and serves as a precursor for bile acid and steroid hormone synthesis. Therefore, it may be ..... know what effect the injection of a tropic hormone would. 1987.
620th Meeting Held at Trinity College, Dublin, on 23-26 September 1986
Receptor-Mediated Endocytosis of Lipoproteins: Its Role in Cholesterol Homoeostasis Lipid Group Colloquium organized by W. C. Love (Trinity College, Dublin) and K. E. Suckling (Smith Kline and French Research Ltd., Welwyn), and edited by K. E. Suckling Sponsored by Smith Kline and French Research Ltd. and Glaxo Group Research Ltd.
Compartmentalization of cholesterol in hepatic and intestinal cells: implications for bile and lipoprotein secretion EDUARD F. STANGE Department of Internal Medicine, University Clinic of Urn, Steinhovelstr. 9, 7900 Ulm, West Germany
output of preformed cholesterol is modulated to exactly balance changes in the output of newly formed cholesterol. This has to be due to tight control of intracellular traffic, since, at least in the rat, cholesterol production and Cholesterol is an essential component of cellular membranes chylomicron (Wade et al., 1984) as well as LDL (Stange & and serves as a precursor for bile acid and steroid hormone Dietschy, 1984; Spady et al., 1 9 8 5 ~ )uptake are indepensynthesis. Therefore, it may be synthesized or, alternatively, dently regulated. In contrast to the rat which is rather taken up in lipoprotein form by virtually every cell in the resistent to the induction of supersaturated bile, in the mammalian body. However, the key role in cholesterol hamster such an event is precipitated by giving a fat-free diet metabolism is played by only two organs, the liver and the (Turley et al., 1983). In this instance, the secretion of intestine. The original concept of a common pool of chol- preformed as well as newly synthesized cholesterol is esterol derived from a variety of sources within a given dramatically increased, but the phenomenon is still hepatic or intestinal cell proved inadequate in many exper- unexplained. Thus, usually the compartment of cholesterol supplying imental situations, suggesting that the fluxes are strictly regulated and directed according to the functional demands. biliary secretion is independent of large fluctuations in Although this concept is by no means new, it has been cholesterol synthesis and lipoprotein uptake. Although its corroborated by several novel findings with important sources vary under different metabolic conditions, most of it is preformed with only minor contributions by LDLimplications. In the liver the pathways of cholesterol are extremely cholesterol ester, at least in the rat (Bhattacharya at al., complex because this organ is composed of a variety of 1986). Another candidate for preferentially delivering biliary heterogeneous cells of different functions in lipoprotein cholesterol is HDL, both in rat hepatocytes (Ford et al., . its attracuptake (Harkes & van Berkel, 1984; Blomhoff et al., 1984; 1985) and man (Schwartz et al., 1 9 7 8 ~ )Despite Lippiello et al., 1985), like endothelial cells, Kupffer cells, tiveness in terms of reverse cholesterol transport from perparenchymal cells, bile duct epithelia and others. Also, there ipheral cells for biliary excretion, such a mechanism is still is some evidence that even hepatic parenchymal cells at dif- unproven. If there is indeed a defined lipoprotein class ferent locations with respect to the portal triad differ with involved as precursor for biliary cholesterol, it remains to be respect to their rate of cholesterol synthesis (Singer et a[., determined. Interestingly, in the rat hepatic cholesteryl ester content, 1984) and probably lipoprotein uptake. In addition, there is evidence that the flow of preformed acyl-CoA acyltransferase (ACAT) activity and biliary cholesterol from chylomicron or very-low-density lipo- cholesterol secretion are inversely related (Nervi et al., protein (VLDL) remnants, low-density lipoprotein (LDL) 1984). This relationship holds under most experimental conor high-density lipoprotein (HDL) once it is taken up via ditions and suggests that the compartments