BRUNO BARRIRGLI, MARIA STELLA MORUZZI, BRUNA TADOLINI AND MARIA GIUSEPPINA MONTI Istituto di Chimica Biologica dell'Università di Bologna, Via Irnerio, 48-40126 Bologna, Itali/ ABSTRACT Ornithine decarboxylase activity was studied during the early phases of liver regeneration after partial hepatectomy in rats adapted to controlled feeding schedules (food available 8 hours per day). Liver ornithine decarboxylase activity ex hibited marked daily oscillations during liver regeneration. The time of day of surgery affected the pattern of enzyme activity after partial hepatectomy. A first postoperative peak of activity was constantly found 6 hours after surgery. Another peak of activity was observed near the end of the dark period independent of the timing of the operation. Food intake was found to induce a further peak of activity during the feeding period. Due to the interaction between controlled feeding schedules and partial hepatectomy, the timing of the operation was found to be essential to obtain a better resolution of the different peaks of activity in our experimental conditions. J. Nutr. 105: 408— 412, 1975. INDEXING KEY WORDS regenerating liver •partial hepatectomy •ornithine decarboxylase •controlled feeding schedules •diurnal variations
One of the earliest biochemical changes in the regenerating rat liver after partial hepatectomy is the intensive stimulation of ornithine decarboxylase (EC 4.1.1.17) ( 1-4 ). This enzyme activity is almost max imally stimulated at 4 hours after partial hepatectomy and remains elevated for several days (2, 5). More recently in a careful time study, Hölttä and Jänne (6) have shown that the increase in ornithine decarboxylase activity after 70% hepatec tomy is biphasic, one peak being at 4 hours, the second at about 11 hours after the operation. Recently it has also been shown that the replacement of liver mass after partial hepatectomy interacts with the biological clock and results in a stepwise phenome non (7, 8) in rats maintained under the controlled feeding schedules of Potter et al. (9). On the other hand, ornithine decarboxyl ase activity is known to undergo diurnal rhythm ( 10 ) in the liver of rats maintained under these conditions, showing a peak of 408
activity stimulated by food and light sig nals (11). Therefore, both regeneration and environmental stimuli are able to en hance this enzyme activity. In the present paper, we report experi ments performed in an attempt to dis tinguish between the increases of ornithine decarboxylase activity due to regeneration and those due to food intake. The results obtained show separate increases of the enzyme activity and further strengthen the need for a precise knowledge and con trol of the feeding schedules on which the animals are maintained to obtain more precise information from the experiments. METHODS Eight-week-old male albino rats of the Wistar strain weighing 230-260 g, obtained from the departmental animal house, were used throughout all experiments. The rats were housed from weaning in an air-con ditioned windowless room with an inverted Received for publication July 15, 1974.
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Ornithine Decarboxylase Activity in Regenerating Liver from Rats Adapted to Controlled Feeding Schedules
ORNITHINE
DECARBOXYLASE
LIVER
409
acidified medium. The center well and its contents were put directly into a scintilla tion vial containing 10 ml of toluene-base scintillation fluid and counted in a liquid scintillation spectrometer4 with a counting efficiency of 76%. Protein was assayed by the method of Lowry et al. ( 14). RESULTS
Figure 1 shows the pattern of ornithine decarboxylase activity after partial hepatectomy performed at different times of the day. The dashed lines represent the daily pattern of ornithine decarboxylase activity in intact rats maintained under controlled feeding schedules (see Ref. 10). When the operation was performed at 0900 (fig. 1A), ornithine decarboxylase activity showed a very quick rise 3 hours after the operation. This first sharp rise of the enzyme activity reached a maximum at 6 hours after the operation and was followed by a rapid de crease during the next 3 hours. On the contrary, between 9 and 12 hours after the operation, ornithine decarboxylase activity showed a plateau around 40 pmoles of "COo/mg protein/45 minutes and declined progressively thereafter during the next 12 hours. This peak of enzyme activity after partial hepatectomy was almost coincident with, and possibly partly reflected, environ mentally entrained ornithine decarboxylase activity (see dashed line, fig. 1A). This first large increase of enzyme activity was also partly due to the operation itself, in fact a smaller increase was found in shamoperated rats 3 hours after the operation. The pattern of ornithine decarboxylase activity in sham-operated rats is reported only for clarity in figure 1C (dotted line). In an attempt to distinguish between food intake effects and partial hepatectomy effects on this enzyme activity, we per formed partial hepatectomies at different times of the day. Figure IB shows the pattern of ornithine decarboxylase activity when the operation was performed at 2400. Also in this case, we found a sharp increase peaking 6 hours after the operation. This first postoperative 'Purina chow (20% protein), Ditta Piccioni, Brescia. Italy. 2 XEX ClM'inlealsOmbll. Frankfurt, Main, Germany. 3 Kontos Olass Co.. Vini-Inni!. N..T. ' Nuclear-Chicago, model Isocap/300.
