collection (1592-CRL) and were used at passage 20-22. Cells were grown as monolayers on 100-ram plastic dishes and were mainrained in 10% Dulbecco's minimum essential medium ( D M E M ) supplemented with L-glutamine (2 raM), Larginine (0.4 mM), 0.1% non-essential amino acids, sodium lactate (2 mM), NaCO 3 (40 mM), glucose (10 raM), 0.3% phenol red, insulin (400 I.U./ml), 1% Pen Strep (Gibco), and 5% lipoprotein free fetal calf serum (FCS) in an atmosphere containing 8% CO2. Subcuttiration for experiments was done on 30 mm Millicell H A filters to allow accessibility of the basolaterai membrane [14]. The cells were routinely used at day 5 after plating, Low density iipoproteins (200 /~g/ml) were added to the medium for 24 h before the expertments. Other medium additions were supplemented as specified.
Preparation of lipoprotems Lipoprotein fractions were separated by ultracentrifugation of human plasma as described previously
l
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1000
o
750
500
250
o. 0
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2
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Fig. 3. IEC-6 cells
were
?
?
6
8
(h)
incubated at 37°C with 10 p.g iodil:ated
HDL~/ml for different time periods up to 8 h under conditions specified in Fig. 1. Unspecific binding was measured with a 50-fold surplus of unlabeled lipoprotein. Specificbinding (e), internalisation (o) and degradation (v) were calculated from the differences between their corresponding values of total and unspecific binding, lntemaSsation and degradation were determined as specified in Materials and Methods.
100
LDL
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ee
¢1 o. 1 2 5 0
. D
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,,D
~. _o
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_c
u
40 o
c
2O
i1
HOL 3 i O
i
i 100
i 200
i 300
,
i 400
i 500
ug llpoprotlln/ml medium Fig. 2. IEC-6 cells at day 5 of -'J!::~;c~'ci-¢incubated at 4°C for 2 h with 10 ~g iod[nated HDL 3 ml in 2 nil of DMEM supplemented with 5% of LPD-FCS. Simultanouslyeither unlabeled LDL or HDL 3 in concentrations up to 5~ ~g/ral were present in the medium. Specific binding was competed for by HDL3 but not by LDL. Percent values refer to the initial [ZZSl]l-lDL3binding without any unlabeled lipoptolein additions.
0
i
i
i
i
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i
20 40 60 B0 100 120 time
(rain)
Fig. 4. IEC-6 cellswere incubated at 4 ° C for 2 h as described in Fig. I together with 10 9-g iodinated H D L 3 / m ] medium. Sorrowly washing on ice was followed by subsequent warm up to 37°C and
incubation for different time periods up to 2 h. At any given time point, protein-bound radioactivity was simultaneously detem~ined in cells and medium. While specific binding shows subsequent loss with time, medium reiea~ of an intact lipoprotcin particle occurred in
increasingamounts. O, specificbinding;o, medium release.
32
A
~
I
¸
33 [15,16]. The fractions isolated were low density lipoproteins (LDL, d = 1.019-1.063 g/ml) as well as high density lipcproteins (HDL 3, d = 1.125-1.2i0 g/ml) HDL3 were rendered apoprotein E free by heparin sepharose affinity chromatography []7] as checked by sodium dodecyl sulfate polyacrylamide gel eleetrophoresis (SDS-PAGE).
Lipoprotein labeling Colloidal gold was prepared as described by Frens et al. [18] using a particle size of 20-40 nm. Conjugation of colloidal gold to HDL 3 was carried out using a modification of a method described for LDL [19]. 5.0 ml of a monodispe~e colloidal gold solution was added to 0.5 ml of the lipoprotein solution (100 F g / m l ) in 50 mM EDTA-buffer. After rapid conjugation the complex was stabile over a wide pH range. The appearance of colloidal gold-lipoprotein complexes was checked by the electron microscopic technique of negative staining. t25I-labeling of HDL 3 was performed by the method of Brown and Goldstein [20] with minor modifications. Final specific radioactivity of [t2SI]HDL~ varied from 400 to 600 e p m / n g protein.
Studies with iodinated lipoproteins All experiments were carried out in Falcon multiwell dishes with cells grown on milliceil HA filters. Cells were preineubated with 200 ~ g LDL-protein/ml medium for 48 h prior to the studies. The incubation mixture contained DMEM, 20 m g / m l lipoprotein-free FCS and 5-10 /~g/ml [tzSI]HDL3. Prior to binding experiments at 4 ° C or 37 ° C, ceils were washed with Tris-albumin buffer. For studies at 4°C, cells were chilled on ice 15 rain prior to the addition of the ice-cold incubation medium. After the incubation period, cells were washed three times with Tris-albumin buffer to remove unspeeifically .round lipoprotein. At the end of the incubation at 37 °C, cells were chilled on ice for 15 rain prior to washing. After the final wash the buffer was removed, cells were incubated with buffer containing 1 mM EDTA and 1 m g / m l trypsin for 1 h at 4 ° C. Cells were harvested from the filters with a rubber policeman and the cell suspension was transferred to microfuge tubes and s;:dimented by centrifugation at 3000 rpm. The counts in the supernatant represented the membrane-bound HDL 3. The washed
cell pellet was digested in 2 ml of 0.1 M NaOH and aliquots of 1.0 ml for counting (representing internalized HDL 3) and 20 ~1 for protein determination were taken. Degradation was measured as the amount of soluble radioactivity after trichloroacetic acid precipitation of the medium and removal of free iodine by oxidation and chloroform extraction according to standard methods [21]. Control incubations were carried out with cell-free medium. The time dependence of binding was studied with 10 /zg lipoprotein/ml medium. Unspecific binding was determined using a 50-fold excess of unlabeled HDL 3.
