Lovastatin, an Inhibitor of Cholesterol Synthesis, Induces

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Proc. Nati. Acad. Sci. USA Vol. 85, pp. 5264-5268, July 1988 Medical Sciences Lovastatin, an inhibitor of cholesterol synthesis, induces hydroxymethylglutaryl-coenzyme A reductase directly on membranes of expanded smooth endoplasmic reticulum in rat hepatocytes (proliferation of hepatocyte smooth endoplasmic reticulum/immunoelectron microscopy) IRWIN 1. SINGER*t, SOLOMON ScoTT*, DIANA M. KAZAZIS*, AND JESSE W. HUFFt Departments of *Biochemical and Molecular Pathology and tBiochemical Regulation, Merck Sharp and Dohme Research Laboratories, Rahway, NJ 07065 Communicated by Edward M. Scolnick, March 30, 1988

ABSTRACT Lovastatin is a potent competitive inhibitor of light microscopy. Direct localization ofHMG-CoA reductase the rate-limiting enzyme of cholesterol synthesis, 3-hydroxy- on these induced SER membrances at high resolution with 3-methylglutaryl-coenzyme A reductase (NADPH) [HMG-CoA immunoelectron microscopy (IEM) would provide strong in reductase; (S)-mevalonate:NADP+ oxidoreductase (CoA-acyl- vivo evidence in support of this concept. Moreover, since ating), EC 1.1.1.34]. We determined the subcellular distribu- SER proliferation is an abnormal change produced by other tion of HMG-CoA reductase at high resolution by means of agents that do not affect cholesterol synthesis (12, 13), it is immunoelectron microscopy on ultrathin frozen liver sections possible that the observed SER expansion might have little to of rats treated with lovastatin and cholestyramine. High do with the intracellular accumulation of HMG-CoA reduc- concentrations of reductase were located on the outer (cyto- tase. Therefore, we have performed an IEM study ofthe SER plasmic) surfaces of smooth endoplasmic reticulum (SER) whorls induced in rat hepatocytes by lovastatin, using spe- membranes induced in hepatocytes by acute drug administra- cific immunoprobes for HMG-CoA reductase. tion. The enzyme was specifically localized over the whorled SER membranes and was absent from nonwhorled SER, rough endoplasmic reticulum, and peroxisomes. Intense HMG-CoA MATERIALS AND METHODS reductase labeling was only observed in hepatocytes containing Materials. Lovastatin (Merck Sharp & Dohme) and cho- high levels of HMG-CoA reductase activity; no staining was lestyramine were obtained from Merck. Rabbit antibodies detected in untreated livers. These observations show that monospecific for rat HMG-CoA reductase were raised HMG-CoA reductase is induced as an integral component of against its 50- to 55-kDa extramembranous active site, which the SER membranes that form in rat hepatocytes subsequent to projects into the cytoplasm (11, 14). Another polyclonal lovastatin treatment and suggest that the formation of SER HMG-CoA reductase antibody was provided by R. K. Pa- whorls in rat hepatocytes is due to mechanism-based effects of thak and R. G. W. Anderson (University of Texas, Dallas). lovastatin. The IgG fractions of both rabbit antisera were isolated on protein A-Sepharose. Purified rat liver HMG-CoA reductase Lovastatin, formerly called mevinolin, is a powerful serum was a gift from G. Ness (University ofSouth Florida, Tampa, cholesterol-lowering agent in humans and other species (1- FL). Affinity-purified goat anti-rabbit IgG conjugated to 6). It is a potent competitive inhibitor of 3-hydroxy-3- fluorescein or 10-nm colloidal gold was from Boehringer- methylglutaryl-coenzyme A reductase [HMG-CoA reduc- Mannheim or Janssen Pharmaceutica (Piscataway, NJ). tase; (S)-mevalonate:NADP+ oxidoreductase (CoA-acyl- Animals. Sprague-Dawley rats (Charles River Breeding ating), EC 1.1.1.34], the rate-limiting enzyme of cholesterol Laboratories; 200-g male, 7 weeks old) were