Enzyme treatment of glycogen particles in rat liver and muscle

Enzyme treatment of glycogen particles in rat liver and muscle

© 1967 by Academic Press Inc. 444 J. ULTRASTRt~CTURERESEARCH18, 444--455 (1967) Enzyme Treatment of Glycogen Particles in Rat Liver and Muscle G. R...

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© 1967 by Academic Press Inc.

444

J. ULTRASTRt~CTURERESEARCH18, 444--455 (1967)

Enzyme Treatment of Glycogen Particles in Rat Liver and Muscle G. ROSATI

Laboratori di Fisica, Istituto Superiore di San#h, Roma, Italy Received August 1, 1966 Liver and striated muscle glycogen particles, both isolated and in tissue sections, were examined in the electron microscope. They were treated enzymatically with diastase, amylase, and pepsin. Pepsin was used because it is believed that glycogen in tissues is bound to proteins. The enzymatic treatment of ultrathin sections gave the following results: (a) Liver glycogen particles disappeared from the sections after digestion with any one of the enzymes used; (b) muscle glycogen particles did not disappear from the sections even after a double treatment with pepsin and diastase. Glycogen particles isolated from either liver or striated muscle were digested by diastase. Pieces of liver and muscle, incubated in 0.1% glucose to synthesize more glycogen, contained glycogen particles which appeared larger than usual, as seen in the electron microscope. Following diastase treatment, these particles returned to their original dimensions in muscle sections, and disappeared completely from the liver sections. Many papers dealing with the identification of glycogen in the electron microscope have been published. Of particular interest are those of Karrer, Drochmans, Revel, Baker, Theman, and others (2-7, 9, 10, 12, 17, 18, 22-24). These investigators studied the appearance of isolated glycogen and of glycogen contained in tissue sections in the electron microscope using various procedures. They observed great differences in the appearance of the glycogen from different kinds of cells. There are two basic types of glycogen particles. Those of the first type are roughly spherical, about 150-300 • in diameter. They have been found in striated muscle and have been described by several authors (7). The second type, the ~ particles of Drochmans (4), consists of complex units formed of a closely packed mass of particles 150-300/~ in diameter [the/5 particles of Drochmans (4)]. These are commonly referred to as "rosettes" (12) and are typical of m a m malian liver. They also occur in various tissues of other animals and vary greatly in size from one tissue to another.

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The w o r k described in this p a p e r c o m p a r e s the effects of a m y l o l y t i c a n d p r o t e o l y t i c enzymes on the two m a i n types of glycogen. W e have e x a m i n e d in p a r t i c u l a r the liver a n d the striated muscle of the rat. T r e a t m e n t s with p r o t e o l y t i c enzymes were p e r f o r m e d because it is believed t h a t glycogen in tissues is b o u n d to proteins (1, 11, 15). These enzymes digest the p r o t e i n s a n d r e n d e r the glycogen soluble. I n o r d e r to d e t e r m i n e w h a t relationship exists between the glycogen a n d the substrate in the tissues, small pieces of liver a n d striated muscle were i n c u b a t e d in a W a r b u r g a p p a r a t u s in the presence of glucose. This resulted in the synthesis of new glycogen.

M A T E R I A L S A N D METHODS

Fixation and embedding. Rat liver and diaphragm tissues were fixed for 1 hour in 2.5 % glutaraldehyde buffered at p H 7.2 with cacodylate (20, 21), rinsed for 2-3 hours with the same buffer, which was changed frequently, postfixed for 1 hour in 1 To OsO~ in cacodylate buffer. Some pieces of diaphragm were fixed in 6 % glutaraldehyde buffered with cacodylate and were not postfixed with OsO4. After fixation the tissues were dehydrated in increasing concentrations of acetone and embedded in Vestopal W (19). The sections, cut with a Porter-Blum or LKB microtome, were stained for 10 minutes with Pb(OH)2 by method A of Karnovsky (8) and examined in a Siemens Elmiskop I microscope. Incubation. Small pieces were taken from liver and muscle and immediately incubated with 0.1% glucose in a Warburg apparatus. After 1 hour of incubation the specimens were fixed in glutaraldehyde, postfixed in OsO4, embedded in Vestopal, stained with lead, and examined in the electron microscope. Isolated glycogen. Glycogen was isolated from both rat liver and rabbit muscle, using the cold water procedure of Orrell and Bueding (16). This method preserves the glycogen better than the more drastic one using alkali, and is meant to free the glycogen from the protein. The amount of protein remaining after this treatment was found to be less than 0.5 %. The isolated glycogen was dissolved in distilled water, and a drop of this solution was placed on a carbon-coated grid and examined in the electron microscope. Both negative staining with 2 % PTA buffered at p H 7.2 and positive staining with Pb(OH)2 by the method of Karnovsky (8), were used. Some specimens of the isolated glycogen were treated with diastase for 15, 30, and 60 minutes before the staining with Pb(OH)~ or the negative staining with PTA. The micro-Kjeldahl method according to Borsook and Dubnoff was used to determine the protein content of the isolated glycogen. Enzymatic treatment. The enzymatic treatments were performed on thin sections of liver and striated muscle prepared by the techniques described above; some of them were performed also on glycogen isolated from both tissues. 2 9 - 671823 J . Ultrastructure Research