Studies with gold labeled lipoproteins Binding and internalisation experiments were carried out as described for [tZSl]HDL3 except for the incubation with trypsin buffer. Cells were fixed in a standard procedure with glutaraldehyde (3.5%) at 4 °C and stained with osmium tetroxide and lead citrate. Embedding was carried out with EPON. To obtain vertical slices through the basolateral membranes a second resin preparation positioned in a right angle was necessary for transmission electron microscopy. For scanning electron microscopy cells were cultured on Thermanox foils which were proved to be resistent to 'critical point "lrying' of the probes. Incubations were carried out with a concentration of 5 / ~ g / m l of gold-labeled lipoproteins, control incubations with a 50-fold excess of li0oprotein or I00/zl of the colloidal gold preparation used for labeling.
Cholesterol effiux Cells were cultured under standard conditions on plastic dishes and five dishes were pooled. Newly synthesized cholesterol was labeled with [t4C]octanoate as precursor. 0.1 mM unlabeled octanoate was present in the culture medium in addition to 0.5 #Ci [t4C]octanoate at a specific activity of 5300 d p m / n m o l at days 7-9. The incubation was stopped by chilling the dishes on ice. The medium was collected together with three washes of phosphate buffered saline plus albumin (1 mg/ml). Cells were sedimented by centfifugation and protein content was determined as described above. Lipids were extracted separately from cells and medium and cholesterol was isolated by thin layer chromatography (TLC) according to previously described meth-
Fig. 5, Binding of gold-labeled HDL 3 to the bcsolateral membrane of IEC-6 cells and vhualisation by transmission electron microscopy. (A) Magnification 250QO-fold. The arrows indicate membrane bound gold.labeled HDL 3. Fragment-like membranes represent invaginadons of
basolateral membrane into the filter pores and are due to the position slices were cut. (B) Magnification63000-fold. In contrast to HDL3 bindiug,bindingof gold labeled LDL to the basolatera[membraneis demonstrated.The str,-cturalpropertiesof the bindingprocessseemsto be similar. (C) Magnification63000-fold.Bindingof gold-labeledHDL3 to a coated pit region of the besolateral membrane.(D) Magnification 63000-fold. Gold-labeledLDL in a coated pit. ldentic morphologicpattern of the membranestructures.Open arrowsare directed on the coating area of the membranebilayer, Closed arrows indicate gold labeled lipoproteins.(E) Magnification20000-fold.Control incubationwith 50-fold excessof unlabeledHDL3. (F) Magnification2501~-fold.Control incubationwithcolloidalgold.
34
Fig. 6. The intraeellular fate of HDL 3 studied by transmission electron microscopy. Magnification 25000-fold in (A), (B), (C) and 31 500-fold in (D). (A) Gold labeled HDL 3 is located in a membrane coated endosomc which obviously shows a partially thicker membrane (small arrows) in the neighbourhood of a Golgi structure. (B) Gold-labeled HDL 3 containing endosomes of normal size. (C) Lysosomes, containing gold-labeled LDL~ in contrast to the HDL3.containing endosomes dark,,i than the sunoundJng cytoplasm. (13) Control incubation with a 50-fold excess of unlabeled HDL 3 shows an endosome similar to the endosomes in B, but no gold labeL
35
FiB. 7. Intracellular I-IDL 3 in contact with lipid droplets. (A) Magnifw,ation 25000-fold° Gold-labeled HDL~ in an cndosom¢ (closed artery) Iocutcd in the vicinity of a lipid droplet (open arrow). (C) Magnification 400~O-fold. Endosome, containing gok~-Iabeled I-IDL3 (cloud arrow) toscthcr with a Tcmnant of a lipid droplet (open arrow) and membrane dctrilus. (B and D) MagniEcation 31500 and 40000-fold, respeclively. Control incubations with a 50-fold excess of unlabeled HDL, Similar endosomes containing lipid droplets ate visible.
36 ods [10]. Cholesterol synthesis was calculated from the C2-ftux from [t4C]octanoate into cholesterol according to Ref. 22. Materials The scanning e[ectron microscope, type EM 500 was from Philips (Eindhoven, The Netherlands) and the transmission electron microsco.~e, Type EM 10 was from Zeiss (Oberkochem, F.R.G.). Plastic dishes were obtained from Nunc GmbH (Wiesbaden, F,R.G.), Millicell HA filters from Millipore GmbH (Esehborn, F.R,G.), Falcon sixwell culture plates from Becton Dickinson GmbH (Heidelberg, F.R.G.). The tissue culture cover slips (foils), Thermanox, were from Miles Scientific (Division of Miles Laboratories Inc., Naperville, U.S.A.). Octanoic acid, sodium salt, [1t4C](NEC-092H), cholesterol, [7-3H(N)](NET-030) and t25I, sodium salt, (NEZ-033) were obtained from NEN Du Pont de Nemours (Dreieieh, F.R.G.). Heparin sepharose CL-6B was from Pharmacia (Uppsala, Sweden). All other reagents in p.a, quality were from Sigma (Munich, F.R.G.).