of free cholesreceptor- or non-receptor-mediated pathways is strictly con- terol at the endoplasmic reticulum serving as substrate for trolled. For example, the amount of preformed as compared esterification or biliary secretion are functionally, and probwith newly synthesized cholesterol secreted into bile may ably also topically, related. Such a mechanism would imply vary between 98 and 66% in animals fasted or fed that rapid esterification of cholesterol entering the hepacholestyramine to suppress or increase hepatic cholesterol tocyte in lipoprotein-bound form protects bile from supersynthesis, respectively (Turley & Dietschy, 1981). Since both saturation and actually overshoots by depleting the comin the rat and hamster (Turley et al., 1983) these manipu- partment for biliary secretion. Finally, the concept that this lations hardly alter total biliary cholesterol secretion, the compartment mixes intracellularly has become questionable, since there is evidence for separate pathways for newly synthesized cholesterol which is not in equilibrium with the Abbreviations used: VLDL. very-low-density lipoprotein; LDL. lowremaining hepatocyte cholesterol and the preformed pool density lipoprotein; HDL. high-density lipoprotein; ACAT. acyl-CoA acyltransferase; HMG-CoA reductase. 3-hydroxy-3-rnethylglutaryl- which is in near-complete equilibrium (Robins et al., 1985). CoA reductase; WHHL, Wanatabe heritable hyperlipidaernic. The data would suggest that newly synthesized biliary VOl. 15
189
190
BIOCHEMICAL SOCIETY TRANSACTIONS
remnants
Esterified
i
I V
cholesterol
svnthesized
cholesterol Cholic
\
u rnicelles
Fig. 1. Proposed compartmentalization andfruxes of intracellular cholesterol in the hepatic parenchymal cell
cholesterol is derived from the interior of the hepatocyte, whereas the preformed cholesterol stems from the blood via the plasma and canalicular membrane. Since bile salts in the enterohepatic circulation recruit cholesterol for biliary secretion, it is probably the availability of free cholesterol for such recruitment that determines the size and source of the compartment secreted into bile. Furthermore, there is considerable evidence that the cholesterol serving as substrate for bile acid synthesis is strictly separated from the pool supplying biliary cholesterol in rat (Normann & Norum, 1976), monkey (Stephan & Hayes, 1985) and man (Schwartz et al., 1975). Moreover, even the precursor pools for the various primary bile acids may be distinct in experimental animals (Mitropoulos et al., 1974; Stephan & Hayes, 1985), but possibly not in man (Schwartz et al., 1977). However, attempts to quantify the contribution of newly synthesized versus preformed cholesterol using a variety of techniques have led to different results. For example, it has been calculated that the proportion of newly synthesized cholesterol amounted to 50-60% in the rat inhaling " 0 (Bjorkhem & Lewenhaupt, 1979) for both cholic acid and chenodeoxycholic acid. However, the data were obtained more than 2 days after establishing the bile fistula under conditions of derepressed bile acid synthesis. Recent studies using 'H,O as a precursor have revealed that in the rat with an acute bile fistula muricholate arose from a pool of cholesterol that was derived 24% from newly synthesized cholesterol, whereas the value equalled 14 and 4 % for cholate and chenodeoxycholate, respectively (Stange et al., 1985b). This contribution was more than doubled after a long-term bile fistula because of derepression of cholesterol synthesis. In conclusion, both biliary cholestcrol and newly synthesized bile acids are derived mostly from preformed cholesterol, but the individual precursor compartments are distinct in man also (Halloran et a[., 1978; Schwartz et al., 1978b). Principally, however, bile acid synthesis may be driven by cholesterol from any source in cultured hepatocytes (Davis et al., 1983), although the validity of these results under conditions in vivo remains to be shown. A similar problem with a concept obtained in cultured cells arises in the regulation of cholesterol synthesis by LDL.
