Proc. Nati. Acad. Sci. USA Vol. 75, No. 10, pp. 5039-5042, October 1978
Cell Biology
Endoplasmic reticulum and autophagy in rat hepatocytes (lysosomes/electron microscopy/liver)
ALEX B. NOVIKOFF AND WOO-YUNG SHIN* Department of Pathology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461
Contributed by Alex B. Novikoff, July 28, 1978
Electron microscopy of hepatocytes in both ABSTRACT normal rat liver and rat liver treated to induce hyperplasia of smooth endoplasmic reticulum shows that autophagic vacuoles and residual bodies (types of lysosomes) are continuous with endoplasmic reticulum. Our laboratory has emphasized the diverse activities of GERL (1, 2) and other regions of the endoplasmic reticulum (ER) in hepatocytes (ref. 3; A. B. Novikoff, P. M. Leuenberger, P. M. Novikoff, and M. Quintana, unpublished results). In 1964 we suggested that in hepatocytes the ER is involved in forming autophagic vacuoles-lysosomes in which intracellular material is degraded: "Smooth endoplasmic reticulum appears to wrap around bits of cytoplasm, often becoming 'compacted' in the process. This may reflect an altered permeability and release of acid hydrolases and other materials to the interior of the autophagic vacuoles. The electron micrographs suggest, but do not firmly establish, that the endoplasmic reticulum thus altered becomes the delimiting membranes of the vacuoles" (ref. 4, p.
188). The growing knowledge of proteolysis in cells (reviewed in ref. 5) has revived interest in the degree to which autophagy is involved in such protein breakdown in liver. Biochemical studies on perfused rat livers, reported recently by Mortimore and his colleagues (listed in ref. 6), implicate lysosomes in the proteolysis subsequent to withdrawal of amino acids from the perfusing medium. Mortimore and Schworer (6) quantitated the "fractional cytoplasmic volumes" of the autophagic vacuoles with various constituents that rapidly develop after amino acid deprivation; the vacuoles containing glycogen were especially emphasized. The purposes of this communication are (i) to show the relation of such autophagic vacuoles to the ER in rat hepatocytes; and (ii) to present a three-dimensional model reconstructed from consecutive serial sections. MATERIALS AND METHODS
Sprague-Dawley rats of both sexes, 150-250 g in weight, were used for the observations on the normal rat. They were fed Purina rat chow ad lib. To induce hyperplasia of the smooth ER, we fed male Sprague-Dawley rats the Miller et al. diet (7) containing 0.06% 4-dimethylaminobenzine for 7 days, and then we partially hepatectomized the rats by the procedure of
Higgins and Anderson (8). The rats were fed the 4-dimethylaminobenzine diet for an additional 5 days and then killed. The animals were anesthetized by ether, and thin slices of liver were placed into cold 1% OS04 in 0.1 M phosphate buffer containing 5% sucrose. Cubes, ca. 1 mm in each dimension,
were dissected and transferred to fresh fixative. After 2 hr of fixation, the cubes were rapidly dehydrated in alcohols, treated with propylene oxide, and embedded in Epon 812. Sections, about 500-600 A thick, were cut with an LKB Ultratome and mounted on copper grids. After they were stained with lead citrate, they were examined in the Siemens Elmiskop I microscope. Serial sections were mounted on parlodion-coated single slit grids. Serial sections were prepared from four untreated rats and three rats fed 4-dimethylaminobenzine and partially hepatectomized. The numbers of consecutive sections photographed ranged from 5 to 31. A plastic model was prepared from 12 consecutive photographs of a hepatocyte with hyperplastic smooth ER. RESULTS By superimposition of plastic cutouts of 12 consecutive sections, 4 of which are shown in Fig. 1 A-D, a model was prepared (Fig. 1E). The criteria used to identify the various kinds of lysosomes are stated elsewhere (9). Five residual bodies (numbered 1-5) and an autophagic vacuole, type 2 (labeled AV2), are seen in this small region of the