membrane (Fig. 5). This binding was abolished in the presence of an excess of unlabeled HDL3 (Fig. 5E). Following incubation of the cells with unbound colloidal gold, no gold particles could be visualized at the cell membrane or interior (Fig. 5F). During further incubation endosomes containing the gold-labeled particles were observed which were clearly distinct from gold-labeled LDL containing tysosomes (Fig. 6C) as visu~.:~-'.edby parallel studies (Fig. 6). In other experiments it could be shown that the incubation of enterocytes with gold-labeled LDL for 24 h led to a massive lysosomal enrichment of colloidal gold. In contrast, after the incubation of cells with gold labeled HDL3 for 24 h, no residual gold label could be detected in endosomes. Most of the HDL 3 containing endosomes were observed in the vicinity of lipid droplets formed after preincubation of the cells with LDL. In some instances the endosomes appeared to contact the lipid droplets (Fig. 7) before subsequent fusion and extru. sion of the gold labeled HDL3 from the cell membrane. Similar endosomes with lipid droplets but with-
Results
Studies with iodinated HDL 3 lodinated apo E free HDL 3 exhibited specific binding to IEC-6 cells at 4 ° C with saturation kinetics (Fig, 1). The maximal binding capacity amounted to 2980 ng/mg cell protein. Scatchard analysis revealed a K d of 36.4 ~g/ml. The specific binding of apo E free HDL 3 was effectively displaced by unlabeled apo E free HDL 3 but not by unlabeled LDL (Fig. 2), suggesting that the binding was not mediated by the LDL receptor. Compared to the amount of HDL 3 bound at any given time during incubation at 37" C, the internalized fraction accumulating over time was low (Fig. 3). Specific exceeded unspecific internalisation as assessed in the presence of excess HDL 3 by a factor of 4 (data not shown). Degradation of HDL 3 increased with time and was equivalent to less than 10% of the maximally bound amount after 8 h of incubation. In a separate series of experiments HDL 3 was prebound to the cells at 4 ° C and its fate was followed during subsequent warm up at 37 °C (Fig. 4). The amount of membrane bound HDL 3 decreased rapidly to approximately half of the initial value within 120 rain. At a similar rate the label appeared in the medium in trichloroacetic acid precipitable form. During this time period the amount of label internalized or degraded was l~ss than 5% of that initially bound. Studies with gold-labeled HDL 3 Gold-labeled HDL3 was found to bind predominantly to the coated pit regions of the basolateral
cells
medium 3O
I
IO
0 corxtro]
HDLs
rnevlnolin
Fig. 8. c2-nux into newly synthesized cholesterol of cells were measured using [14C]octanoate as precursor, Incubation time for controls, HDL 3 and mevinolin were 48 h. The bottom parts of bars represents the C2-flu~ into newly synthesized cholesterol, recovered in the cell pellet, while the upper pads of bars reflects the amount of rlewly synthesized cholesterol extracted from the medium after the indicated incubation period. With HDL 3 (50 ~ g / m l medium), cholesterol synthesis was effectively slimutated and most of the surplus of cholesterol, compared to c0ntmls, was recovered in the medium portion, therefore enhancing the cholesterol efflux. The effect on the cholesterol content of cells was minima[. In contrast, mevinolin (0.3 ~g/mI medium), a potent HMG-CoA reductase inhibitor, caused a significant reduction of cholesterol synthesis and loss into the medium as expected from its role as reductase inhibitor.
37 out gold-label could be observed in incubations containing a 50-fold excess of unlabeled lipoprotein (Fig. 7).
Cholesterol efflux When IEC-6 cells were incubated with 14C-labeled octanoate, the C2-fi~ into newly synthesized cholesterol recovered from cells and medium, rose from 13.9 nmol/mg cell protein per 48 h under control coi:~lions to 28.5 in the presence of HDL 3. Interestingly, the amount of cell associated labeled cholesterol was stable, but the surplus of the newly synthesized cholesterol was transported into the medium by the action of HDL 3 (Fig. 8).
Discussion Specific binding of HDL 3 has been observed in a variety of cells such as fibroblasts [12], arterial smooth muscle cells [12], endothelial cells [23], as well as macrophages [13] and in these instances the ability to interact via a putative receptor protein was associated with the efflux of cholesterol. On the other hand, in endocrine cells [24] and hepatocytes [25] the same lipoprotein fraction also exhibits specific binding, but this may be associated with cholesterol influx. Principally, two mechanisms responsible for this HDL 3 mediated export of cholesterol have been proposed: a docking receptor promoting the translccation of cholesterol from the interior of the cell to the cell membrane with subsequent release of HDL3 without prior internalisation [12,26] or, alternatively, a pathway of endosomal internalisation and subsequent retroendooJtosis after enrichment with cholesterol [13,27]. Previous work in isolated intestinal ceils of rat [4,5] and human [7,8] origin has described a high binding affinity for HDL and active internalisation and degradation [4,7] of the particles by intestinal proteinases. Surprisingly, the degradation of HDL 3 did not suppress hydroxymethylglutaryl CoA rcductase like LDL [16,28] and rather resulted in an increase in sterol synthesis, a decrease in cholesterol estetification and a secretion of labeled cholesterol into the medium [7]. However, freshly isolated intestinal cells are metabolically active and viable only for a very short time [29], have a high unspecific pretense activity [30] and undergo rapid cell lysis. Thus, it remained to be determined whether the interaction of HDL a, and, in particular, the rapid degradation of these lipoprotein particles occurred also in more stable enterocytes in monolayer culture. The increase in sterol synthesis mediated by HDL3, in 1EC-6 cells was comparable to that observed in freshly isolated cells [7]. However, despite specific internalisation of HDL 3 the particles were apparently degraded only to a minor extent and resecreted into the medium in trichloroacetic acid precipi-
table form. It should be emphasized that the small amount of measured intemalisation at any given time point represents the balance between uptake and medium release. Minimal degradation seems to occur, but the accumulation of degradation products reached onfy 10% of the amount bound specifically after 8 h. This pathway was confirmed in the electron microscopic studies which delineated a sequence of enclosereal uptake, interaction with lipid droplets and final resecretion by retroendocytt-~;s. This route is reminiscent of the mechanism of retrocndoc~osis described initially in macrophages [13] and recently also in hepatocytes [27]. A similar pathway was described in human CaCo-2 cells [31]. It is therefore conceivable that enterocytes release cholesterol from absorption, local synthesis or LDL uptake via a pathway of binding, internalisation, lipid enrichment and retroendocytosis of HDL The hypothesis is compatible with previous findings on a high flux of preformed as well as newly synthesized HDL in the mesenteric lymph from the gut [32,33]. It is also consistent with the stimulation by exogeneous cholesterol of HDL3 binding in human isolated intestinal epithelial cells [8] as well as in CaCo-2 cells (unpublished data). It should be emphasized that the retroendocytotic pathway in these cultured cells supplements and quantitatively exceeds basal cholesterol effltLx mediated by lipoprotein syntbesis as evidenced by the 3-fold stimulation of cholesterol secretion in the presence of HDL3 in the medium. Possibly this mechanism mediates the absorption of biliary cholesterol in the postabsorptive state when chylomicron synthesis is minimal. The quantitative significance of this pathway in "he intact organism remains to be evaluated.