Originally, it was postulated that only LDL-cholesterol entering a cell through the lipoprotein receptor system effectively down regulates the rate-limiting enzyme 3-hydroxy3-methylglutaryl-CoA reductase (HMG-CoA reductase) (Brown & Goldstein, 1974). Subsequent work in cultured hepatocytes of WHHL rabbits deficient in LDL receptors (Attie et al., 1981) and human hepatocytes (Edge et al., 1986) appeared to support this contention. On the other hand, human LDL which is not effectively bound by the rat LDL receptor suppressed reductase activity in cultured rat hepatocytes (Stange et al., 1982). This is in agreement with a normal rate of hepatic sterol formation in WHHL rabbits (Dietschy et al., 1983) and homozygous familial hypercholesterolaemic subjects lacking functional LDL receptors (Hoeg et al., 1984). Therefore, the present data obtained in vivo (Spady et al., 198%) refute the hypothesis that LDLcholesterol is compartmentalized into a regulatory and a non regulatory pool depending on its receptor-mediated or non-mediated mechanism of uptake. However, at a given rate of lipoprotein cholesterol uptake, chylomicron remnants may be very efficient (Andersen et al., 1979) and VLDL very inefficient (Dashti et al., 1984) suppressors of hepatic cholesterol synthesis. Thus, it becomes evident that within the hepatocyte the flux of cholesterol into the distinct pools for biliary secretion, bile acid synthesis, esterification and suppression of cholesterol production is tightly regulated by compartmentalization (Fig. 1). It should be emphasized that much less is known about the source of cholesterol leaving the liver incorporated into lipoproteins, rendering the present concept rather incomplete. In the second organ to be discussed, the small intestine, there is again cellular heterogeneity with respect to anatomical and functional structure (Fig. 2). The regenerative compartment is located in the crypts, suggesting that cholesterol demand serves mostly membrane synthesis, whereas cholesterol absorption takes place in the more differentiated villus cells. Indeed, there is a definite gradient of both sterol synthesis (Stange & Dietschy, 1 9 8 3 ~as ) well as LDL uptake (Stange & Dietschy, 19836) from the crypts towards the villus tip in the live rat. Thus, although by no means completely separate, endogenous cholesterol from synthesis or 1987
620th MEETING, DUBLIN
191
Luminal triglyceride
Luminal
Fig. 2. Proposed compartmentalization andjuxes of intracellular cholesterol in the intestinal epithelial cell
LDL uptake and absorbed cholesterol occur at different levels of the mucosa. In addition, it was suggested on the basis of work on intestinal ACAT that the pool of cholesterol esterified within the enterocyte is derived mostly from absorbed cholesterol (Stange et al., 1983). In later work, this hypothesis was corroborated by the low incorporation of mevalonate into esterified cholesterol in organ cultured mucosa even when cholesterol synthesis was stimulated over 90-fold (Herold et al., 1984). More importantly, the strict separation of newly synthesized cholesterol from all preformed cholesterol within the mucosal cell was also evident in viva Thus, when rats were given 'H,O essentially no intestinal derived newly synthesized cholesterol was recovered in mesenteric lymph during the infusion of a glucose and amino acid solution only (Stange & Dietschy, 1985). In contrast, during the infusion of triglyceride fat into the duodenum a small but sizable proportion of the amount formed in the gut was secreted into lymph, probably in the