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Endoplasmic Reticulum and Autophagy in Rat Hepatocytes (Lysosomes/Electron Microscopy/Liver) ALEX B

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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 ABSTRACT Electron microscopy of hepatocytes in both were dissected and transferred to fresh fixative. After 2 hr of normal rat liver and rat liver treated to induce hyperplasia of fixation, the cubes were rapidly dehydrated in alcohols, treated smooth endoplasmic reticulum shows that autophagic vacuoles and residual bodies (types of lysosomes) are continuous with with propylene oxide, and embedded in Epon 812. Sections, endoplasmic reticulum. about 500-600 A thick, were cut with an LKB Ultratome and mounted on copper grids. After they were stained with lead Our laboratory has emphasized the diverse activities of GERL citrate, they were examined in the Siemens Elmiskop I micro- (1, 2) and other regions of the endoplasmic reticulum (ER) in scope. hepatocytes (ref. 3; A. B. Novikoff, P. M. Leuenberger, P. M. Serial sections were mounted on parlodion-coated single slit Novikoff, and M. Quintana, unpublished results). In 1964 we untreated rats suggested that in hepatocytes the ER is involved in forming grids. Serial sections were prepared from four autophagic vacuoles-lysosomes in which intracellular material and three rats fed 4-dimethylaminobenzine and partially is degraded: "Smooth endoplasmic reticulum appears to wrap hepatectomized. The numbers of consecutive sections photo- around bits of cytoplasm, often becoming 'compacted' in the graphed ranged from 5 to 31. A plastic model was prepared process. This may reflect an altered permeability and release from 12 consecutive photographs of a hepatocyte with hyper- of acid hydrolases and other materials to the interior of the plastic smooth ER. 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. RESULTS 188). By superimposition of plastic cutouts of 12 consecutive sections, The growing knowledge of proteolysis in cells (reviewed in 4 of which are shown in Fig. 1 A-D, a model was prepared (Fig. ref. 5) has revived interest in the degree to which autophagy 1E). The criteria used to identify the various kinds of lysosomes is involved in such protein breakdown in liver. Biochemical are stated elsewhere (9). Five residual bodies (numbered 1-5) studies on perfused rat livers, reported recently by Mortimore and an autophagic vacuole, type 2 (labeled AV2), are seen in and his colleagues (listed in ref. 6), implicate lysosomes in the this small region of the hepatocyte in which hyperplasia of proteolysis subsequent to withdrawal of amino acids from the smooth perfusing medium. Mortimore and Schworer (6) quantitated smooth ER has been induced. All are interconnected by the "fractional cytoplasmic volumes" of the autophagic vacuoles ER (arrows). Indeed, one may consider them as swollen areas with various constituents that rapidly develop after amino acid of smooth ER. That the smooth membranes are indeed part of deprivation; the vacuoles containing glycogen were especially the ER is shown by numerous continuities with rough ER (i.e., emphasized. The purposes of this communication are (i) to show ribosome-studded ER), seven of which are indicated by ar- the relation of such autophagic vacuoles to the ER in rat hep- rowheads in Fig. 1B. Within the residual bodies, grains and atocytes; and (ii) to present a three-dimensional model recon- other electron-opaque materials, presumably the result of in- structed from consecutive serial sections. tracellular digestion (9), are seen. Membranes, appearing as tubules and vesicles within the autophagic vacuole, type 2, are interpreted as internalized smooth ER (9). MATERIALS AND METHODS Similar continuities of residual bodies and autophagic vac- Sprague-Dawley rats of both sexes, 150-250 g in weight, were uoles with ER in normal hepatocytes are illustrated in Figs. 2 used for the observations on the normal rat. They were fed and 3. Such continuities are found with regularity in the serial Purina rat chow ad lib. To induce hyperplasia of the smooth ER, sections but their existence may fail to be revealed in random we fed male Sprague-Dawley rats the Miller et al. diet (7) thin sections. containing 0.06% 4-dimethylaminobenzine for 7 days, and then In normal hepatocytes, unlike those