Acknowledgements We gratefully appreciate the technical assistence by Ch. Lindaner and I. Crastrock.
References I Stang¢, E.F. and Dietschy, J.M. 0985) in Sterols and nile Acids (Danielsson, H. and Sjfvall, J., eds.), pp. 121-149, Elsevier Science Publishers B.V., Amsterdam. 2 Field, F.J., Kam, N.T.P. and Mathur, S.N. (1990) GaslroenterologF 99, 539-551. 3 Stange, E.E and Dielschy, J.M. (1983) Prec. Natl. Acad. Sci. USA 80, 5739-5743. 4 Fidge. N.H., Nestel. P.J. and Suzuki, N. (lq83) Biochim. Biophys. Acla 753, 14-21. 5 Kagami, A., Fidge, N., Suzuki, N. and Nestel, P. (1~4 -) niochlm. niQph~. Acta 795, 179-190. 6 Suzuki, N., Fidge, N., Nestel, P. and Yin, J. (1983) J. Lipid Res. 24, 253-264. 7 Svifidov, D.D., Safonova, I.G., Gusev, V.A., Talalaev, A.G., Tsi-
bulsky,V.P.. Ivanov.V.O., Preobrazensky,S.N., Repin,V.S. and Smirnov,V.N. (1986)M¢labolism35, 588-595.
38 8 Sviridov. D.O., Safonova, I.G., Tsybulsky, V.P.. Talalaev, A.G., Preobrazensky, S.N, Repin, V.S. and Smirnov. V~N, (19871 Bioehim. Biophys. Acta 919. 266-274. 9 Stange, E.F. (19871 Bioehem, Soe. Trans. 15, 189-192. lfl Heroid, G., Schneider, A.. Ditsehuneit. H. and Stange. E.F. (I984) Biochim. Biophys. Acta 796, 27-33. 11 Stange, E.F. and Dietschy, LM. (19851J. Lipid Res. 26, 175-184. 12 Oram, J.F., Brinton, E.A. and Bierman E.L, (19831 J. Clin. Iovcst, 72, 1611-t621, 13 Schmitz, G., Robenek, H., Lohmann. U. and Assmann, G. (19851 EMBO J. 4, 613-622. 14 Traber, M.G., Kayden. H.J. and Rindler. M.J. (19871 J. Lipid Res. 28, 1350-1363. 15 Havel. R.J.. Eder, H,A. and Bragdon, LH. (19551 J. Ctin. Invest, 34, 1345-1353. 16 Stange, E.F., Alavi, M., Schneider, A., Prectik, G. and Ditsehuneit, It, (19801 Biochim. Biophys. Acta 620, 520-527. 17 Weisgraber, K.H, and Mahley, R.W. (19801 J, Lipid Res. 21, 316-325, 18 Frens. G. (19731 Nature Phys. Sci. 241, 20-22. 19 Handley, P.A., Arheeny, C.M., Witte, L.D. and Chien, S. (19811 Proc. Natl. Aead. Sci. U.S.A. 78. 368-371. 20 Brown, M.8. and Goldstein, J.L. (19861 Science 23. 23-47. 21 Goldstein, J.L.. Basu. S.H. and Brown, M.S. (19831 Methods Enz'ymol. 98, 241-260.
22 Andersen, J.M. and Dietschy, J.M, (19791 J, Lipid Res. 20, 740-752, 23 Martin-Nizard, F., Meresse, S., Cecchelli, R., Fruchart, J.C. and Delbart, C. (19891 Biochim. Biophys, Acta I005. 210-208. 24 Chen, Y.-D.I., Kraemer, F.B. and Reaven, G.M. (19801 J. Biol. Chem. 255, 9162-9167. 25 Chaeko. G.K, (19821 Biochim. Biophys. Acta 712, 129-141, 26 Slotte, J,P., Oram. J,F. and Bierman, E.L. 0987) J. Biol. Chem. 262, 12904-12907. 27 DeLamatre, J.G., Sarphie, T.G,, Archihold. R.C. and Hornick, C.A. (19901J. Lipid Rcs, 31. 191-202. 28 Nano, J,-L., Barbaras, R., N~grel, R. and Rampal. P, ([986) Bicehim. Binphys. Acta 876, 72-79. 29 Stange, E.F. and Dietschy, J.M. (19831J. Lipid Res. 24, 72-82. 30 Suzuki, N., Fidge, N, and Nestel. P. (19831 Biochim. Biophys. Acta 750, 457-464. 31 Herold, G., Rogler, G.. Reimann, F.M. and Stange, E.F, (19901 Klin. Wochenschr. 68 (Suppl. 22), 1-6. 32 Green, P.H.R. and Gliekman, R.H. (19811 J. Lipid Res. 22, 1153-1173. 33 Bisgaier. C.L. and Glickman, R.M. (19831Annu. Rev. PhysioL 45, 625-636.