chylomicron surface as free cholesterol. Therefore, the data were compatible with a concept of separate functional compartments of absorbed and LDL-derived preformed cholesterol being secreted in lipoprotein form and a pool from synthesis supplying the local needs of membrane formation. Thus, during blockade of cholesterol synthesis by HMG-CoA reductase inhibitors mucosal growth was arrested (Stange et a[., 1985~). However, the pathways seem to be even more complex. Although enterocyte sterol formation may be suppressed by newly synthesized (Stange et al., 1981a), absorbed (Stange et al., 19816) and LDL- (Stange et al., 1980) cholesterol, it may even be induced by H D L (Stange et al., 1980). Recent work suggested that HDL, may protect cultured intestinal epithelial cells from the growth- arresting effect of mevinolin, a HMG-CoA reductase inhibitor (A. Schneider & E. F. Stange, unpublished work). This rescue was due to the induction of the reductase by depleting cellular cholesterol rather than cholesterol supply and, interestingly, LDL was totally ineffective in this respect, although it efficiently delivered sterol to the cells. From these studies it was concluded that not only were newly synthesized and LDLderived cholesterol kept in separate compartments within
Vol. 15
these cultured cells, but most surprisingly only locally formed cholesterol may be incorporated into cellular membranes and support growth. Finally, it became evident that the binding of HDL, to a receptor at the enterocyte basolateral membrane as described previously (Kagami et al., 1984) serves to release cholesterol into the circulation bound to H D L rather than the supply of this molecule. Again, little is known about the source of cholesterol secreted in the various classes of lipoproteins into lymph, but it is probably mostly preformed (Stange & Dietschy, 1985). It is of interest to note that such a separation of endogenous and exogenous pools is supported by early work in the rat (Sodhi et al., 1973) and has been defined topically to some degree by modulating brush-border but not basolateral membrane fluidity as a consequence of alterations in intestinal cholesterol synthesis (Brasitus & Schachter, 1982). In conclusion, evidence is rapidly accumulating for both liver and small intestine that cholesterol from different sources is not mixing at random intracellulary but rather is strictly compartmentalized into different functional pools subserving specific functions. The precise mechanisms involved remain to be identified, but the concept emerging is certainly much more complex than previously anticipated. Andersen, J . M., Turley, S. D. & Dietschy, J. M. (1979) Proc. Nail. Acad. Sci. U.S.A. 76, 165-169 Attie, A. D., Pittman, R. S., Watanabe, Y. & Steinberg, D. (1981) J . Bid. Chem. 156,9789-9792 Bhattacharya, S., Balasubramaniam, S. & Simons, L. A. (1986) Biochim. Biophys. Actu 876, 413-416 Bjorkhem, I. & Lewenhaupt. A. (1979) J . B i d . Chem. 254, 5252--5256 Blomhoff, R . , Drevon. C. A,. Eskild, W., Helgerud. P.. Norurn, K. R. & Berg, T. ( I 984) J . B i d . Chrm. 259. 8898 8903 Brasitus, T. A. & Schachter, D . (1982) Biochemistry 21. 2241 2246 Brown, M . S. & Goldstein, J. L. (1974) Proc. Nail. Acud. Sci. U.S.A. ~
71. 788 792
Dashti, N . & Wolfbauer, G. (1986) Biochim. Biophys. Aria 875, 473-486
Dashti, N . , Wolfbauer, G., Koren. E., Knowles, B. & Alavagoric, P. ( I 984) Biochim. Biuphys. Aciu 794. 373-384 Davis, R. A,, Hyde, P. M., Kuan, J-C. W., Malone-McNeal, M. & Archambault-Schexnayder, J. ( I 983) J . Biol. Chem. 268, 3661--3667