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and displaced lighting schedule in which lights were on from 2100 to 0900 in a 24hour cycle. A stock diet1 was supplied just before the lights were switched off and was removed 8 hours schedule later according to the "8 + 16" feeding developed by Potter et al. (9). Water was supplied ad libitum. Partial hepatectomies were performed under ether anesthesia, with removal of the main lobes (68-70% of liver was excised) as described by Higgins and Anderson (12). The operations were performed at 0900 ± 30 minutes, at 2400 ±30 minutes, or at 2100 ±30 minutes as indicated in the figures. At intervals ranging between 3 and 48 hours after partial hepatectomies, and at different times during the 24-hour day, the rats were killed by decapitation, and the livers were quickly removed, promptly frozen, and stored at —80° for a maximum of 2 days. No difference in ornithine de carboxylase activity was found in livers stored for over 1 week. Livers were ho mogenized in 25 HIM Tris-HCl buffer, pH 7.2, containing 0.1 mM EDTA and 5 mM dithiothreitol with a Potter-Elvehjem homogenizer fitted with a Teflon pestle. The homogenates (15% w/v) were centrifuged for 90 minutes at 105,000 X g in an ultracentrifuge. The resulting supernatant frac tion was used for assay of ornithine decarboxylase activity. Ornithine decarboxylase activity was measured as described by Janne and Williams-Ashman (13). The standard in cubation mixture contained in a final vol ume of 1.0 ml: 100 /¿molesof glycylglycine buffer, pH 7.2; 0.2 /¿moleof pyridoxal 5-phosphate; 5 /¿molesof dithiothreitol; 0.5 /¿moleof L-ornithine; 0.6 /¿Ciof DL-[114C]ornithine (12 mCi/mmole),2 and 0.4 ml of liver extract. The incubations were car ried out in vials fitted with a disposable polypropylene center well attached to a rubber stopper 3 for 45 minutes in a shak ing water bath at 37°.The released 1JCO2 was trapped into 0.3 ml of l M Hyamine hydroxide in the center well. The reaction was stopped by injecting 1 ml of 40% (w/v) trichloroacetic acid into the reaction mixture, and the vials were left for an additional 60 minutes at room temperature to ensure the release of all 14COofrom the
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Fig. 1 Liver ornithine decarboxylase activity after partial hepatectomy as a function of time of day. The operations were carried out (A) at 0900, (B) at 2400, and (C) at 2100. Rats were killed at the time reported in the figure. Solid lines represent the average ornithine decarboxylase activity in the regenerating liver, dashed lines in the normal liver (see Ref. 10), and dotted line (C) sham-operated animals. Each open symbol represent a single rat. The pattern of shamoperated rats (C) represents the average pattern from rats operated at three different times of the day and obtained from three rats at every time point studied. The abscissa showing time after partial hepatectomy (lower scale) comes at a different clock time (upper scale) in each ex periment.
peak at 6 hours after the operation was followed at a lower level (around 28 pmoles "CO^/mg protein/45 minutes) by a plateau between 8 and 10 hours. After a progressive decrease between 10 and 14 hours after the operation, the enzyme activity showed a second peak at 15 to 17 hours and decreased thereafter to low levels of activity between 18 and 30
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Fig. 2 Liver ornithine decarboxylase activity after partial hepatectomy as a function of time of day in rats starved on the day of experiment. (A) Operation was carried out at 2400 or (B) at 2100, and the rats were starved on the next day; (C) operation was performed at 2100, and rats were starved on the second postoperative day. Rats were killed at the times reported in the figure. Solid lines represent the average ornithine de carboxylase activity and dashed line in (C) repre sents the pattern of enzyme activity in the con ditions of figure 1C. Each open symbol represents a single rat. The abscissa showing time after partial hepatectomy (lower scale) comes at a dif ferent clock time ( upper scale ) in each experiment.