36
BBAMCR 13024
HDL3-retroendocytosis in cultured small intestinal crypt cells: a novel mechanism of cholesterol efflux Gerhard Rogler, Gerhard Herold and Eduard F. Stange Department of Internal Medicine ii, University of UIra, Ulm (F.K G.)
(Receiveu 29 March 1991) (Revisedmanuscriptreceived8 July 1991) Key words: High density ]ipoprotein; Receptor; Retroendocytosis; Cholesterol; IEC-6; (Rat intestinal crypt cell)
The present study in IEC-6 crypt.derived rat epithelial cells describes a retroendocytotic pathway for HDL~. These intestinal cells exhibited specific binding of apoE free HDL~ with a maximal binding capacity of 2980 n g / m g cell protein and a K a of 36A / z g / m l . Specific binding was competed for by HDL 3 but not by I,DL. Apparent internalisation of HDL~ was low, degradation was negligible and intact particles were rescereted into the medium within 2 h. Electron microscopic studies showed binding and Internalisation of gold-labeled HDL 3 in coated pit regions and transport in endosomes distinct from lysosomes to lipid droplets. De nero cholesterol sTnthosis from [~4C]octanoate was enhanced nearly 2-fold by HDL 3 and the surplus of newly formed cholesterol was recovered in the medium. It was concluded that intact HDL 3 was bound specifically to intestinal cells and was resecreted through a process of retroendocytasis probably mediating effiux of cellular cholesterol. Introduction The small intestinal mucosa is actively involved both in the absorption and synthesis of cholesterol [1,2]. Enterocytes also display receptors for low [3] and high density lipoproteins [4-8] which were reported to mediate rapid uptake and subsequent degradation of these particles. Thus, the mucosal cell is exposed to cholesterol from a multitude of sources and current evidence suggests that these pools of cholesterol are compartmentalized intracellulady [9,10]. The export of intestinal cholesterol is similarly complex because it may occur via sloughing of ceils from the villus tip or by formation and lymphatic secretion of lipoproteins, particularly chylomicrons [1]. A recent study in the live rat found absorption and lymphatic secretion of bile derived cholesterol in the absence of triaeylglyeerois in the lumen, i.e., under circumstances of limited ehyiomicron iormation [11]. Since HDL partides were suggested to mediate reverse cholesterol
transport in other tissues and cells [12,13], we hypothesized a similar role for this iipoprotein fraction in the gut, The present work describes the HDL3,-pathway in
Correspondence:G. Hero|d, Departmentof Internal Medicine It, Universityof UIm, Robert Koch Sir. 8, 7900 UIm, F.R.G.
eooo 0o 5000
~ 40 20
~4000
o
0
10002 0 0 0 3 0, 0 0 /
Y
,3000
1000
0
20
40
60
80
",tOO
~g HDLa / ml m e d i u m
Fig, 1, lEe-6 cells at day 5 of culture were incubatedat 4oC for 2 h in 2 ml of DMEM supplementedwith 5% of lipoprotvin-deficient (LPD) FC$ and iodinated HDL3 in concentrations up iv 100 p,g lipoprotein prvtein/mL Unspecific binding was measured using a 50-fold surplus of unlabeled lipoprotein and binding procedures were stopped by chilling on ice for 15 rain. Specific binding was calculated from the difference between total and unspecificbinding. Maximal binding and Kd were determined using Scatchard plotting as shown above, o, unspecificbinding; v, specificbinding;o, total binding,
3i cultured small intestinal cells and its putative role in mediating cholesterol efflux from the gut.
2000
Methods and Materials
1750
Cell culture
1500
Rat intestinal epithelial cells IEC-6 were obtained from American Type Culture Collection (1592-CRL) and were used at passage 20-22. Cells were grown as monolayers on 100-ram plastic dishes and were mainrained in 10% Dulbecco's minimum essential medium ( D M E M ) supplemented with L-glutamine (2 raM), Larginine (0.4 mM), 0.1% non-essential amino acids, sodium lactate (2 mM), NaCO 3 (40 mM), glucose (10 raM), 0.3% phenol red, insulin (400 I.U./ml), 1% Pen Strep (Gibco), and 5% lipoprotein free fetal calf serum (FCS) in an atmosphere containing 8% CO2. Subcuttiration for experiments was done on 30 mm Millicell H A filters to allow accessibility of the basolaterai membrane [14]. The cells were routinely used at day 5 after plating, Low density iipoproteins (200 /~g/ml) were added to the medium for 24 h before the expertments. Other medium additions were supplemented as specified.
Preparation of lipoprotems Lipoprotein fractions were separated by ultracentrifugation of human plasma as described previously
l
i
1000
o
750
500
250
o. 0
t
~
-
+
2
4 time
Fig. 3. IEC-6 cells
were
?
?
6
8
(h)
incubated at 37°C with 10 p.g iodil:ated
HDL~/ml for different time periods up to 8 h under conditions specified in Fig. 1. Unspecific binding was measured with a 50-fold surplus of unlabeled lipoprotein. Specificbinding (e), internalisation (o) and degradation (v) were calculated from the differences between their corresponding values of total and unspecific binding, lntemaSsation and degradation were determined as specified in Materials and Methods.