hepatocyte in which hyperplasia of smooth ER has been induced. All are interconnected by smooth ER (arrows). Indeed, one may consider them as swollen areas of smooth ER. That the smooth membranes are indeed part of the ER is shown by numerous continuities with rough ER (i.e., ribosome-studded ER), seven of which are indicated by arrowheads in Fig. 1B. Within the residual bodies, grains and other electron-opaque materials, presumably the result of intracellular digestion (9), are seen. Membranes, appearing as tubules and vesicles within the autophagic vacuole, type 2, are interpreted as internalized smooth ER (9). Similar continuities of residual bodies and autophagic vacuoles with ER in normal hepatocytes are illustrated in Figs. 2 and 3. Such continuities are found with regularity in the serial sections but their existence may fail to be revealed in random thin sections. In normal hepatocytes, unlike those where hyperplasia of smooth ER has been induced, much electron-opaque glycogen is present in areas of smooth ER. This provides an endogenous "marker" for "microautophagy" (11), as seen in Figs. 2 and 3. The regions of smooth ER that acquire glycogen within their cisternae are strikingly pleomorphic (Figs. 2 and 3; see also figures 10 and 11 in ref. 9).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: ER, endoplasmic reticulum. * Present address: Department of Laboratories, Veterans Administration Hospital, 16111 Plummer Street, Sepulveda, CA 91343. 5039
Proc. Nati. Acad. Sci. USA 75 (1978)
Cell Biology: Novikoff and Shin
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FIG. 1. Portion of a hepatocyte with hyperplasia of smooth ER. Sections 2, 3, 4, and 6 of 12 consecutive serial sections, are shown in A, B, C, and D, respectively, (X25,000.) Five residual bodies are numbered 1-5, and an autophagic vacuole, type 2 (see ref. 9), is labeled AV,. Arrowheads in B show seven areas in which smooth ER continues with rough ER (note ribosomes). The arrows indicate some continuities of smooth ER with the residual bodies and the autophagic vacuole; see also a continuity of smooth ER with a peroxisome (P) in D. Areas of hyperplastic smooth ER are labeled SER; in A and D, rough ER is labeled RER; and in B, a mitochondrion is labeled M. (E) The two surfaces of a plastic model reconstructed from 12 consecutive sections of the hepatocyte seen in A-D. The cut surfaces of the AV2 and the five residual bodies appear lighter in the photographs because their interiors have been filled with light-colored rubber. Arrows indicate smooth ER. Other cytoplasmic structures visible in A-D are not shown in the model.
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Cell Biology
Endoplasmic reticulum and autophagy in rat hepatocytes (lysosomes/electron microscopy/liver)
ALEX B. NOVIKOFF AND WOO-YUNG SHIN* Department of Pathology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461
Contributed by Alex B. Novikoff, July 28, 1978
Electron microscopy of hepatocytes in both ABSTRACT normal rat liver and rat liver treated to induce hyperplasia of smooth endoplasmic reticulum shows that autophagic vacuoles and residual bodies (types of lysosomes) are continuous with endoplasmic reticulum. Our laboratory has emphasized the diverse activities of GERL (1, 2) and other regions of the endoplasmic reticulum (ER) in hepatocytes (ref. 3; A. B. Novikoff, P. M. Leuenberger, P. M. Novikoff, and M. Quintana, unpublished results). In 1964 we suggested that in hepatocytes the ER is involved in forming autophagic vacuoles-lysosomes in which intracellular material is degraded: "Smooth endoplasmic reticulum appears to wrap around bits of cytoplasm, often becoming 'compacted' in the process. This may reflect an altered permeability and release of acid hydrolases and other materials to the interior of the autophagic vacuoles. The electron micrographs suggest, but do not firmly establish, that the endoplasmic reticulum thus altered becomes the delimiting membranes of the vacuoles" (ref. 4, p.