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hours. This second peak of activity, found between the removal of food ( 1700 ) and the beginning of the light period (2100) seemed to correspond to the plateau found at the same time of day when rats were operated on at 0900 instead of at 2400. To confirm the pattern reported in figure IB, one lot of rats was operated on 3 hours earlier (at 2100); the results are reported in figure 1C. Again as in the two previous experiments, we found a sharp rise of en-
ORNITHINE DECARBOXYLASE IN REGENERATING LIVER
enzyme activity showed a second peak be tween 18 and 21 hours after partial hepatectomy corresponding to 1500 and 1800 in our conditions. Also in this case we were unable to find any increased activity at the time of day when both intact and par tially hepatectomized rats ( fig. 1C ) showed a peak of activity (i.e., during the daily feeding period). To further confirm this behavior, we followed the ornithine decarboxylase activ ity during the second postoperative day. In this experiment rats operated on at 2100 were fed during day 1 (from 0900 to 1700) and were denied food on day 2 (fig. 2C). The results again point out the dis appearance of the food-entrained peak found when the rats were fed (fig. 1C), although the peak due to the operation seemed to be somewhat anticipated (be tween 1200 and 1500). DISCUSSION The present results show two phenomena in the regulation of ornithine decarboxylase activity after partial hepatectomy. The first is the constant appearance of the first post operative peak of enzyme activity 6 hours after the operation independent of the tim ing of the operation in the controlled feed ing schedule. This result is in agreement with previous work (3, 6) using uncon trolled feeding schedules. The first increase of ornithine decarboxylase activity was due mainly to the process of regeneration, which exceeded and overlapped the small increase due to the operation itself (fig. 1C, clotted line). Recent results ( 15) have provided some evidence for a connection between the rises in ornithine decarboxylase activity and DNA synthesis. Theseas increases enzyme activity indicated "prereplicain tive" (15) anticipate DNA synthesis by several hours. In our experimental condi tions, there was a lag of 15 hours between the first increase of ornithine decarboxylase activity and the first peak of DNA syn thesis (7, 8). The other peaks reveal the presence of constant diurnal variations of ornithine de carboxylase activity in response to the stimuli of the controlled feeding schedules to which the rats were subjected before the operation. These peaks are insensitive
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zyme activity peaking at 6 hours after the operation, the increase being of the same extent as the two previous ones. This first postoperative peak and subse quent low level of activity (about 10 pmoles llCO2/mg protein/45 minutes), maintained from 9 to 12 hours after the operation, were followed by a second peak at the time of day (between 1200 and 1500) when intact rats showed the environmen tally entrained diurnal peak of activity. This was followed by a third peak be tween the time of removal of food cups and the onset of light. During the light period the level of ornithine decarboxylase activity was maintained at low levels and increased again during day 2 after the operation. Between 36 and 48 hours after the operation, ornithine decarboxylase ac tivity showed a pattern very similar to that found during the first postoperative day; i.e., a peak of activity corresponding to the one induced by food intake and a second one after the removal of food ( 1700 ) and before the onset of light (2100). In a second series of experiments, orni thine decarboxylase activity was measured during the early phases of regeneration in rats denied food on the first or on the second postoperative day. The results are reported in figure 2. When the operation was performed at 2400 and the food was denied on the first postoperative day (fig. 2A), an early peak of activity was found 6 hours after the operation. This first postoperative peak de creased rapidly to a low level of activity and no shoulder was found between 8 and 12 hours after the operation, as was found in rats operated at the same time of day and fed since 0900 (fig. IB). After a low level maintained between 8 and 14 hours after the operation, ornithine decarboxylase showed a second peak of activity between 16 and 17 hours after the operation corre sponding to 1500 and 1600, respectively (actual time in our experimental condi tions ). By operating on the rats at 2100 and denying the food on the first postoperative day, a very similar pattern was found (fig. 2B). Again after the first postoperative peak at 6 hours after the operation, there was a low level of activity from 9 to 15 hours after the operation. Thereafter, the
411