100
LDL
100 .~.
ee
¢1 o. 1 2 5 0
. D
-,r 80 = o
,,D
~. _o
60
6o
_c
u
40 o
c
2O
i1
HOL 3 i O
i
i 100
i 200
i 300
,
i 400
i 500
ug llpoprotlln/ml medium Fig. 2. IEC-6 cells at day 5 of -'J!::~;c~'ci-¢incubated at 4°C for 2 h with 10 ~g iod[nated HDL 3 ml in 2 nil of DMEM supplemented with 5% of LPD-FCS. Simultanouslyeither unlabeled LDL or HDL 3 in concentrations up to 5~ ~g/ral were present in the medium. Specific binding was competed for by HDL3 but not by LDL. Percent values refer to the initial [ZZSl]l-lDL3binding without any unlabeled lipoptolein additions.
0
i
i
i
i
i
i
20 40 60 B0 100 120 time
(rain)
Fig. 4. IEC-6 cellswere incubated at 4 ° C for 2 h as described in Fig. I together with 10 9-g iodinated H D L 3 / m ] medium. Sorrowly washing on ice was followed by subsequent warm up to 37°C and
incubation for different time periods up to 2 h. At any given time point, protein-bound radioactivity was simultaneously detem~ined in cells and medium. While specific binding shows subsequent loss with time, medium reiea~ of an intact lipoprotcin particle occurred in
increasingamounts. O, specificbinding;o, medium release.
32
A
~
I
¸
33 [15,16]. The fractions isolated were low density lipoproteins (LDL, d = 1.019-1.063 g/ml) as well as high density lipcproteins (HDL 3, d = 1.125-1.2i0 g/ml) HDL3 were rendered apoprotein E free by heparin sepharose affinity chromatography []7] as checked by sodium dodecyl sulfate polyacrylamide gel eleetrophoresis (SDS-PAGE).
Lipoprotein labeling Colloidal gold was prepared as described by Frens et al. [18] using a particle size of 20-40 nm. Conjugation of colloidal gold to HDL 3 was carried out using a modification of a method described for LDL [19]. 5.0 ml of a monodispe~e colloidal gold solution was added to 0.5 ml of the lipoprotein solution (100 F g / m l ) in 50 mM EDTA-buffer. After rapid conjugation the complex was stabile over a wide pH range. The appearance of colloidal gold-lipoprotein complexes was checked by the electron microscopic technique of negative staining. t25I-labeling of HDL 3 was performed by the method of Brown and Goldstein [20] with minor modifications. Final specific radioactivity of [t2SI]HDL~ varied from 400 to 600 e p m / n g protein.
Studies with iodinated lipoproteins All experiments were carried out in Falcon multiwell dishes with cells grown on milliceil HA filters. Cells were preineubated with 200 ~ g LDL-protein/ml medium for 48 h prior to the studies. The incubation mixture contained DMEM, 20 m g / m l lipoprotein-free FCS and 5-10 /~g/ml [tzSI]HDL3. Prior to binding experiments at 4 ° C or 37 ° C, ceils were washed with Tris-albumin buffer. For studies at 4°C, cells were chilled on ice 15 rain prior to the addition of the ice-cold incubation medium. After the incubation period, cells were washed three times with Tris-albumin buffer to remove unspeeifically .round lipoprotein. At the end of the incubation at 37 °C, cells were chilled on ice for 15 rain prior to washing. After the final wash the buffer was removed, cells were incubated with buffer containing 1 mM EDTA and 1 m g / m l trypsin for 1 h at 4 ° C. Cells were harvested from the filters with a rubber policeman and the cell suspension was transferred to microfuge tubes and s;:dimented by centrifugation at 3000 rpm. The counts in the supernatant represented the membrane-bound HDL 3. The washed
cell pellet was digested in 2 ml of 0.1 M NaOH and aliquots of 1.0 ml for counting (representing internalized HDL 3) and 20 ~1 for protein determination were taken. Degradation was measured as the amount of soluble radioactivity after trichloroacetic acid precipitation of the medium and removal of free iodine by oxidation and chloroform extraction according to standard methods [21]. Control incubations were carried out with cell-free medium. The time dependence of binding was studied with 10 /zg lipoprotein/ml medium. Unspecific binding was determined using a 50-fold excess of unlabeled HDL 3.
Studies with gold labeled lipoproteins Binding and internalisation experiments were carried out as described for [tZSl]HDL3 except for the incubation with trypsin buffer. Cells were fixed in a standard procedure with glutaraldehyde (3.5%) at 4 °C and stained with osmium tetroxide and lead citrate. Embedding was carried out with EPON. To obtain vertical slices through the basolateral membranes a second resin preparation positioned in a right angle was necessary for transmission electron microscopy. For scanning electron microscopy cells were cultured on Thermanox foils which were proved to be resistent to 'critical point "lrying' of the probes. Incubations were carried out with a concentration of 5 / ~ g / m l of gold-labeled lipoproteins, control incubations with a 50-fold excess of li0oprotein or I00/zl of the colloidal gold preparation used for labeling.