where hyperplasia of we partially hepatectomized the rats by the procedure of smooth ER has been induced, much electron-opaque glycogen Higgins and Anderson (8). The rats were fed the 4-dimeth- is present in areas of smooth ER. This provides an endogenous ylaminobenzine diet for an additional 5 days and then "marker" for "microautophagy" (11), as seen in Figs. 2 and 3. killed. The regions of smooth ER that acquire glycogen within their The animals were anesthetized by ether, and thin slices of cisternae are strikingly pleomorphic (Figs. 2 and 3; see also liver were placed into cold 1% OS04 in 0.1 M phosphate buffer 10 11 in ref. containing 5% sucrose. Cubes, ca. 1 mm in each dimension, figures and 9). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviation: ER, endoplasmic reticulum. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate * Present address: Department of Laboratories, Veterans Adminis- this fact. tration Hospital, 16111 Plummer Street, Sepulveda, CA 91343. 5039 Downloaded by guest on September 27, 2021 5040 Cell Biology: Novikoff and Shin Proc. Nati. Acad. Sci. USA 75 (1978) AA "I i .4 I I .4 -.t"illo I.. N,-- . it I I 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 con- tinues 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 in- dicate smooth ER. Other cytoplasmic structures visible in A-D are not shown in the model. *' a..I% .{ W X >. ' -. s (,,,<' * 9 * 4:~~~~~~~~~' * . # S It.* *44 B FIG. 2 (Legend appears cat the bottom of the next page.) Downloaded by guest on September 27, 2021 Cell Biology: Novikoff and Shin Proc. Natl. Acad. Sci. USA 75 (1978) 5041 fps A AX ~~~~A~~~~~" ar:f'tAVs s t&~~a. 4~~~~~~~~~~~~~~~~~4 h94~' FIG. 3. Portion of a normal hepatocyte. Sections 4 (A) and 5 (B) from a series of 15 consecutive serial sections. (X35,000.) Residual bodies are numbered 1-4 AV2 indicates probable autophagic vacuoles, type 2. The membrane of the lower AV2 appears compacted in areas and is therefore more electron-opaque. The possibility cannot be ruled out that the upper body labeled AV2 is a residual body derived from GERL, containing lipoprotein particles, in the manner described by Novikoff and Yam (10). The paired arrowheads indicate areas interpreted as glycogen's being internalized by microautophagy (9). Free glycogen (G) is also present in the cytosol. Glycogen is seen in the residual bodies; it is also present in dilated areas of smooth ER (crossed arrow). Arrows indicate continuities of smooth ER with residual bodies, with the lower AV2 in A and, in A, with a peroxisome (P). Also labeled are portions of the Golgi stack (GO) and GERL (GE) and a mitochondrion (M). DISCUSSION cluding not only soluble materials but also glycogen and other particulate mailer. The latter could include degraded proteins. As suggested in our 1964 paper (4) and extended in 1973 by one For example, Allison (12) suggests that the a-hemoglobin chains of us (9), the ER of rat hepatocytes appears to be involved in present in excess over (3 chains in maturing erythrocytes of forming autophagic vacuoles of three kinds: (i) AV1, the type patients with (3 thalassemia are denatured in the cytosol and find generally identified because recognizable cytoplasmic organ- their way into lysosomes. elles are present within them; (ii) AV2, which seems to involve Interconnections of residual bodies and autophagic vacuoles internalization of smooth ER membrane; and (iii) microauto- with ER are sufficiently common that questions may be raised phagy, which apparently involves engulfment of small areas about how long they remain attached to the ER and whether of cytosol by ER. Both AV2 formation and microautophagy and when they separate from the ER. Without suitable tracers, could bring cytosolic constituents into the ER cisternae, in- answers to such questions cannot be gained from static electron FIG. 2 (on preceding page). Portion of a normal hepatocyte. One of 10 consecutive serial sections. (x44,000.) Cytoplasmic body, labeled AV1, is an autophagic vacuole, type 1, sectioned tangentially. The mitochondrion (or mitochondria?) within it is clearly evident in other sections (in ref. 9 see figure SC, another in the series of 10 consecutive serial sections, which shows a mitochondrion's delimiting membrane and the membrane's continuity with other regions of smooth ER).
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