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BARBIROLI, MORUZZI, TADOLINI AND MONTI 2. Russell, D. & Snyder, S. H. (1968) Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity in regenerating rat liver, chick embryo, and various tumors. Proc. Nat. Acad. Sci. USA 60, 1420-1427. 3. Fausto, N. (1969) Studies on ornithine de carboxylase activity in normal and regenerat ing livers. Biochim. Biophys. Acta J90, 193201. 4. Schrock, T. R., Oakrnan, N. J. & Bucher, N. L. R. (1970) Ornithine decarboxylase activ ity in relation to growth of rat liver. Effects of partial hepatectomy, hypertonic infusions, celite injection or other stressful procedures. Biochim. Biophys. Acta 204, 564-577. 5. Raina, A., Jänne,J., Hannone, P. & Hölttä, E. (1970) Synthesis and accumulation of polyamines in regenerating rat liver. Ann. N.Y. Acad. Sci. 171, 697-708. 6. Hölttä,E. & Jänne, J. (1972) Ornithine decarboxylase activity and the accumulation of putrescine at early stages of liver regenera tion. FEBS Lett. 23, 117-121. 7. Barbiroli, B. & Potter, V. R. (1971) DNA synthesis and interaction between controlled feeding schedules and partial hepatectomy in rats. Science 172, 738-741. 8. Hopkins, H. A., Campbell, H. A., Barbiroli, B. & Potter, V. R. ( 1973 ) Thymine kinase and deoxyribonucleic acid metabolism in growing and regenerating livers from rats on controlled feeding schedules. Biochem. J. 136, 955-966. 9. Potter, V. R., Baril, E. F., Watanabe, M. & Whittle, E. D. (1968) Systematic oscilla tions in metabolic functions in liver from rats adapted to controlled feeding schedules. Fed eration Proc. 27, 1238-1245. 10. Tadolini, B., Monti, M. giornalieri G. & Moruzzij M. S. (1973) Livelli epatici dell'attivitÃ
11.
12.
13.
ACKNOWLEDGMENTS
We are grateful to Professor G. Moruzzi for his interest in this research and for pro viding all the facilities and the financial support, and to Mr. G. Negroni for his excellent technical assistance.
14.
15.
LITERATURE CITED 1. Jänne, J. & Raina, A. (1968) Stimulation of spermidine synthesis in the regenerating rat liver: relation increased ornithine de carboxylase activity. Acta Chem. Scand. 22, 1349-1351.
16.
ornitina-decarbossilasica in ratti mantenuti ad assunzione controllata di cibo. Boll. Soc. Ital. Biol. Sper. 49, 1379-1382. Hopkins, H. A., Bonney, J. R., Walker, P. R., Yager, J. D., Jr. & Potter, V. R. (1973) Food and light as separate entrainment signals for rat liver enzymes. Advan. Enzyme Regul., vol. 11. Higgins, G. M. & Anderson, R. M. (1931) Experimental pathology of liver. I. Restoration of liver of white rat following partial surgical removal. Arch. Pathol. 12, 186-190. Jänne,J. & Williams-Ashman, H. G. (1970) Mammalian ornithine decarboxylase: activa tion and alteration of physical behaviour by thiol compounds. Biochem. J. 119, 595—597. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) Protein measure ment with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Gaza, D. J., Short, J. & Lieberman, I. (1973) On the possibility that the prereplicative in crease in ornithine decarboxylase activity are related to DNA synthesis in liver. FEBS Lett. 32, 251-253. Gelfant, S. & Smith, J., Jr. (1972) Aging: noncycling cells an explanation. Science 178, 357-361.
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to the length of time after the operation, but nevertheless at least one of them rep resents a response to the operation. This is represented by a peak of activity con stantly present in the 4-hour period be tween the removal of food and the begin ning of the light and in the present feeding schedules around 1900. This peak of activ ity seemed to be in connection with the peak of DNA synthesis found at the same time the following day (7, 8). In this case the lag between the rise of ornithine decarboxylase activity and DNA synthesis appeared to be somewhat increased to about 20-24 hours after the first postopera tive day. When the operation was per formed at 0900, the first peak of enzyme activity occurred at a time of day when there was a peak of activity due to diurnal variations (fig. LA), but it was possible to separate the single peaks due to différent stimuli (i.e., food intake and operation) by operating at earlier times of the day, (i.e., 2400 or 2100, figs. IB and C). Our present findings give further sup port to an earlier model (7, 8) that postu lated the presence of two groups or lots of cells in the rat liver. "The duplication of the first lot of cells has a high priority" and responds to the operation with a peak of ornithine decarboxylase activity 6 hours after the operation independent of the timing of the operation. On the other hand, when the operation is performed at certain operating times (i.e., 2400 or 2100), a second lot of cells responds rapidly to the operation and results in a second peak of ornithine decarboxylase activity between 1700 and 2100. Finally, cells that possibly are in a non replicative cycle (Gì-or Go-blocked [16]) show the diurnal environmentally entrained peak of activity during the feeding period.