caged under synthesis (1). A similar compound termed mevastatin (ML- reversed lighting (12 hr dark/12 hr light) and fed Purina Rat 236B, compactin, mevastatin) exhibits an analogous mode of Chow supplemented with 3% (wt/wt) cholestyramine for 9 action (7), but lovastatin is a more active inhibitor (K, = 6 x days ad libitum, followed by Rat Chow containing 0.25% 10"1 M) than mevastatin (Ki = 1.4 x 10' M) (1, 8). The lovastatin plus 3% cholestyramine for 3 days prior to sacri- cholesterol-lowering effects of these agents are probably due fice. This regimen was selected because it stimulated high in part to increased production of hepatic low density levels of rat liver HMG-CoA reductase activity and wide- lipoprotein receptors (9, 10). spread SER proliferation, which were qualitatively similar to Acute treatment of rats with lovastatin (alone or with the effects of lovastatin administered alone (11). Control cholestyramine) induces large increases in hepatic HMG- animals were fed standard Purina Rat Chow. Compound- CoA reductase activity and a simultaneous appearance of treated rats exhibited normal weight gain throughout the prominent intracytoplasmic reductase immunofluorescence dosage period. The animals were sacrificed at the diurnal high microscopy (IFM) staining (11). The induction and distribu- point (10 a.m.) for HMG-CoA reductase activity. Liver micro- tion of this HMG-CoA reductase labeling correlates with the somes were isolated, and their HMG-CoA reductase activity appearance of massive whorls of smooth endoplasmic retic- was measured as before (11). ulum (SER) membranes detected by electron microscopy in vivo (11). While this correlation suggests that SER prolifer- IEM. We localized HMG-CoA reductase on ultrathin ation occurs to provide a scaffold for the increased quantities frozen sections of rat liver using ImmunoGold labeling. This ofHMG-CoA reductase synthesized, there has been no direct IEM method was selected because it renders all sectioned proof of this hypothesis because of the limited resolution of Abbreviations: HMG-CoA reductase, 3-hydroxy-3-methylglutaryl- coenzyme A reductase; IEM, immunoelectron microscopy; IFM, The publication costs of this article were defrayed in part by page charge immunofluorescence microscopy; RER, rough endoplasmic reticu- payment. This article must therefore be hereby marked "advertisement" lum; SER, smooth endoplasmic reticulum. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5264 Downloaded by guest on September 26, 2021 Medical Sciences: Singer et al. Proc. Natl. Acad. Sci. USA 85 (1988) 5265 intracellular compartments accessible to immunoprobes (Fig. 2 A and B), in peroxisomes (Fig. 2B), over adjacent without using permeabilizing agents that disrupt organelle portions of the cytoplasm containing glycogen deposits (Fig. structure, and it avoids the use of organic solvents and 2C, or on the outer membrane of the nuclear envelope (Fig. plastics which may denature antigens and obstruct labeling. 2C). HMG-CoA reductase immunostaining was totally elim- Effects of various fixatives on HMG-CoA reductase antigen- inated by treatment of the reductase antibodies with purified icity were monitored by indirect IFM as described (14) on antigen (Figs. 2D and 3B). Upon higher magnification, the 1-,um semithin frozen liver sections of compound-treated ImmunoGold labeling for HMG-CoA reductase appeared to rats. Optimal fixation was obtained with a mixture of 3.5% be located over the external surfaces of the whorled SER paraformaldehyde and 0.1% glutaraldehyde in 0.1 M membranes, while their inner aspects were mostly unlabeled sucrose/0.1 M sodium cacodylate, pH 7.2/4.5 mM CaC12 (see (Fig. 3A). HMG-CoA reductase immunolabeling was also Fig. 1). For IEM, fixed liver tissues were infiltrated