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The effect of fasting on the in vitro synthesis of amylase in rat exocrine pancreas E)epartnzent of Plzcmrmacology and Tlaerapeutics, McGilH &Tnitversidy, Montreal, P.Q., Ctxnada $136 1 Y6 Received July 14, 1976 VIEWA-MATOS, A. N., and TENENHOUSE, A. 1977. The effect of fasting on the in vitro synthesis of amylase in rat exocrine pancreas. Can. 5. Physiol. Pharmacol. 55,90-97. Immunological methods were used to study the effect of fasting on the in vitro synthesis of amylase (EC 3.2.1.1) in rat exocrine pancreas. After 72 h of fasting, the amylase enzyme activity of the pancreas and the rate of amylase synthesis were reduced 58%. No significant change in the activities of trypsin or chymotrypsin were detected. The decrease in leucine incorporation in total pancreas protein was accounted for by the decreased amylase synthesis. No change in the rate of amylase breakdown was detected. These results indicate that the rate of synthesis of amylase is controlled by food intake and is not directly related to the tissue content of enzyme. VIEIRA-M~ass, A. $I. et TENENHOUSE, A. 1979. The effect of fasting on the in vitro synthesis of amylase in rat exocrine pancreas. Can. 3. Physisl. Pharmacol. 55, 90-97. Des mkthodes immunologiques sont utiliskes pour Ctudier l'cffet du jeQne sur la synthkse de l'amylase, (EC 3.2.1.1 ) dans le pancrCas exocrine du rat in vitro. Apsks un jeQne de 72 h, B'activitC enzymatique de l'amylase et la vitesss de synthkse sont rkduites de 50%. Aucun changement significatif des activitks de la trypsin ou de la chy~notrypsinen'est relevk. La diminution d'incorporation de la leucine dans les protkines pancrkatiques pent Ctre expliquke par la diminution de synthkse de l'amylase. Abncun changemelat du taux de dkgradation de l'amylase n'est observk. Ces rksultats indiquent que la vitesse de synthkse de l'amylase est contr6lke par l'alimcntation et n'est gas directement like au eontenu d'enzyme tissulaire. [Traduit par le journal]
InQroduc~on The effect of starvation on the synthesis of the digestive enzymes of the exocrine pancreas has been studied by several investigators, with contradictory results. When enzyme activity was used as a measure of synthesis, some workers reported a decreased rate of synthesis of all enzymes (Veghelyl and Kerneny 1962) while others reported an increased rate of synthesis of certain enzymes (Christophe et a&. 1971) . When amino acid incorporation into total pancreatic protein was used as a measure sf protein synthesis, it was found that starvation either had no effect on the rate of synthesis (Allfrey et a&. 1954; Lee and Fisher 1971 ) or decreased the rate of synthesis (Webster 1969; Meldolesi 1970; Morisset and Webster 1971, 1972; Webster et al. 1972; ABBREVIATHONS: TCA, trichloroacetic acid; TAME, p-toluene sulfonyl-L-arginine methyl ester; BTEE, benzoyl-L-tyrosine ethyl ether. 'Present address: Bept. de ciencias fisiologicas, Escuela de Medicha U.B.O., Cd. Bolivar, Venezuela.
Danielsson et ak. 1974). A major reason for these conflicting results is that only in the studies of Morisset and Webster ( 1 972) and Danielsson et al. ( 1974) was degradation of a specific enzyme measured. It has been established in studies with several organs (Akikusa 1971; Reid et al. 1978; Evans and Scholtz 1971) that starvation effects the metabolism of various proteins differently. It is clear, therefore, that the behaviour of a specific protein cannot be inferred from changes in metabolism of total protein. Nor can the results for one protein be extrapolated to any other in the same organ. The experiments described in this report were designed to measure the effects sf starvation on the metabolism of pancreatic amylase using immunological techniques which have proven useful in the study of specific proteins in a number of organs (Shimke and Doyle 1970).
Materials and Methods Female Wistar rats weighing 250-386 g were used.
