Folyamines and pancreatic growth induced by caerulein

Life Sciences, Vol. 35, pp. Printed in the U.S.A.

FOLYAMINES

2471-2480

AND PANCREATIC Jean Morisset

Pergamon

GROWTH

and Ouhida

Centre de recherche sur les D6partement de biologie, Universit6 de Sherbrooke, Qu6bec, (Received

in final

INDUCED

Press

BY CAERULEIN

Benrezzak

m6canismes de s6cr6tion Facult6 des sciences Sherbrooke Canada, JIK 2RI

form October

2, 1984)

Summary Activation of polyamine metabolism may be important to initiation of pancreatic cell growth. We are reporting that such activation did occur during pancreatic growth initiation by caerulein, a cholecystokinin analog. Maximal increases in total putrescine (]19%), spermidine (63%) and spermine (50%) were observed 12, 96 and 96 hr respectively after the beginning of the caerulein treatment. This time period coincides with pancreatic hypertrophy and hyperplasia as characterized by increased cell mass and DNA content. Rates of pancreatic weight and DNA content increases were significantly correlated with total spermidine and spermine contents. These data suggest that polyamine biosynthesis is closely associated with pancreatic growth. It is now well recognized that cholecystokinin or its analogue caerulein have important trophic effects on the pancreas (I-4). However, there is very little information available about the mechanisms by which this gastrointestinal hormone can regulate DNA synthesis and pancreatic growth. It has been shown in a variety of mammalian tissues that growth promoting hormones or factors change markedly the metabolism of polyamines by increasing the enzyme activities involved in their biosynthesis (5). More particularly, the activity of ornithine decarboxylase, the enzyme responsib]e for the synthesis of putrescine, can be increased many fold within a few hours of exposure to trophic stimuli (6). In this study, we have evaluated the effects of chronic administration of caerulein on the changes in putrescine, spermidine and spermine contents and concentrations in rat pancreas to determine if the m e t a b o l i s m of these polyamines might be involved in pancreatic tissue stimulated to grow. Materials

and Methods

Caeru]ein, a gift from Prof. R. de Castiglione (Farmitalia, Milan, Italy), was injected subcutaneously in hydrolyzed gelatin to prolong its absorption (3). ~ - d i f l u o r o m e t h y l o r n i t h i n e (DFMO) a gift from Dr. P. McCann (Merrell Research Center, Cincinnati, Ohio) was given as a 2 percent solution in drinking water. The experiments were performed on male Sprague-Dawley rats weighing between 150 and 155 g at the beginning of the experiment. Three groups of rats were used: a) control, b) caerulein, and c) caerulein + DFMO. A DFMO 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press

Ltd.