with 2.3 absent from the hepatocyte RER of control livers (Fig. 3C). M sucrose and frozen rapidly, and -80-nm ultrathin frozen These cells totally lacked SER whorls, even in the periportal sections were cut, mounted on grids, and immunolabeled as hepatic regions where HMG-CoA reductase-specific immu- described (15). HMG-CoA reductase staining was performed nofluorescent labeling was detected with IFM in unfixed on the section surface with 100 ,ug of rabbit anti-reductase frozen samples (11). IgG per ml followed by goat anti-rabbit IgG conjugated to 10-nm colloidal gold. Controls were either anti-HMG-CoA reductase IgG preincubated with purified HMG-CoA reduc- DISCUSSION tase or nonimmune rabbit IgG substituted for the primary We localized HMG-CoA reductase in the livers ofrats treated step in our protocol. After refixation with 2% glutaraldehyde, with lovastatin/cholestyramine using IEM and found that the sections were negatively stained with 1% phosphotung- high concentrations of reductase were concentrated on the stic acid (pH 7.0). membranes of SER whorls induced in hepatocytes. The HMG-CoA reductase ImmunoGold label was preferentially RESULTS located on the external surface of these SER membranes, as opposed to their intracisternal surfaces. This distribution IFM. Since livers from rats treated with lovastatin and presumably occurred because our antibodies were raised cholestyramine exhibited maximum levels of HMG-CoA re- against the 50- to 55-kDa extramembranous carboxyl-termi- ductase and SER proliferation (11), this material was selected nal domain of HMG-CoA reductase (11). for further study. Liver microsomes from the compound- To perform this study, special fixation and counter-staining treated rats described herein exhibited a mean HMG-CoA methods were used to maximize the preservation of both reductase activity of 5200 pmol/min per mg of protein (SEM HMG-CoA reductase antigenicity and subcellular hepatocyte = 634; n = six rats), which represents a 20-fold induction ultrastructure. In addition, IEM localization of HMG-CoA relative to the controls (mean = 266 pmol/min per mg; SEM reductase was performed on the surfaces of ultrathin frozen = 40; n = six rats). After fixation with glutaraldehyde and sections. Thus, we are confident that the HMG-CoA reduc- formaldehyde, significant anti-HMG-CoA reductase IFM tase antibodies had access to all subcellular compartments staining was observed in the hepatocyte cytoplasm (Fig. LA). exposed by the sectioning process, so that unlabeled organ- This labeling was abolished by preincubating the HMG-CoA elles (e.g., RER, nuclear membrane, peroxisomes) probably reductase antibodies with purified enzyme (Fig. 1B) or by the have low amounts of reductase, or lack this enzyme entirely. use of nonimmune IgG (not shown). No IFM labeling was Furthermore, we believe that this IEM labeling is specific for detected in livers of control animals. HMG-CoA reductase because (i) similar labeling patterns IEM. Conspicuous SER whorls were present in ultrathin were observed with monospecific reductase antibodies pro- frozen sections of hepatocytes treated with lovastatin and duced by another laboratory (16); (it) ImmunoGold staining cholestyramine. These expanded SER membranes were was eliminated by preincubation of reductase IgG with heavily labeled for HMG-CoA reductase by using our IEM purified HMG-CoA reductase; and (iii) the use ofnonimmune protocol (Fig. 2A). No significant immunostaining was de- IgG produced negligible background labeling. The ability to tected in the nearby rough endoplasmic reticulum (RER) stain HMG-CoA reductase by IEM also correlated with the