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Dietschy, J. M., Kita, T., Suckling, K. E., Goldstein, J. L. & Brown, M. S . (1983) J . Lipid Res. 24, 469-480 Edge, S. B., Hoeg, J. M., Triche, T., Schneider, P. D. & Brewer, H. B. (1986) J . B i d . Chem. 261, 38063806 Ford, R. P.. Botham, K. M., Suckling, K. E. & Boyd, G. S. (1985) FEBS Lert. 179, 177-180 Halloran, L. G., Schwartz, C. C., Vlahcevic, Z. R., Nisman, R. M. & Swell, L. (1978) Surgery 84, 1L7 Harkes, L. & van Berkel, T. J. C . (1984) Biochim. Biophys Acra 794, 340 -347 Herold, G.. Schneider, A,, Ditschuneit, H. & Stange, E. F. (1984) Biochim. Biophys. Actu 796. 2 7 3 3 Hoeg, J. M., Demosky, S. J., Jr., Schaefer, E. J., Starzl. T. E. & Brewer, H. B.. Jr. (1984) J . Clin. Invest. 73, 429-436 Kagami, A,, Fidge, N., Suzuki, N. & Nestel, P. (1984) Biochim. Biophys. Ac/u 795, 179-190 Lippiello, P. M., Sisson, P. J. & Waite, M. (1985) Biochem. J . 232, 395~40 I Mitropoulos, K. A,, Myant, N. B., Gibbons, G. F., Balasubramaniam, S. & Reeves, B. E. A. (1974) J. Biol. Chem. 249, 6052-6056 Nervi, F., Bronfman, M., Allalbn, W., Depiereux, E. & Del Pozo, R. (1984) J . Clin. Invest. 74, 2226-2237 Normann, P. T. & Norum, K. R. (1976) Scand. J . Gastroenrerol. 11, 427- 432 Robins. S. J., Fasulo, J. M., Collins, M. A. & Patton, G. M. (1985) J . Biol. Chem. 260, 651 1-6513 Schwartz, C. C., Vlahcevic, Z. R., Halloran, L. G., Gregory, D. H., Meek, J. B. & Swell, L. (1975) Gastroenterology 69, 1379-1382 Schwartz. C. C., Vlahcevic, Z. K., Halloran, L. G . , Nisman, R. & Swell, L. (1977) Proc. Soc. Exp. Biol. Med. 156, 261-264 Schwartz, C. C., Halloran, L. G., Vlahcevic, Z. R., Gregory, D. H. & Swell, L. ( 1 9 7 8 ~ )Science 200, 62-64 Schwartz, C. C., Berman. M., Vlahcevic, Z. R., Halloran, L. G., Gregory. D. H. & Swell. L. (19786) J . Clin. Invest. 61, 408-423 Singer, I . I . , Kawka, D. W., Kazazis, D. M., Alberts, A. W., Chen, J . S.. Huff, J. W. & Ness, G. C . (1984) Proc. Narl. Acad. Sci. U.S.A. 81. 5556-5560
Sodhi, H. S., Orchard, R. C., Agnish, N. D., Varughese. P. V. & Kudchodkar, B. J. (1973) Arherosclerosis 17, 197-210 Spady, D. K., Turley, S. D. & Dietschy, J. M . (1985a) J . Lipid Res. 26, 465-472 Spady, D. K., Turley, S. D. & Dietschy. J. M. (19856) J . Clin. Invrsr. 76, 1113-1122 Stange. E. F. & Dietschy, J. M. ( 1 9 8 3 ~ J) . Lipid Res. 24 72-82 Stange, E. F. & Dietschy, J. M. (1983h) Proc. Narl. Acud. Sci. U . S . A . 80, 5739-5743 Stange, E. F. & Dietschy, J. M . (1984) J . Lipid Res. 25, 703 713 Stange, E. F. & Dietschy, J. M . (1985) J . Lipid. Res. 26, 175-184 Stange, E. F., Suckling, K. E. & Dietschy. J. M . (1983) J . Biol. Chwn. 258, 12868-.12875 Stange, E. F., Alavi, M.. Schneider, A,, Preclik, G. & Ditschuneit, H . (1980) Biochim. Biophys. Acra 620. 520-527 Stange, E. F., Preclik, G., Schneider, A,, Alavi, M . Ditschuneit, H. (1981~)Biochim. Biophys. Acra 663, 613.- 620 Stange, E. F., Alavi, M., Schneider, A,, Ditschuneit. H. & P o k y , J. R. (I98 I b) J . Lipid. Res. 22, 47-56 Stange, E. F., Fleig, W. E., Schneider, A,. Nother-Fleig. G.. Alavi. M., Preclik, G. & Ditschuneit. H. (1982) A/herosclerosis 41. 67 80 Stange, E. F., Schneider. A,. GrGsch. G. & Ditschuneit. H. (19851) Z. Gu.s/roenterol. 23, 22 I -227 Stange, E. F., Spady, D. K. & Dietschy, J. M. (19856) in En/erohrpo/ic. Circulution of Bile A d s and Sterol Mc~ruholism(Paumgartner, G.. Gerok, W. & Stiehl, A., eds.), pp. 29-36. M T P Press Ltd. Lancastcr Stephan, Z . F. & Hayes, K. C. (1985) lipid.^ 20. 343 349 Turley, S. D. & Dietschy, J. M. (1981) J . Biol. Chem. 256, 2438 2446 Turley, S. D., Spady, D. K. & Dietschy, J. M . (1983) Gustroc.nterol/)~~ 84, 253-264 Wade, D. P., Soutar, A. K. & Gibbons, G. F. (1984) Biochem. J . 218. 203-2 1 1