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group alone was omitted since we have p r e v i o u s l y shown that the ODC inhibitor had no effect of its own on p a n c r e a t i c growth (7) and did not affect po]yamine levels after 12 days of treatment (8). As suggested by Bartolome e t a ] . (9), the DFMO treatment was begun 2 days prior to caerulein for a complete saturation of existing ornithine d e c a r b o x y l a s e (ODC) activity before initiation of pancreatic growth, and c o n t i n u e d during caerulein treatment. Caerulein was given three times dai]y at a dose of I ~g kg -1 and the contro] group received saline. Water c o n s u m p t i o n was recorded daily for e s t i m a t i o n of DFMO intake. Animals were s a c r i f i c e d 12, 24, 48 and 96 hrs after the first caerulein injection; they remained fed overnight before sacrifice to avoid a reduction in DFMO consumption. After sacrifice, the pancreas was c a r e f u l l y excised, trimmed free of fat, mesentary, and lymph nodes and weighed. A small piece (100-120 mg) was saved for DNA d e t e r m i n a t i o n and the remainder was h o m o g e n i z e d in i0 ml of 0.4 M perchloric acid (PCA), left overnight at 4°C and c e n t r i f u g e d at 15 000 xg for 15 min. The p r e c i p i t a t e was then w a s h e d once with 5 ml of PCA 0.4 M, recentrifuged and the supernatant combined to the first one. Polyamine contents (putrescine, sperm±dine and sperm±he) were analyzed as described by K i n g s n o r t h et al. (10). DNA was e x t r a c t e d a c c o r d i n g to Mainz et al. (I) and d e t e r m i n e d a c c o r d i n g to Volkin and Cohn (11) using calf thymus DNA as the standard. Statistical significance of observed differences among control and treated groups was d e t e r m i n e d as p r e v i o u s l y described (4) using analysis of variance and a She±re test. For the c o r r e l a t i o n studies, linear regression analysis was used. Results In the course of these experiments, there was no significant difference in water c o n s u m p t i o n between rats drinking water or 2% DFMO. Including the two days prior to the first c a e r u l e i n injection, rats in the caerulein + DFMO group had ingested a total of 1.33 ±0.04, 1.89 ±0.07, 2.43 ± 0 . O 9 and 4.29 ± 0.3 g of DFMO when killed at 12, 24, 48 and 96 hr respectively. As shown in Table 1, DFMO c o n s u m p t i o n s i g n i f i c a n t l y reduced the animals' body weights at all the times studied; a significant decrease of 7% was observed after 24 hr, and decreases of 15 and 13% after 48 and 96 hr. Significant increases of 50 and 53% in p a n c r e a t i c weight were observed after 48 and 96 hr of c a e r u l e i n treatment. These increments were s i g n i f i c a n t ly reduced by 26 and 21% with DFMO treatment. The pancreatic cellular mass was also s i g n i f i c a n t l y increased by c a e r u l e i n after 48 and 96 hr by 24 and 25%. DFMO was able to reduce this p a n c r e a t i c h y p e r t r o p h y only at 96 hr with a 14% significant inhibition. Significant increases of 22% in total DNA contents were also observed after 48 and 96 hr of caerulein treatment (data not shown), with DFMO significantly reducing this effect on p a n c r e a t i c hyperplasia. In summary, these data indicate that p a n c r e a t i c h y p e r p ] a s i a and h y p e r t r o p h y are well e s t a b l i s h e d after 2 and 4 days of c a e r u l e i n treatment but can be reduced by inhibition of ornithine d e c a r b o x y l a s e activity. Figure i shows the time course of the changes in total amounts of putrescine, sperm±dine, and sperm±he occurring within 12 and g6 hr of caerulein treatment. One can observe that each polyamine has only one control group riot the 4 different time periods studied. We did have control groups for each period but as an analysis of variance indicated that there was no significant difference between all controls we decided to set only one control group.

Vol. 35, No. 24, 1984

Polyamines and Induced Pancreatic Growth

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1984

Vol. 35, No. 24, 1984

Polyamines and Induced Pancreatic Growth

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Polyamines

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Accumulation of putrescine (Fig. IA) in the pancreatic gland was appreciable, reaching a maximum within the first 12 hr after the first caerulein injection (319% of control) which in turn was reduced 39% by DFMO. At the 24 hr period, the caerulein effect on putrescine content was still significant with a 217% increase over control values, while DFMO reduced these levels by 61%. After 48 and g6 hr, caerulein and no significant effect on putrescine levels. The spermidine content (Fig. IB) was increased by 24% after 48 hr of caerulein treatment but 96 hr were necessary to see a significant increase of 63%. At these time periods, DFMO significantly reduced caerulein induced polyamine increases by 33 and 30% respectively. Surprisingly, significant increases in spermine levels (50%) were observed after 96 hr of caerulein treatment, increases which were not affected by DFMO (Fig. IC). These results suggest that ornithine decarboxylase induction by caerulein in the pancreatic gland is accompanied by increases in putrescine and spermidine levels which can be abrogated by DFMO. Later increases in spermine were also observed but were not affected by DFMO. When the three polyamine data are expressed on a cellular basis (nmol/ I00 ~g DNA), we observed the same changes in concentration as we did with total content. The only difference observed was in the spermine concentration which still showed an increase above control values but even this was not significant after 96 hr of caerulein (Table 2). To evaluate any correlation between pancreatic growth rate and cellular polyamine contents, we statistically correlated the pancreatic weight and DNA content with total contents of putrescine, spermidine and spermine during the course of the caerulein treatment. As shown in Fig. 2, highly significant positive correlations were found between pancreatic weight and total pancreatic contents of spermidine and spermine; the correlation was significantly negative with putrescine. With regard to the correlations between total DNA content and the three polyamine total contents, Fig. 3, they remained significantly positive with spermidine and spermine with r values much lower than those obtained with pancreatic weight. The correlation with putrescine remained negative but not significant. The statistical analyses of these data are presented in Table 3. TABLE 3

Correlation between Increases in Pancreatic Weight Total DNA Content and Polyamine Total Contents

Polyamines

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These correlations were established in the caerulein treated group and included all values obtained after 12, 24, 48 and 96 hr of caerulein treatment.

Vol.