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VIEIRA-MATOS AND TENENHOUSE
One group of rats ( F D ) had free access to water and a commercial balanced diet (22% proteins, 69% carbohydrates, 4% fat. salts and vitamin supplement). The fasted rats (FT) were kept in metabolic cages with free access to watcr only, and were fasted for 72 h unless otherwise indicated. The rats were killed by decapitation, the dorsal part of the pancreas was excised, and placed in a petri dish containing cold ( 4 " 6 ) incubation medium. The incubation medium was KrebsRinger phosphate solution, supplemented with 10 mill glucose and Lamino acids at the concentrations described by Eagle (1959). The tissue was cleaned free of fat and connective tissue, and cut with scissors into 5- to 10-mg pieces. All the operations were done at 4 "C. The tissue (200 mg wet weight) was added to Erlenmeyer flasks containing 3 ml of the incubation medium (which had been bubbled with 100% 0, for 10 min) and preincubated for 10 min at 37 "C, before the addition of I.-["Haleucine. The final specific activity of the L-[3H]leucine was 10 pCi/mol. The incubation was terminated by homogenizing the total contents of the incubation flasks at 4 "C. The homogenate was filtered through gauze, and an aliquot of the filtrate (0.5 ml) precipitated with TCA (final concentration, 5 % ) containing 20 m M 'nonradioactive' ['Hlleucine. The precipitate was treated as decribed by Siekevitz (1952) for measurement of incorporation of radioactive amino acid into total protein. The remainder of the homogenate was dialysed at 4 " C for 8-12 h against phosphate saline buffer (0.01 M NaH,PO,-Na2HP04, 0.85% NaCl, pH 7.4). After dialysis the homogenate was centrifuged at 105 000 x g for 30 min, and the supernatant ('crude pancreatic extract') assayed for enzyme activities and [3H]leucine incorporation into amylase using the immunological techniques described below. Amylase was assayed using the method of Bernfield (1955), trypsin and chymotrypsin using the method of Hummel (1959). Both enzymes were activated by incubating homogenate supernatant with enterokinase ( E C 3.4.21.9) (20 mg/ml) at 37 " C for 30 min. Specific synthetic substrates were used: TAME for the trypsin assay and BTEE for the chymotrypsin assay. Purified bovine trypsin (217 U/mg) and chymotrypsin (47 U/mg) (Worthington Biochemical Corp.) were used as standards, and the enzyme activities are expressed as microgram equivalents of the bovine standards. In preliminary experiments, this dialysis procedure was found to be as effective as treatment with 0.5% Lubrol in releasing amylase activity and radioactive protein from particulate fractions of the rat pancreas homogenate. Rat pancreatic amylase was purified by the chromatographic method described by Vandermeers and Christophe (1968). The specific activity of the purified amylase varied between 500 and 700 U/mg protein (unit as defined by Bernfield 1955). Antibodies against the eleetrophoretically pure enzyme were produced in New Zealand rabbits by direct intrasplenic injections of 200-250 pg of the enzyme protein, mixed with complete Freund's adjuvant (1: 1, v/v). Booster injections were given intramuscularly. The y-globulin present in the serum of the immunized rabbits was
91
partially purified by repeated (three times) precipitation with arnnlonium sulphate ( 3 3 % saturation), King test, immunodiffusion, and immranoelcctrophoresis were performed as described by Campbell et al. (1978). Each antibody preparation was tested by immunodiffusion and immunoclectrophorcsis. and was used only if a single precipitin band was found by both methods. Optimal quantities of antibody preparation and of amylase contained in the crude pancreatic extract were mixed in I ml of phosphate saline buffer. The samples were incubated for 30 min at 37 " C and then overnight at 4 " 6 . The precipitate formed was iscslated by centrifugation 10 000 x I: for 20 min at 4 " 6 , washed three times with phosphate saline buffer containing 10 n1M L-flH]leucine, and used to measure the radioactivity incorporated into amylase. The supernatant was saved to test for antigen excess by ring test or measr~rement of amylase activity. The samples in which antigen was found in the supernatant were discarded. Nucleic acids were extracted from the TCA precipitate of the tissue homogenate by heating the samples which had been resuspended in 2 ml of TCA for 20 min at 90 " C . RNA was measured by the orcinol method described by Dische (1955) using purified yeast MNA (Worthington Co.) as standard, DNA as described by Burton (1956) using purified rat thymus DNA (Worthington Co.) as standard, and protein according to Lowry et al. ( 1951 ) .
Results Preliminary experiments were done to determine optimal conditions for immunoprecipitation of amylase and to assess the specificity of the immunochemical method. The crude pancreatic extract sf tissue which had been incubated in the presence of L-[~P%]leucinc was prepared as described in Materials and Methods. Aliquots containing increasing amounts of amylase activity were incubated in the presence of a fixed volume of the antibody preparation (1 0 p l ) , as described in Materials and Methods for quantitative immunoprecipitation. The radioactivity associated with the antigen-antibody complex was measured. The results of a typical experiment are shown in Fig. I . The increasing amount of radioactivity associated with the antigen-antibody complex indicated that the newly synthesized amylase reacted to form an insoluble complex with the antibody. A point was obtained where neither antigen nor antibody excess was found (equivalence point). At the equivalence point and in thc zone of slight antibody excess the precipitation of amylase was quantitative. In all the following experiments the antigen-anti-
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CAN. J. FHYSBOE. P H A M A W L . VQL. 55, 1977
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TABLE 1. Radioactivity specifical%yand nonspecifically associated with the antigen-antibody complex
Incubation of L- [3H]leucine, min
TCAprecipitable radioactivity from crude homogenate, dpnlm
I st antibody treatment Antibodyprecipitable radioactivity, dl?m
2nd antibody treatment
Antibody TCA,
c a p /dpm
Antibodyprecipitable radioactivity,
An tibody TCA, dpm /dpm
ase was then added to the supernatant after removal of the antigen-antibody precipitate and the procedure repeated. Less than 2 % of the original total TCA-precipitable radioactivity was recovered with the antigen-antibody precipitate.
8 3 Ag Excess -
25
I5
38
60
90
AMY.