Cholesterol effiux Cells were cultured under standard conditions on plastic dishes and five dishes were pooled. Newly synthesized cholesterol was labeled with [t4C]octanoate as precursor. 0.1 mM unlabeled octanoate was present in the culture medium in addition to 0.5 #Ci [t4C]octanoate at a specific activity of 5300 d p m / n m o l at days 7-9. The incubation was stopped by chilling the dishes on ice. The medium was collected together with three washes of phosphate buffered saline plus albumin (1 mg/ml). Cells were sedimented by centfifugation and protein content was determined as described above. Lipids were extracted separately from cells and medium and cholesterol was isolated by thin layer chromatography (TLC) according to previously described meth-
Fig. 5, Binding of gold-labeled HDL 3 to the bcsolateral membrane of IEC-6 cells and vhualisation by transmission electron microscopy. (A) Magnification 250QO-fold. The arrows indicate membrane bound gold.labeled HDL 3. Fragment-like membranes represent invaginadons of
basolateral membrane into the filter pores and are due to the position slices were cut. (B) Magnification63000-fold. In contrast to HDL3 bindiug,bindingof gold labeled LDL to the basolatera[membraneis demonstrated.The str,-cturalpropertiesof the bindingprocessseemsto be similar. (C) Magnification63000-fold.Bindingof gold-labeledHDL3 to a coated pit region of the besolateral membrane.(D) Magnification 63000-fold. Gold-labeledLDL in a coated pit. ldentic morphologicpattern of the membranestructures.Open arrowsare directed on the coating area of the membranebilayer, Closed arrows indicate gold labeled lipoproteins.(E) Magnification20000-fold.Control incubationwith 50-fold excessof unlabeledHDL3. (F) Magnification2501~-fold.Control incubationwithcolloidalgold.
34
Fig. 6. The intraeellular fate of HDL 3 studied by transmission electron microscopy. Magnification 25000-fold in (A), (B), (C) and 31 500-fold in (D). (A) Gold labeled HDL 3 is located in a membrane coated endosomc which obviously shows a partially thicker membrane (small arrows) in the neighbourhood of a Golgi structure. (B) Gold-labeled HDL 3 containing endosomes of normal size. (C) Lysosomes, containing gold-labeled LDL~ in contrast to the HDL3.containing endosomes dark,,i than the sunoundJng cytoplasm. (13) Control incubation with a 50-fold excess of unlabeled HDL 3 shows an endosome similar to the endosomes in B, but no gold labeL
35
FiB. 7. Intracellular I-IDL 3 in contact with lipid droplets. (A) Magnifw,ation 25000-fold° Gold-labeled HDL~ in an cndosom¢ (closed artery) Iocutcd in the vicinity of a lipid droplet (open arrow). (C) Magnification 400~O-fold. Endosome, containing gok~-Iabeled I-IDL3 (cloud arrow) toscthcr with a Tcmnant of a lipid droplet (open arrow) and membrane dctrilus. (B and D) MagniEcation 31500 and 40000-fold, respeclively. Control incubations with a 50-fold excess of unlabeled HDL, Similar endosomes containing lipid droplets ate visible.
36 ods [10]. Cholesterol synthesis was calculated from the C2-ftux from [t4C]octanoate into cholesterol according to Ref. 22. Materials The scanning e[ectron microscope, type EM 500 was from Philips (Eindhoven, The Netherlands) and the transmission electron microsco.~e, Type EM 10 was from Zeiss (Oberkochem, F.R.G.). Plastic dishes were obtained from Nunc GmbH (Wiesbaden, F,R.G.), Millicell HA filters from Millipore GmbH (Esehborn, F.R,G.), Falcon sixwell culture plates from Becton Dickinson GmbH (Heidelberg, F.R.G.). The tissue culture cover slips (foils), Thermanox, were from Miles Scientific (Division of Miles Laboratories Inc., Naperville, U.S.A.). Octanoic acid, sodium salt, [1t4C](NEC-092H), cholesterol, [7-3H(N)](NET-030) and t25I, sodium salt, (NEZ-033) were obtained from NEN Du Pont de Nemours (Dreieieh, F.R.G.). Heparin sepharose CL-6B was from Pharmacia (Uppsala, Sweden). All other reagents in p.a, quality were from Sigma (Munich, F.R.G.).
membrane (Fig. 5). This binding was abolished in the presence of an excess of unlabeled HDL3 (Fig. 5E). Following incubation of the cells with unbound colloidal gold, no gold particles could be visualized at the cell membrane or interior (Fig. 5F). During further incubation endosomes containing the gold-labeled particles were observed which were clearly distinct from gold-labeled LDL containing tysosomes (Fig. 6C) as visu~.:~-'.edby parallel studies (Fig. 6). In other experiments it could be shown that the incubation of enterocytes with gold-labeled LDL for 24 h led to a massive lysosomal enrichment of colloidal gold. In contrast, after the incubation of cells with gold labeled HDL3 for 24 h, no residual gold label could be detected in endosomes. Most of the HDL 3 containing endosomes were observed in the vicinity of lipid droplets formed after preincubation of the cells with LDL. In some instances the endosomes appeared to contact the lipid droplets (Fig. 7) before subsequent fusion and extru. sion of the gold labeled HDL3 from the cell membrane. Similar endosomes with lipid droplets but with-
Results
Studies with iodinated HDL 3 lodinated apo E free HDL 3 exhibited specific binding to IEC-6 cells at 4 ° C with saturation kinetics (Fig, 1). The maximal binding capacity amounted to 2980 ng/mg cell protein. Scatchard analysis revealed a K d of 36.4 ~g/ml. The specific binding of apo E free HDL 3 was effectively displaced by unlabeled apo E free HDL 3 but not by unlabeled LDL (Fig. 2), suggesting that the binding was not mediated by the LDL receptor. Compared to the amount of HDL 3 bound at any given time during incubation at 37" C, the internalized fraction accumulating over time was low (Fig. 3). Specific exceeded unspecific internalisation as assessed in the presence of excess HDL 3 by a factor of 4 (data not shown). Degradation of HDL 3 increased with time and was equivalent to less than 10% of the maximally bound amount after 8 h of incubation. In a separate series of experiments HDL 3 was prebound to the cells at 4 ° C and its fate was followed during subsequent warm up at 37 °C (Fig. 4). The amount of membrane bound HDL 3 decreased rapidly to approximately half of the initial value within 120 rain. At a similar rate the label appeared in the medium in trichloroacetic acid precipitable form. During this time period the amount of label internalized or degraded was l~ss than 5% of that initially bound. Studies with gold-labeled HDL 3 Gold-labeled HDL3 was found to bind predominantly to the coated pit regions of the basolateral
cells
medium 3O
I
IO
0 corxtro]
HDLs
rnevlnolin
Fig. 8. c2-nux into newly synthesized cholesterol of cells were measured using [14C]octanoate as precursor, Incubation time for controls, HDL 3 and mevinolin were 48 h. The bottom parts of bars represents the C2-flu~ into newly synthesized cholesterol, recovered in the cell pellet, while the upper pads of bars reflects the amount of rlewly synthesized cholesterol extracted from the medium after the indicated incubation period. With HDL 3 (50 ~ g / m l medium), cholesterol synthesis was effectively slimutated and most of the surplus of cholesterol, compared to c0ntmls, was recovered in the medium portion, therefore enhancing the cholesterol efflux. The effect on the cholesterol content of cells was minima[. In contrast, mevinolin (0.3 ~g/mI medium), a potent HMG-CoA reductase inhibitor, caused a significant reduction of cholesterol synthesis and loss into the medium as expected from its role as reductase inhibitor.