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G. ROSATI

The sections were picked up in Mylar or polyethylene rings (13) and floated on the enzymatic solutions, at 37°C. The enzymatic solutions used were: 0.5% pepsin in 0.1 N HC1; 1% diastase in distilled water; saliva. A double treatment was also performed on muscle sections. They were first treated with pepsin, then washed in water, and finally treated with diastase, using the solutions described above. In every case, some sections were incubated in the solvent used for the enzymes, following the same procedures and for the same period of time as the control specimens. In some cases the sections were oxidized for 30 minutes with H202 before treatment. After a treatment of 6-8 hours, the sections were rinsed, mounted on grids, stained, and examined in the electron microscope.

OBSERVATIONS In liver sections fixed in glutaraldehyde, postfixed in OsO4, and stained with lead, glycogen appears in the f o r m of "rosettes" which vary greatly in size (Fig. 1). In muscle sections, fixed and stained in the same way, glycogen particles appear to be smaller and roughly spherical in shape, 150-250 A in diameter (Fig. 3).

Enzymatic treatment of liver The examination of liver sections, after digestion for 6-8 hours with 0.5% pepsin in 0.1 HC1, even if not oxidized previously, showed the presence in the cytoplasm of clear zones with borders which were not well defined (Fig. 2). The zones corresponded to the areas which contained glycogen in the control sections. Similar clear zones in the cytoplasm could also be seen in the sections which had been treated with diastase or amylase for the same period of time (Fig. 5).

Enzymatic treatment of striated muscle Typical particles were always observed in diaphragm sections treated with pepsin (Fig. 4), diastase (Fig. 6), or amylase. Muscle glycogen particles were also seen in sections which had been treated with pepsin first and then with diastase. N o glycogen particles could be f o u n d in sections of striated muscle fixed with glutaraldehyde alone and stained with lead. The particles seen in these specimens were ribosomes and were attached by RNase.

Fro. 1. Rat liver, fixed in 2.5 % glutaraldehyde, postfixed in OsO4, and stained with Pb(OH)~ according to Karnovsky. Glycogen particles appear in the form of "rosettes." x 82,000. FI~. 2. Electron micrograph of a section of the liver, fixed and stained as described above, following a 7-hour treatment with pepsin, without previous oxidation. Clear zones in the cytoplasm, which correspond to the areas containing glycogen in the control sections, can be seen. × 44,000.

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Enzymatic treatment of incubated tissues Specimens of incubated muscle contained glycogen particles which were larger than usual (Fig. 7). Their average diameter was 350-400 •. When these specimens were treated only with diastase the glycogen particles decreased in size, returning to their original proportions (Fig. 8). In the case of the incubated liver specimens, there was not a significant increase in the size of the rosettes. The glycogen completely disappeared from the sections following diastase treatment. Isolated glycogen Liver and muscle glycogen, isolated by the techniques described above, were examined in the electron microscope. The specimens were either negatively stained with PTA or stained with Pb(OH)2 according to the method of Karnovsky. The appearance of the isolated particles from both tissues corresponded perfectly to that observed in the sections. Liver glycogen had the typical "rosette" configuration (Figs. 12 and 13), and muscle glycogen was in the form of smaller particles which were very compact, even in negative staining, when their subunits could sometimes be seen (Figs. 9 and 10). The protein content of these isolated glycogen samples was less than 0.5 %. But to obtain isolated glycogen from muscle containing such a small amount of proteins, more extractions with chloroform than in the case of liver had to be applied. Diastase treatment of isolated glycogen The glycogen isolated from liver, when treated with 1% diastase, progressively decomposed. The extent of this decomposition depended upon the time of exposure to the action of the enzyme. After an exposure of 15 minutes, the "rosettes" appeared disaggregated. After 30 minutes, the rosettes were no longer recognizable and the glycogen appeared in the form of spherical particles (Fig. 14). After 1 hour, no more: glycogen could be found either in the specimens stained with Pb(OH)z or in those negatively stained with PTA. Muscular glycogen is decomposed more quickly by diastase (Fig. 11). After digestion for 30 minutes, no structure which could be identified as glycogen could be found.