Received 30 October 1986
Receptor-mediated endocytosis in steroid hormone-producing tissue KEITH E. SUCKLING* and BEGONA OCHOAt *Smith Kline and French Rcwarch Ltd., The Frythe, Welwyn, Herts., AL6 9 A R , U . K . , and t Department of Physiological Biochemistry, University of the Basque Country, Leioa ( Viscaya), Spain
Steroid hormone-producing tissue has been important in the study of receptor-mediated endocytosis of lipoproteins from a very early stage. Although relatively little of the total body turnover of cholesterol is accounted for by its conversion into steroid hormones, it is generally agreed that most steroid hormone-producing, cells, especially those of the adrenal cortex, are dependent on plasma lipoproteins for the supply of cholesterol for steroidogenesis (Gwynne & Strauss, 1982). Many lines of evidence support the concept that receptor-mediated endocytosis is important in the supply of cholesterol to these tissues. Perhaps one of the clearest is in WHHL rabbits, in which the low-density lipoprotein (LDL) receptor is not functional. Here the adrenal gland shows a five-fold increase in cholesterol synthesis over normal rabbits as a response to maintain the cholesterol supply for synthesis of adrenal cortical hormones (Dietschy et a/., 1983). In the rat LDL is not the main carrier of cholesterol in the blood and much evidence suggests that cholesterol is delivered by high-density lipoprotein (HDL) (reviewed in Gwynne & Strauss, 1982). Thus in steroid hormoneproducing tissues, within a broad general pattern of cholesAbbreviations used: LDL, low-density lipoprotein; HDL, highdensity lipoprotein; WHHL, Watanabe heritable hyperlipidaemic.
terol metabolism, several sources of cholesterol are available and different mechanisms of uptake of cholesterol from the blood may operate. In addition to the sources mentioned already, plasma lipoproteins and intracellular synthesis, steroid hormone-producing tissues frequently contain a store of cholesteryl ester that can be mobilized by the action of the appropriate tropic hormone (Boyd et a/., 1983). The effect of these hormones is to initiate a dramatic change in the intracellular organization of cholesterol metabolism. When considering the function of steroid hormone-producing tissue and the control of its cholesterol metabolism, we have to understand the role of these three sources of cholesterol and how they relate to the overall fluxes of cholesterol within the cell. In order to determine the importance of cholesterol synthesis and receptor-mediated uptake of lipoproteins in the adrenal cortex, Spady & Dietschy (1985) have made an extensive series of studies in vivo in the rat, hamster and rabbit. In all three species receptor-mediated uptake accounted for over 93% of the total uptake of cholesterol from the blood, but the extent to which cholesterol synthesis and uptake of lipoprotein was dominant depended on the species. The hamster derived 10 times more cholesterol from intracellular synthesis than from LDL uptake. The opposite was the case in the rabbit. Spady & Dietschy consider that man is most closely modelled by the situation in the hamster. The importance of receptor-mediated uptake is as great in the adrenal as in any tissue. These studies were performed by continuous influsion of lipoproteins into the animals over a period of several hours. It would be interesting to know what effect the injection of a tropic hormone would 1987
