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