37 out gold-label could be observed in incubations containing a 50-fold excess of unlabeled lipoprotein (Fig. 7).
Cholesterol efflux When IEC-6 cells were incubated with 14C-labeled octanoate, the C2-fi~ into newly synthesized cholesterol recovered from cells and medium, rose from 13.9 nmol/mg cell protein per 48 h under control coi:~lions to 28.5 in the presence of HDL 3. Interestingly, the amount of cell associated labeled cholesterol was stable, but the surplus of the newly synthesized cholesterol was transported into the medium by the action of HDL 3 (Fig. 8).
Discussion Specific binding of HDL 3 has been observed in a variety of cells such as fibroblasts [12], arterial smooth muscle cells [12], endothelial cells [23], as well as macrophages [13] and in these instances the ability to interact via a putative receptor protein was associated with the efflux of cholesterol. On the other hand, in endocrine cells [24] and hepatocytes [25] the same lipoprotein fraction also exhibits specific binding, but this may be associated with cholesterol influx. Principally, two mechanisms responsible for this HDL 3 mediated export of cholesterol have been proposed: a docking receptor promoting the translccation of cholesterol from the interior of the cell to the cell membrane with subsequent release of HDL3 without prior internalisation [12,26] or, alternatively, a pathway of endosomal internalisation and subsequent retroendooJtosis after enrichment with cholesterol [13,27]. Previous work in isolated intestinal ceils of rat [4,5] and human [7,8] origin has described a high binding affinity for HDL and active internalisation and degradation [4,7] of the particles by intestinal proteinases. Surprisingly, the degradation of HDL 3 did not suppress hydroxymethylglutaryl CoA rcductase like LDL [16,28] and rather resulted in an increase in sterol synthesis, a decrease in cholesterol estetification and a secretion of labeled cholesterol into the medium [7]. However, freshly isolated intestinal cells are metabolically active and viable only for a very short time [29], have a high unspecific pretense activity [30] and undergo rapid cell lysis. Thus, it remained to be determined whether the interaction of HDL a, and, in particular, the rapid degradation of these lipoprotein particles occurred also in more stable enterocytes in monolayer culture. The increase in sterol synthesis mediated by HDL3, in 1EC-6 cells was comparable to that observed in freshly isolated cells [7]. However, despite specific internalisation of HDL 3 the particles were apparently degraded only to a minor extent and resecreted into the medium in trichloroacetic acid precipi-
table form. It should be emphasized that the small amount of measured intemalisation at any given time point represents the balance between uptake and medium release. Minimal degradation seems to occur, but the accumulation of degradation products reached onfy 10% of the amount bound specifically after 8 h. This pathway was confirmed in the electron microscopic studies which delineated a sequence of enclosereal uptake, interaction with lipid droplets and final resecretion by retroendocytt-~;s. This route is reminiscent of the mechanism of retrocndoc~osis described initially in macrophages [13] and recently also in hepatocytes [27]. A similar pathway was described in human CaCo-2 cells [31]. It is therefore conceivable that enterocytes release cholesterol from absorption, local synthesis or LDL uptake via a pathway of binding, internalisation, lipid enrichment and retroendocytosis of HDL The hypothesis is compatible with previous findings on a high flux of preformed as well as newly synthesized HDL in the mesenteric lymph from the gut [32,33]. It is also consistent with the stimulation by exogeneous cholesterol of HDL3 binding in human isolated intestinal epithelial cells [8] as well as in CaCo-2 cells (unpublished data). It should be emphasized that the retroendocytotic pathway in these cultured cells supplements and quantitatively exceeds basal cholesterol effltLx mediated by lipoprotein syntbesis as evidenced by the 3-fold stimulation of cholesterol secretion in the presence of HDL3 in the medium. Possibly this mechanism mediates the absorption of biliary cholesterol in the postabsorptive state when chylomicron synthesis is minimal. The quantitative significance of this pathway in "he intact organism remains to be evaluated.
Acknowledgements We gratefully appreciate the technical assistence by Ch. Lindaner and I. Crastrock.
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