One of the earliest biochemical changes in the regenerating rat liver after partial hepatectomy is the intensive stimulation of ornithine decarboxylase (EC 4.1.1.17) ( 1-4 ). This enzyme activity is almost max imally stimulated at 4 hours after partial hepatectomy and remains elevated for several days (2, 5). More recently in a careful time study, Hölttä and Jänne (6) have shown that the increase in ornithine decarboxylase activity after 70% hepatec tomy is biphasic, one peak being at 4 hours, the second at about 11 hours after the operation. Recently it has also been shown that the replacement of liver mass after partial hepatectomy interacts with the biological clock and results in a stepwise phenome non (7, 8) in rats maintained under the controlled feeding schedules of Potter et al. (9). On the other hand, ornithine decarboxyl ase activity is known to undergo diurnal rhythm ( 10 ) in the liver of rats maintained under these conditions, showing a peak of 408
activity stimulated by food and light sig nals (11). Therefore, both regeneration and environmental stimuli are able to en hance this enzyme activity. In the present paper, we report experi ments performed in an attempt to dis tinguish between the increases of ornithine decarboxylase activity due to regeneration and those due to food intake. The results obtained show separate increases of the enzyme activity and further strengthen the need for a precise knowledge and con trol of the feeding schedules on which the animals are maintained to obtain more precise information from the experiments. METHODS Eight-week-old male albino rats of the Wistar strain weighing 230-260 g, obtained from the departmental animal house, were used throughout all experiments. The rats were housed from weaning in an air-con ditioned windowless room with an inverted Received for publication July 15, 1974.
Downloaded from https://academic.oup.com/jn/article-abstract/105/4/408/4763453 by University of Glasgow user on 15 February 2019
Ornithine Decarboxylase Activity in Regenerating Liver from Rats Adapted to Controlled Feeding Schedules
ORNITHINE
DECARBOXYLASE
LIVER
409
acidified medium. The center well and its contents were put directly into a scintilla tion vial containing 10 ml of toluene-base scintillation fluid and counted in a liquid scintillation spectrometer4 with a counting efficiency of 76%. Protein was assayed by the method of Lowry et al. ( 14). RESULTS
Figure 1 shows the pattern of ornithine decarboxylase activity after partial hepatectomy performed at different times of the day. The dashed lines represent the daily pattern of ornithine decarboxylase activity in intact rats maintained under controlled feeding schedules (see Ref. 10). When the operation was performed at 0900 (fig. 1A), ornithine decarboxylase activity showed a very quick rise 3 hours after the operation. This first sharp rise of the enzyme activity reached a maximum at 6 hours after the operation and was followed by a rapid de crease during the next 3 hours. On the contrary, between 9 and 12 hours after the operation, ornithine decarboxylase activity showed a plateau around 40 pmoles of "COo/mg protein/45 minutes and declined progressively thereafter during the next 12 hours. This peak of enzyme activity after partial hepatectomy was almost coincident with, and possibly partly reflected, environ mentally entrained ornithine decarboxylase activity (see dashed line, fig. 1A). This first large increase of enzyme activity was also partly due to the operation itself, in fact a smaller increase was found in shamoperated rats 3 hours after the operation. The pattern of ornithine decarboxylase activity in sham-operated rats is reported only for clarity in figure 1C (dotted line). In an attempt to distinguish between food intake effects and partial hepatectomy effects on this enzyme activity, we per formed partial hepatectomies at different times of the day. Figure IB shows the pattern of ornithine decarboxylase activity when the operation was performed at 2400. Also in this case, we found a sharp increase peaking 6 hours after the operation. This first postoperative 'Purina chow (20% protein), Ditta Piccioni, Brescia. Italy. 2 XEX ClM'inlealsOmbll. Frankfurt, Main, Germany. 3 Kontos Olass Co.. Vini-Inni!. N..T. ' Nuclear-Chicago, model Isocap/300.