188). The growing knowledge of proteolysis in cells (reviewed in ref. 5) has revived interest in the degree to which autophagy is involved in such protein breakdown in liver. Biochemical studies on perfused rat livers, reported recently by Mortimore and his colleagues (listed in ref. 6), implicate lysosomes in the proteolysis subsequent to withdrawal of amino acids from the perfusing medium. Mortimore and Schworer (6) quantitated the "fractional cytoplasmic volumes" of the autophagic vacuoles with various constituents that rapidly develop after amino acid deprivation; the vacuoles containing glycogen were especially emphasized. The purposes of this communication are (i) to show the relation of such autophagic vacuoles to the ER in rat hepatocytes; and (ii) to present a three-dimensional model reconstructed from consecutive serial sections. MATERIALS AND METHODS
Sprague-Dawley rats of both sexes, 150-250 g in weight, were used for the observations on the normal rat. They were fed Purina rat chow ad lib. To induce hyperplasia of the smooth ER, we fed male Sprague-Dawley rats the Miller et al. diet (7) containing 0.06% 4-dimethylaminobenzine for 7 days, and then we partially hepatectomized the rats by the procedure of
Higgins and Anderson (8). The rats were fed the 4-dimethylaminobenzine diet for an additional 5 days and then killed. The animals were anesthetized by ether, and thin slices of liver were placed into cold 1% OS04 in 0.1 M phosphate buffer containing 5% sucrose. Cubes, ca. 1 mm in each dimension,
were dissected and transferred to fresh fixative. After 2 hr of fixation, the cubes were rapidly dehydrated in alcohols, treated with propylene oxide, and embedded in Epon 812. Sections, about 500-600 A thick, were cut with an LKB Ultratome and mounted on copper grids. After they were stained with lead citrate, they were examined in the Siemens Elmiskop I microscope. Serial sections were mounted on parlodion-coated single slit grids. Serial sections were prepared from four untreated rats and three rats fed 4-dimethylaminobenzine and partially hepatectomized. The numbers of consecutive sections photographed ranged from 5 to 31. A plastic model was prepared from 12 consecutive photographs of a hepatocyte with hyperplastic smooth ER. RESULTS By superimposition of plastic cutouts of 12 consecutive sections, 4 of which are shown in Fig. 1 A-D, a model was prepared (Fig. 1E). The criteria used to identify the various kinds of lysosomes are stated elsewhere (9). Five residual bodies (numbered 1-5) and an autophagic vacuole, type 2 (labeled AV2), are seen in this small region of the hepatocyte in which hyperplasia of smooth ER has been induced. All are interconnected by smooth ER (arrows). Indeed, one may consider them as swollen areas of smooth ER. That the smooth membranes are indeed part of the ER is shown by numerous continuities with rough ER (i.e., ribosome-studded ER), seven of which are indicated by arrowheads in Fig. 1B. Within the residual bodies, grains and other electron-opaque materials, presumably the result of intracellular digestion (9), are seen. Membranes, appearing as tubules and vesicles within the autophagic vacuole, type 2, are interpreted as internalized smooth ER (9). Similar continuities of residual bodies and autophagic vacuoles with ER in normal hepatocytes are illustrated in Figs. 2 and 3. Such continuities are found with regularity in the serial sections but their existence may fail to be revealed in random thin sections. In normal hepatocytes, unlike those where hyperplasia of smooth ER has been induced, much electron-opaque glycogen is present in areas of smooth ER. This provides an endogenous "marker" for "microautophagy" (11), as seen in Figs. 2 and 3. The regions of smooth ER that acquire glycogen within their cisternae are strikingly pleomorphic (Figs. 2 and 3; see also figures 10 and 11 in ref. 9).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: ER, endoplasmic reticulum. * Present address: Department of Laboratories, Veterans Administration Hospital, 16111 Plummer Street, Sepulveda, CA 91343. 5039
Proc. Nati. Acad. Sci. USA 75 (1978)
Cell Biology: Novikoff and Shin
5040
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i
.4
I
I
.4
N,--
-.t"illo
I..
it
.
I
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FIG. 1. Portion of a hepatocyte with hyperplasia of smooth ER. Sections 2, 3, 4, and 6 of 12 consecutive serial sections, are shown in A, B, C, and D, respectively, (X25,000.) Five residual bodies are numbered 1-5, and an autophagic vacuole, type 2 (see ref. 9), is labeled AV,. Arrowheads in B show seven areas in which smooth ER continues with rough ER (note ribosomes). The arrows indicate some continuities of smooth ER with the residual bodies and the autophagic vacuole; see also a continuity of smooth ER with a peroxisome (P) in D. Areas of hyperplastic smooth ER are labeled SER; in A and D, rough ER is labeled RER; and in B, a mitochondrion is labeled M. (E) The two surfaces of a plastic model reconstructed from 12 consecutive sections of the hepatocyte seen in A-D. The cut surfaces of the AV2 and the five residual bodies appear lighter in the photographs because their interiors have been filled with light-colored rubber. Arrows indicate smooth ER. Other cytoplasmic structures visible in A-D are not shown in the model.
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