F~G. 3. Rat diaphragm fixed in 2.5 % glutaraldehyde, postfixed in OsO4, and stained with Pb(OH)~ according to Karnovsky. Glycogen particles appear as spherical particles 150-300 • in diameter. x 40,000. FIO. 4. Electron micrograph of a diaphragm section, following a 6-hour treatment with pepsin, Glycogen particles are still present, x 60,000.

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G. ROSATI TABLE I

ELECTRON MICROSCOPIC OBSERVATIONS OF UNTREATED AND OF ENZYME-TREATED GLYCOGEN PARTICLES IN TISSUE SECTIONS AND ISOLATED Liver glycogen Treatment

No treatment

Muscle glycogen

Observations

Treatment

Glycogen in No treatment form of "rosettes" Pepsin diasDisappearPepsin, diastase, amyance of the tase, amylase, H202 + glycogen lase, H~O~ + pepsin, H~O~ particles pepsin, H~O~ + diastase from the sec+ diastase, tions pepsin + diastase

Observations

Isolated glycogen Treatment

Observations

Particles 150- No treatment Liver particles in 300 .~ in form of "rosettes" diameter muscle particles Glycogen parroughly spherical ticles remain Diastase Progressive disapclearly visible pearance of particles

DISCUSSION AND CONCLUSIONS

Our observations are summarized in Table I. Liver and muscle glycogen, seen in tissue sections, show evident differences in morphology (Figs. 1-3) and behave differently when treated with certain enzymes. After digestion with diastase for 6-8 hours, liver glycogen particles disappear from the sections, while the muscle glycogen particles remain intact (Figs. 5 and 6). The same is true of digestion with pepsin; again the liver glycogen disappears and the muscle glycogen remains (Figs. 2-4).

FIG. 5. Electron micrograph of a liver section following diastase treatment for 7 hours. It can be seen that a considerable amount of glycogen has been removed, x 30,000. FIG. 6. Electron micrograph of a diaphragm section treated for 7 hours with diastase. In this case, too, glycogen particles are still evident x 70,000. FIG. 7. Diaphragm incubated for 1 hour with 0.1% glucose at 37°C. It can be seen that the average diameter of the glycogen granules has increased, x 50,000. FIo. 8. Electron micrograph of the diaphragm incubated for 1 hour in 0.1% glucose at 37°C and treated with diastase for 7 hours. The average diarneter of the granules of glycogen has returned to normal, x 50,000. FIG. 9. Glycogen particles, isolated from the striated muscle of the rabbit, seen in negative staining with PTA. x 160,000. FIG. 10. Glycogen, isolated from the striated muscle of the rabbit, stained with Pb(OH)~ according to Karnovsky. x 155,000. Fla. 11. Muscle glycogen particles after a 15-minute digestion with diastase. They appear smaller. x 160,000. FIG. 12. Rat liver glycogen isolated and seen in negative staining with PTA. x 160,000. FIo. 13. Rat liver glycogen isolated and stained with Pb(OH)2 according to Karnovsky. x 160,000. FIG. 14. Liver glycogen particles after a 30-minute diastase treatment. Glycogen is no longer recognizable in the form of rosettes but now appears as spherical particles, x 160,000.