35, No. 24, 1984

Polyamines

and Induced Pancreatic Growth

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24, 1984

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Discussion There are some reports on a variety of biochemical alterations occurring during the prereplicative phase in the rat pancreas stimulated with caerulein. Metabolic changes were observed in protein and RNA contents as well as in their synthesis rates (II). These events are intimately related to subsequent stimulation of DNA synthesis whose rate peaked 48 hr after the beginning of caerulein treatment (4). The present study demonstrated for the first time in the pancreas that polyamine metabolism is increased when the tissue is stimulated to grow with caerulein. We were able to show that cellular putrescine, spermidine, and spermine increased progressively as the pancreatic cells traversed the cell cycle from G 1 to mitosis. The increase was seen first for putrescine, coincident with observed increases in protein and RNA contents (11), as well as with initiation of the S phase as evidenced by increased rates of DNA synthesis (4). The delay observed for increases in spermidine and spermine reflects the order of their biosynthesis. These changes were however seen only after the DNA synthesis rates had reached their maximum. Our data differ in some ways with those of Inoue et al. (13) who showed significant increases in spermidine concentration, 12 and 16 hr after isoproterenol treatment in parotid and submaxillary glands, two exocrine glands like the pancreas. However, they were unable to detect any increase in spermine 28 hr after the drug administration. The time courses of putrescine and spermidine accumulation after partial hepatectomy in regenerating rat liver was quite comparable to that seen in the pancreas. There was a burst in putrescine observed at 28 hr with significant decline by 52 hr, while the observed increase in spermidine at 28 hr was still maintained at 52 hr; spermine concentrations remained at the control levels during this period (14). Our data also do not agree with that of Danzin et al. (15) who found that caerulein at high doses (300 ng to 5~g/kg) had no effect on ODC and on the pancreatic polyamine concentrations. In their study however, secretin, a much less potent trophic factor for the pancreas (3-4), was associated with increased putrescine concentration within 4 hr of its administration with only a slight increase in ODC activity. They believe that the accumulated putrescine was formed from spermidine, via acetylation, rather than by decarboxylation of ornithine. These differences between the two studies cannot be ascribed to analytical differences in polyamine determinations since their putrescine levels, 25 nmol/g of tissue, are quite similar to ours 40 nmol/g of tissue. However, their rats were fasted for 24 hr before and during treatment, and the hormones were given i.p. Our animals were fed and caerulein was injected s.c. in gelatin to prolong its absorption, thus a11owing a longer period of stimulation, up to 6 hr (3), an i.p. stimulation lasting no more than 60 min (16). The pattern of polyamine variations observed in stimulated pancreatic growth (this study), and in intestinal growth after jejunectomy (17) presents some differences, although the concentrations of the three polyamines were increased in both tissues. In the intestinal mucosa, Luk and Baylin (17) have demonstrated that peak levels of putrescine, spermidine and spermine were observed two days after resection, while in the pancreas putrescine total content and concentration were at their highest at least 84 hr before spermidine and spermine (Fig. l). Moreover, in the intestine study, villus and crypt growth rates, DNA synthesis and content and intestinal weight, all showed a significantly positive correlation with mucosal putrescine content. In the pancreas, positive correlations could be obtained only with spermidine and spermine levels regarding pancreatic weight and DNA content. Spermidine

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and spermine were not used to establish such correlations in the intestine (17) but since [heir c o n c e n t r a t i o n s p a r a l l e l e d putrescine, one can assume that they would have been positive as well. Even though we did not measure ODC activity, we feel that it was induced by caerulein since putrescine, increased by the treatment, was significantly lowered after 12 hr and totally reduced down to control values after 24 hr of DFMO treatment. Moreover, in other tissues such as in the intestine (17), increases in ODC activity and putrescine were parallel. Our results demonstrate that polyamine synthesis and a c c u m u l a t i o n do occur in the pancreas in response to a t r o p h i c factor such as caerulein. These data also indicate that these polyamines are needed [or p a n c r e a t i c growth, as m e a s u r e d by DNA content and cellular mass, since these two parameters were s i g n i f i c a n t l y reduced following the inhibition of ornithine decarboxylase by DFMO. It is not yet known if only one or perhaps more of these polyamines are needed for specific cellular functions, and at what particular point during the cell cycle. Studies are now in progress to answer these questions. Acknowledsements The authors wish to express their thanks to M. Vanier for her technical assistance, C. Rancourt for typing the manuscript and J. Loiselle for statistical analysis. Special thanks are also addressed to Dr. Peter P. McCann for the analysis of the polyamine levels in pancreatic tissue. This work was supported and m i n i s t a t e de l'Education

by Grants A6369 du QuEbec.

and EQ733

from NSERC

of Canada

References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. Ii. 12. 13. 14. 15. 16. 17.

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