FIG. 1. IFM staining of HMG-CoA reductase on 1-,um frozen sections of optimally fixed lovastatin/cholestyramine-treated rat liver. (A) Anti-HMG-CoA reductase-positive staining is found in the hepatocyte cytoplasm (arrow), where it sometimes exhibits a perinuclear distribution (arrowhead); nuclei (N) are unstained. (B) No staining is observed when reductase antibodies are pretreated with HMG-CoA reductase. (Bar = 10,m). Downloaded by guest on September 26, 2021 5266 Medical Sciences: Singer et al. Proc. Natl. Acad. Sci. USA 85 (1988)

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FIG. 2. IEM localization of HMG-CoA reductase on ultrathin (80 nm) frozen sections of lovastatin/cholestyramine-treated rat liver with an indirect ImmunoGold labeling method. (A) Hepatocytes with expanded whorls of SER membranes (ser) exhibit intense anti-HMG- CoA reductase ImmunoGold staining (arrowheads); adjacent RER (er) and mitochondrion (m) are unstained. (B) Portion of another hepatocyte labeled for HMG-CoA reductase as in A exhibits no staining in peroxisomes (p; arrowhead indicates crystalloid) and little labeling in the RER (er). (Bar in A = 250 nm for A and B). (C) Anti-HMG-CoA reductase labeling is confined to the induced SER whorls (ser) of hepatocytes and is largely absent from the nuclear membrane (arrowheads) and nearby cytoplasm containing glycogen (g). (D) Pretreatment of HMG- CoA reductase IgG with purified HMG-CoA reductase abolishes the ImmunoGold staining of SER whorls (ser). (Bar in D = 250 nm for C and D.) Downloaded by guest on September 26, 2021 Medical Sciences: Singer et al. Proc. Natl. Acad. Sci. USA 85 (1988) 5267

FIG. 3. Higher magnification micrographs of lovastatin/cholestyrmine-induced rat liver SER whorls immunolabeled for HMG-CoA reductase as in Fig. 2. (A) HMG-CoA reductase ImmunoGold labeling appears localized to the external surfaces of the SER membranes (arrowheads), rather than at the intracisternal surface (arrow). (B) Minimal background is observed when specific staining is blocked with HMG-CoA reductase. (C) Little immunolabeling is seen in the hepatocyte RER ofcontrol rats stained with anti-HMG-CoA reductase IgG. (Bar in C = 250 nm for A-C.) levels of reductase activity measured biochemically in cor- Because it is widely recognized that proliferation of SER responding samples from compound-treated and control membranes and accompanying induction of microsomal en- animals. Therefore, we believe that the IEM localization of zymes occurs in hepatocytes after exposure to various toxins HMG-CoA reductase on the whorled SER membranes of or phenobarbital (12, 13, 19), the appearance of SER whorls hepatocytes accurately reflects the native distribution of this after lovastatin administration might imply that this com- enzyme in lovastatin/cholestyramine-treated rats. pound exerts a toxic subcellular side effect in hepatocytes. Our results are similar to those of previous studies, where However, quantitative rocket immunoelectrophoretic anal- different IEM methods were used to localize HMG-CoA ysis has shown that lovastatin does not induce cytochrome reductase in cultured UT-1 cells (a Chinese hamster ovary P450 ble or epoxide hydrolase in rat hepatocyte micro- line adapted for growth in mevastatin) (16, 17). Mevastatin- somes, while phenobarbital does (C. B. Pickett, personal treated cells exhibited HMG-CoA reductase localized on communication). In addition, only 15% ofa rat cohort treated SER membranes structurally similar to those that we ob- with high doses of lovastatin (180 mg/kg per day) in a served in vivo, but reductase was also concentrated in the 6-month-to-2-yr chronic electron microscopic study exhib- adjacent membranes of the nuclear envelope and on tubular ited hepatic SER proliferation (unpublished data). Taken and sinusoidal derivatives of the SER (16). Since the hepa- together with these data, the direct IEM observation of