Downloaded from https://academic.oup.com/jn/article-abstract/105/4/408/4763453 by University of Glasgow user on 15 February 2019
and displaced lighting schedule in which lights were on from 2100 to 0900 in a 24hour cycle. A stock diet1 was supplied just before the lights were switched off and was removed 8 hours schedule later according to the "8 + 16" feeding developed by Potter et al. (9). Water was supplied ad libitum. Partial hepatectomies were performed under ether anesthesia, with removal of the main lobes (68-70% of liver was excised) as described by Higgins and Anderson (12). The operations were performed at 0900 ± 30 minutes, at 2400 ±30 minutes, or at 2100 ±30 minutes as indicated in the figures. At intervals ranging between 3 and 48 hours after partial hepatectomies, and at different times during the 24-hour day, the rats were killed by decapitation, and the livers were quickly removed, promptly frozen, and stored at —80° for a maximum of 2 days. No difference in ornithine de carboxylase activity was found in livers stored for over 1 week. Livers were ho mogenized in 25 HIM Tris-HCl buffer, pH 7.2, containing 0.1 mM EDTA and 5 mM dithiothreitol with a Potter-Elvehjem homogenizer fitted with a Teflon pestle. The homogenates (15% w/v) were centrifuged for 90 minutes at 105,000 X g in an ultracentrifuge. The resulting supernatant frac tion was used for assay of ornithine decarboxylase activity. Ornithine decarboxylase activity was measured as described by Janne and Williams-Ashman (13). The standard in cubation mixture contained in a final vol ume of 1.0 ml: 100 /¿molesof glycylglycine buffer, pH 7.2; 0.2 /¿moleof pyridoxal 5-phosphate; 5 /¿molesof dithiothreitol; 0.5 /¿moleof L-ornithine; 0.6 /¿Ciof DL-[114C]ornithine (12 mCi/mmole),2 and 0.4 ml of liver extract. The incubations were car ried out in vials fitted with a disposable polypropylene center well attached to a rubber stopper 3 for 45 minutes in a shak ing water bath at 37°.The released 1JCO2 was trapped into 0.3 ml of l M Hyamine hydroxide in the center well. The reaction was stopped by injecting 1 ml of 40% (w/v) trichloroacetic acid into the reaction mixture, and the vials were left for an additional 60 minutes at room temperature to ensure the release of all 14COofrom the
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Fig. 1 Liver ornithine decarboxylase activity after partial hepatectomy as a function of time of day. The operations were carried out (A) at 0900, (B) at 2400, and (C) at 2100. Rats were killed at the time reported in the figure. Solid lines represent the average ornithine decarboxylase activity in the regenerating liver, dashed lines in the normal liver (see Ref. 10), and dotted line (C) sham-operated animals. Each open symbol represent a single rat. The pattern of shamoperated rats (C) represents the average pattern from rats operated at three different times of the day and obtained from three rats at every time point studied. The abscissa showing time after partial hepatectomy (lower scale) comes at a different clock time (upper scale) in each ex periment.
peak at 6 hours after the operation was followed at a lower level (around 28 pmoles "CO^/mg protein/45 minutes) by a plateau between 8 and 10 hours. After a progressive decrease between 10 and 14 hours after the operation, the enzyme activity showed a second peak at 15 to 17 hours and decreased thereafter to low levels of activity between 18 and 30
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Fig. 2 Liver ornithine decarboxylase activity after partial hepatectomy as a function of time of day in rats starved on the day of experiment. (A) Operation was carried out at 2400 or (B) at 2100, and the rats were starved on the next day; (C) operation was performed at 2100, and rats were starved on the second postoperative day. Rats were killed at the times reported in the figure. Solid lines represent the average ornithine de carboxylase activity and dashed line in (C) repre sents the pattern of enzyme activity in the con ditions of figure 1C. Each open symbol represents a single rat. The abscissa showing time after partial hepatectomy (lower scale) comes at a dif ferent clock time ( upper scale ) in each experiment.