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None of the above enzyms heas an effect on striated muscle glycogen, even from sections which have been previously oxidized to remove the reduced osmium. In the case of newly synthesized glycogen, on the other hand, treatment with diastase alone is sufficient to remove it from the muscle tissue sections, as shown by the fact that the particles return to their normal dimensions of 150-250 A. This may be due to the fact that the newly synthesized glycogen is not bound to the protein of the substrate and is more easily removable by the enzyme. F r o m these first results it seems quite clear that there are differences in the reactions of the two types of glycogen examined. Since pepsin sets free the liver glycogen, it appears reasonable to say that in this tissue there is a protein component which is bound to the glycogen. This has already been suggested by other investigators (1, 11, :5, 18). In regard to the muscle glycogen we can suppose that it is bound more tightly to the substrate in such a way as to be inaccessible to diastase. This hypothesis is supported by the fact that it is more difficult to obtain muscle glycogen free from proteins. It is not easy to explain the differences in the behavior of glycogen from tissues sections of liver and of striated muscle following pepsin digestion. These may be due to differences in the chemical nature of the protein bound to the two tissues, which would in turn affect the binding between these proteins and the fixatives and the embedding material. It should be noted that these differences were observed only in the tissue sections. The action of pepsin on isolated glycogen could not be studied because the latter decomposes at the low p H of the 0.1 N HC1 solution. On the other hand, the biochemical data concerning isolated glycogen show that most of proteins bound to it are removed during extraction. This is further confirmed by the fact that diastase alone can digest the glycogen isolated from either liver and muscle. Since our experiments were performed only on isolated glycogen which had not been fixed, we cannot say to what extent the action of diastase can be prevented by fixation. The author is very indebted for suggestions and criticism to Professor D. Bocciarelli, Professor V. Marinozzi, Professor P. Drochmans, and to Professor F. Pocchiari for their collaboration in preparing incubated tissues and isolated glycogen.

REFERENCES 1. ANDRI~,J. and PERSONNE,P., J. Microscopie 4, 122 (1965). 2. BAKER,R. F., J. Histochem. Cytochem. 11, 284 (1963). 3. BIAVA, C., Lab. Invest. 12, 1179 (1963).

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4. DROCHMANS,P., J. Biophys. Biochem. Cytol. 8, 553 (1960). 5. - Proe. 2nd European Regional Conf. Electron Microscopy, Delft, 1960 Vol. 2, p. 645. Almqvist & Wiksell, Uppsala, 1961. 6. - Y. Ultrastruct. Res. 6, 141 (1962). 7. FAWCETT,D. W. and SELBY, C. C., Y. Biophys. Biochem. Cytol. 4, 63 (1958). 8. KARNOVSKY,M. J., J. Biophys. Biochem. Cytol. 11, 729 (1961). 9. KaRRER, H., J. Ultrastruct. Res. 4, 191 (1960). 10. - ibid. 5, 116 (1961). 11. LISON, L., Histochimie et Cytochimie animale, Paris 1960. 12. LUrT, J. H., J. Biophys. Biochem. Cytol. 2, 799 (1956). 13. MARINOZZr,V., J. Ultrastruct. Res. 10, 433 (1964). 14. MILLON~G,G. and PORTER, K. R., Proe. 2nd European Regional Conf. Electron Microscopy, Delft, 1960 Vol. 2, p. 655. Almqvist & Wiksell, Uppsala, 1961. 15. MINIO, F., personal communication (1966). 16. Om~ELL, S. A., BUEDING, E., and REISSIG, M., Ciba Found. Symp. Control Glycogen Metabolism. Little, Brown, Boston, Massachusetts, 1964. 17. REVEL, J. P., NAPOLrrANO, L. and FAWCETT, O. W., J. Biophys. Biochem. Cytol. 8, 575 (1960). 18. - J. Histochem. Cytochem. 12, 104 (1964). 19. RYTER, A. and KELLENBERGER,E., J. UItrastruct. Res. 2, 200 (1958). 20. SABATIM,D. D., BENSCH, K. and BAm~Z,rZTT,R. J., J. Cell. Biol. 17, 19 (1963). 21. SABATINI, D. D., MILLER, F. and BARRNETT, R. J., J. Histochem. Cytochem. 12, 57 (1964). 22. STEINER,J. W., CARRUTIaERS,J. S. and KALIVAT,S. R., Z. Zellforsch. Mikroskop. Anat. 58, 141 (1962). 23. THEMAN, I-I., J. Ultrastruct. Res. 4, 401 (1960). 24. THEMAN, H., Proc. 2nd European Regional Conf. Electron Microscopy, Delft, 1960 Vol. 2, p. 650. Almqvist & Wikse11, Uppsala, 1961.