high tocytes in our study lacked substantial tubular SER and did concentrations of HMG-CoA reductase on SER membranes not exhibit HMG-CoA reductase in the nuclear envelope, it induced by lovastatin strongly suggests that this SER induc- is possible that the mechanism of formation of reductase- tion is caused by mechanism-based effects of lovastatin laden SER membranes in the UT-1 cells in vitro differs from rather than by hepatic cytotoxicity. that operative in rat hepatocytes in vivo. In another related We thank Drs. R. K. Pathak, R. G. W. Anderson, and G. C. Ness study, 5% ofthe hepatocyte HMG-CoA reductase was found for antibodies and purified HMG-CoA reductase, J. S. Chen for in peroxisomes of control rats, and the peroxisomal content determinations of HMG-CoA reductase activity, and A. W. Alberts of reductase increased 6-fold after cholestyramine treatment and C. B. Pickett for helpful discussions. (18). In contrast, we did not find HMG-CoA reductase in peroxisomes of either control or compound-treated hepa- 1. Alberts, A. W., Chen, J., Kuron, G., Hunt, V., Huff, J., tocytes. At this time, we see no clear reason for this Hoffman, C., Rothrock, J., Lopez, M., Joshua, H., Harris, E., discrepancy, but the disagreement may be due to the use of Patchett, A., Monaghan, R., Currie, S., Stapley, E., Albers- Schonberg, G., Hensens, O., Hirshfield, J., Hoogsteen, K., different reductase antibodies (monoclonal versus polyclo- Liesch, J. & Springer, J. (1980) Proc. Natl. Acad. Sci. USA 77, nal) or to sampling diverse time points. In any event, the 3957-3961. precise role played by peroxisomes in regulating cholesterol 2. Bilheimer, D. W., Grundy, S. M., Brown, M. S. & Goldstein, metabolism is unclear. J. L. (1983) Proc. Nat!. Acad. Sci. USA 80, 4124-4128. Downloaded by guest on September 26, 2021 5268 Medical Sciences: Singer et al. Proc. Nat!. Acad. Sci. USA 85 (1988)

3. Edwards, P. A., Lan, S.-F. & Fogelman, A. M. (1983) J. Biol. Chen, J. S., Huff, J. W. & Ness, G. C. (1984) Proc. Natl. Chem. 258, 10219-10222. Acad. Sci. USA 81, 5556-5560. 4. Illingworth, R. D. (1984) Ann. Intern. Med. 101, 598-604. 12. Bolender, R. P. & Weibel, E. R. (1973) J. Cell Biol. 56, 746- 5. Illingworth, R. D. & Sexton, G. J. (1984) J. Clin. Invest. 74, 751. 1972-1978. 13. Jones, A. L. & Fawcett, D. W. (1966) J. Histochem. Cyto- 6. Tobert, J. A., Bell, G. D., Birtwell, J., James, J., Kukovetz, chem. 14, 215-232. W. R., Pryor, J. S., Buntinx, A., Holmes, I. B., Chao, Y. S. & 14. Singer, I. I., Kawka, D. W., McNally, S. E., Scott, S., Al- Bolognese, J. A. (1982) J. Clin. Invest. 69, 913-919. berts, A. W., Chen, J. S. & Huff, J. W. (1987) Arteriosclerosis 7. Endo, A., Tsujita, Y., Kuroda, M. & Tanzawa, K. (1979) 7, 144-151. Biochim. Biophys. Acta 575, 266-276. 15. Singer, I. I., Kazazis, D. M., Kawka, D. W., Rupp, E. A. & 8. Slater, E. E., Alberts, A. W. & Smith, R. L. (1987) in The Role Bayne, E. K. (1985) Arthritis Rheum. 28, 1105-1116. of Cholesterol in Atherosclerosis: New Therapeutic Opportu- 16. Pathak, R. K., Lusky, K. L. & Anderson, R. G. W. (1986) J. nities, eds. Grundy, S. M. & Beam, A. G. (Hanley & Belfus, Cell Biol. 102, 2158-2168. Philadelphia), pp. 35-50. 17. Orci, L., Brown, M. S., Goldstein, J. L., Garcia-Segura, L. M. 9. Ma, P. T. S., Gil, G., Sudhof, T. C., Bilheimer, D. W., Gold- & Anderson, R. G. W. (1984) Cell 36, 835-845. stein, J. L. & Brown, M. S. (1986) Proc. Natl. Acad. Sci. USA 18. Keller, G. A., Pazirandeh, M. & Krisans, S. (1986) J. Cell Biol. 83, 8370-8374. 103, 875-886. 10. Brown, M. S. & Goldstein, J. L. (1986) Science 232, 34-47. 19. Pickett, C. B., Jeter, R. L., Morin, J. & Lu, A. Y. H. (1981) J. 11. Singer, I. I., Kawka, D. W., Kazazis, D. M., Alberts, A. W., Biol. Chem. 256, 8815-8820. Downloaded by guest on September 26, 2021