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hours. This second peak of activity, found between the removal of food ( 1700 ) and the beginning of the light period (2100) seemed to correspond to the plateau found at the same time of day when rats were operated on at 0900 instead of at 2400. To confirm the pattern reported in figure IB, one lot of rats was operated on 3 hours earlier (at 2100); the results are reported in figure 1C. Again as in the two previous experiments, we found a sharp rise of en-
ORNITHINE DECARBOXYLASE IN REGENERATING LIVER
enzyme activity showed a second peak be tween 18 and 21 hours after partial hepatectomy corresponding to 1500 and 1800 in our conditions. Also in this case we were unable to find any increased activity at the time of day when both intact and par tially hepatectomized rats ( fig. 1C ) showed a peak of activity (i.e., during the daily feeding period). To further confirm this behavior, we followed the ornithine decarboxylase activ ity during the second postoperative day. In this experiment rats operated on at 2100 were fed during day 1 (from 0900 to 1700) and were denied food on day 2 (fig. 2C). The results again point out the dis appearance of the food-entrained peak found when the rats were fed (fig. 1C), although the peak due to the operation seemed to be somewhat anticipated (be tween 1200 and 1500). DISCUSSION The present results show two phenomena in the regulation of ornithine decarboxylase activity after partial hepatectomy. The first is the constant appearance of the first post operative peak of enzyme activity 6 hours after the operation independent of the tim ing of the operation in the controlled feed ing schedule. This result is in agreement with previous work (3, 6) using uncon trolled feeding schedules. The first increase of ornithine decarboxylase activity was due mainly to the process of regeneration, which exceeded and overlapped the small increase due to the operation itself (fig. 1C, clotted line). Recent results ( 15) have provided some evidence for a connection between the rises in ornithine decarboxylase activity and DNA synthesis. Theseas increases enzyme activity indicated "prereplicain tive" (15) anticipate DNA synthesis by several hours. In our experimental condi tions, there was a lag of 15 hours between the first increase of ornithine decarboxylase activity and the first peak of DNA syn thesis (7, 8). The other peaks reveal the presence of constant diurnal variations of ornithine de carboxylase activity in response to the stimuli of the controlled feeding schedules to which the rats were subjected before the operation. These peaks are insensitive
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zyme activity peaking at 6 hours after the operation, the increase being of the same extent as the two previous ones. This first postoperative peak and subse quent low level of activity (about 10 pmoles llCO2/mg protein/45 minutes), maintained from 9 to 12 hours after the operation, were followed by a second peak at the time of day (between 1200 and 1500) when intact rats showed the environmen tally entrained diurnal peak of activity. This was followed by a third peak be tween the time of removal of food cups and the onset of light. During the light period the level of ornithine decarboxylase activity was maintained at low levels and increased again during day 2 after the operation. Between 36 and 48 hours after the operation, ornithine decarboxylase ac tivity showed a pattern very similar to that found during the first postoperative day; i.e., a peak of activity corresponding to the one induced by food intake and a second one after the removal of food ( 1700 ) and before the onset of light (2100). In a second series of experiments, orni thine decarboxylase activity was measured during the early phases of regeneration in rats denied food on the first or on the second postoperative day. The results are reported in figure 2. When the operation was performed at 2400 and the food was denied on the first postoperative day (fig. 2A), an early peak of activity was found 6 hours after the operation. This first postoperative peak de creased rapidly to a low level of activity and no shoulder was found between 8 and 12 hours after the operation, as was found in rats operated at the same time of day and fed since 0900 (fig. IB). After a low level maintained between 8 and 14 hours after the operation, ornithine decarboxylase showed a second peak of activity between 16 and 17 hours after the operation corre sponding to 1500 and 1600, respectively (actual time in our experimental condi tions ). By operating on the rats at 2100 and denying the food on the first postoperative day, a very similar pattern was found (fig. 2B). Again after the first postoperative peak at 6 hours after the operation, there was a low level of activity from 9 to 15 hours after the operation. Thereafter, the
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BARBIROLI, MORUZZI, TADOLINI AND MONTI 2. Russell, D. & Snyder, S. H. (1968) Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity in regenerating rat liver, chick embryo, and various tumors. Proc. Nat. Acad. Sci. USA 60, 1420-1427. 3. Fausto, N. (1969) Studies on ornithine de carboxylase activity in normal and regenerat ing livers. Biochim. Biophys. Acta J90, 193201. 4. Schrock, T. R., Oakrnan, N. J. & Bucher, N. L. R. (1970) Ornithine decarboxylase activ ity in relation to growth of rat liver. Effects of partial hepatectomy, hypertonic infusions, celite injection or other stressful procedures. Biochim. Biophys. Acta 204, 564-577. 5. Raina, A., Jänne,J., Hannone, P. & Hölttä, E. (1970) Synthesis and accumulation of polyamines in regenerating rat liver. Ann. N.Y. Acad. Sci. 171, 697-708. 6. Hölttä,E. & Jänne, J. (1972) Ornithine decarboxylase activity and the accumulation of putrescine at early stages of liver regenera tion. FEBS Lett. 23, 117-121. 7. Barbiroli, B. & Potter, V. R. (1971) DNA synthesis and interaction between controlled feeding schedules and partial hepatectomy in rats. Science 172, 738-741. 8. Hopkins, H. A., Campbell, H. A., Barbiroli, B. & Potter, V. R. ( 1973 ) Thymine kinase and deoxyribonucleic acid metabolism in growing and regenerating livers from rats on controlled feeding schedules. Biochem. J. 136, 955-966. 9. Potter, V. R., Baril, E. F., Watanabe, M. & Whittle, E. D. (1968) Systematic oscilla tions in metabolic functions in liver from rats adapted to controlled feeding schedules. Fed eration Proc. 27, 1238-1245. 10. Tadolini, B., Monti, M. giornalieri G. & Moruzzij M. S. (1973) Livelli epatici dell'attivitÃ
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ACKNOWLEDGMENTS
We are grateful to Professor G. Moruzzi for his interest in this research and for pro viding all the facilities and the financial support, and to Mr. G. Negroni for his excellent technical assistance.
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LITERATURE CITED 1. Jänne, J. & Raina, A. (1968) Stimulation of spermidine synthesis in the regenerating rat liver: relation increased ornithine de carboxylase activity. Acta Chem. Scand. 22, 1349-1351.
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ornitina-decarbossilasica in ratti mantenuti ad assunzione controllata di cibo. Boll. Soc. Ital. Biol. Sper. 49, 1379-1382. Hopkins, H. A., Bonney, J. R., Walker, P. R., Yager, J. D., Jr. & Potter, V. R. (1973) Food and light as separate entrainment signals for rat liver enzymes. Advan. Enzyme Regul., vol. 11. Higgins, G. M. & Anderson, R. M. (1931) Experimental pathology of liver. I. Restoration of liver of white rat following partial surgical removal. Arch. Pathol. 12, 186-190. Jänne,J. & Williams-Ashman, H. G. (1970) Mammalian ornithine decarboxylase: activa tion and alteration of physical behaviour by thiol compounds. Biochem. J. 119, 595—597. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) Protein measure ment with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Gaza, D. J., Short, J. & Lieberman, I. (1973) On the possibility that the prereplicative in crease in ornithine decarboxylase activity are related to DNA synthesis in liver. FEBS Lett. 32, 251-253. Gelfant, S. & Smith, J., Jr. (1972) Aging: noncycling cells an explanation. Science 178, 357-361.
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to the length of time after the operation, but nevertheless at least one of them rep resents a response to the operation. This is represented by a peak of activity con stantly present in the 4-hour period be tween the removal of food and the begin ning of the light and in the present feeding schedules around 1900. This peak of activ ity seemed to be in connection with the peak of DNA synthesis found at the same time the following day (7, 8). In this case the lag between the rise of ornithine decarboxylase activity and DNA synthesis appeared to be somewhat increased to about 20-24 hours after the first postopera tive day. When the operation was per formed at 0900, the first peak of enzyme activity occurred at a time of day when there was a peak of activity due to diurnal variations (fig. LA), but it was possible to separate the single peaks due to différent stimuli (i.e., food intake and operation) by operating at earlier times of the day, (i.e., 2400 or 2100, figs. IB and C). Our present findings give further sup port to an earlier model (7, 8) that postu lated the presence of two groups or lots of cells in the rat liver. "The duplication of the first lot of cells has a high priority" and responds to the operation with a peak of ornithine decarboxylase activity 6 hours after the operation independent of the timing of the operation. On the other hand, when the operation is performed at certain operating times (i.e., 2400 or 2100), a second lot of cells responds rapidly to the operation and results in a second peak of ornithine decarboxylase activity between 1700 and 2100. Finally, cells that possibly are in a non replicative cycle (Gì-or Go-blocked [16]) show the diurnal environmentally entrained peak of activity during the feeding period.