Units
- - _+ + +' + If Ab Excess + FIG.1 . Determination of optimal antigen-antibody proportions for quantitative precipitation of amylase synthesized by rat pancreas pieces incubated in vitro. Radioactivity (dprn) associated with the antigen-antibody complex is plotted against the Bog of the units of amylase atsed in the reaction mixture. One amylase unit was defined as the amount of enzyme that hydrol y z d H rng sf maltose in 3 min at 24 "C. Details of the procedure are given in the text.
body proportion which gave slight antibody excess was used to determine the rate of incorporation of [aH]leucine into amylase. The specificity of the immunoprecipitation method was tested by deternaining whether any amylaseantibody-precipitable radioactivity remained after quantitative precipitation of amylase enzymatic activity. These results are shown in Table 1. In these experiments pancreas pieces were incubated in the presence of L-["Ileucine for 10, 20, or 40 min. The tissue was then extracted and precipitated with amylase antibody as described in Materials and Methods. AS can be seen from Table 1, about 30% of the total TCA-precipitable radioactivity was precipitated by the antibody. Urnlabelled amyl-
Effect of Starvation on RNA and Protein Content and Digestive Enzyme Activities in the Rat Pancreas Table 2 summarizes the results of experiments in which the pancreatic content of RNA and protein, as well as the tissue chymotrypsin, trypsin, and amylolytic activities were measured in fed and fasted rats. Although a slight decrease in all parameters was observed, only amylase was significantly changed after fasting for 72 h. The enzyme activity was decreased about 50%. The possibility that this decrease was due to inhibition of the enzyme activity rather than to a decrease in the enzyme protein content was considered. That this was unlikely was shown by measuring the equivalence point of a preparation of amylase antibody using the tissue homogenate from both fasted and fed rats as the source of amylase (Fig. 2 ) . Twenty microlitres of amylase antibody preparation was incubated with 20-90 U of amylase from crude pancreatic extract derived from fasted and fed rats. T o provide the same amount of amylolytic activity in samples from fasted and fed animaIs, the volume of 'fasted' extract was twice that of 'fed' extract. After centrifugation of the antigen-antibody complex the amylolytic activity in the supernatant was measured and plotted against the antigen concentration added to the incubation mixture. Amylase activity appeared in the supernatant from both samples when the amount of amylase activity added exceeded 60 U; i.e., the equivalence point was the same using amylase f r ~ mthe pancreas of
VIEIRA-MATBS AND TENENHOUSE
TABLE 2. RNA and protein content, amylase, trypsin, and chymotrypsin activities in
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pancreas from fed and fasted rats
Fed Fasted
yochange
RNA, mg /rng DNA
Protein, rng /mg DNA
Amylase, U /mg DNA
Trypsin, , ~ /mg g DNA
Chymotrypsin, pg /mg DNA
2.53 + 0.32 (13) 2.02 f 0.28 (1 3) -20 NS
21.25 f 2 . 7 (10) 17.33 f 2 . 4 (109 -20 NS
1596.2 f 150 (30) 709.0 f 57 (30) -55 p < 0.01
145 f 28 (9) 109 f IS) (9) -25 NS
483 f 55 (9) 395 _+ 46 (9) -18 NS
NOTE:The values given are mean values -b SE; the number of determinations is given in parentheses. NS, not significant.
-
A M Y units added Supern. v o l . FD
5
10
6Q 15
-1 28
I00 25
0
FIG. 2. Determination of the equivalence point of the amylase antibody. Crude pancreatic extract from fed ( 0 ) and fasted ( x ) rats was used as antigen. Details of the procedure are given in the text.
fasted or fed rats. Thus, the decrease in the amylase activity found in the extract of pancreas from fasted rats was paralleled by a decrease in the amount of immunoprecipitable protein. The rate of incorporation of L-[3H]leucine into TCA-precipitable protein and immunoprecipitable amylase of rat pancreas tissue was measured as described in Materials and Methods. There was no difference between the rate of incorporation of L-[3M]leucine into total protein of pancreas from fasted and fed rats (Fig. 3.) Incorporation into immunoprecipitable amylase, however, was decreased almost 50% (Fig. 4). An attempt was made to determine the in v i t r ~rate of breakdown of newly synthesized total protein and irnmunoprecipitable amylase by measuring the rate of disappearance of radioactivity incorporated during pulse labelling of pancreatic tissue pieces from both fed and fasted rats. The tissue was incubated as described in Materials and Methods, except that
10
30
40
120
Time (mimB
FIG.3. Rate of incorporation of L-['H]leucine into total protein by pancreatic tissue pieces from fed ( 0 ) and fasted ( 0 ) rats. Each point is the mean ? SE of five experiments. The results are expressed as wanomoles of L-leucine incorporated into TCA-precipitable protein per milligram DNA.
a
.p_
5
a
a
I
9
O
W
30
68
126
FIG.4. Rate of incorporation of [3H]leucine into immunoprecipitable amylase by pancreatic tissue pieces from fed ( 8 ) and fasted ( ) rats. Each point is the mean + SE of five experiments. The results are expressed as nanomoles of L-leucine associated with irnmunoprecipitable amylase per milligram DNA.
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CAN. B. PHYSIOL. PHARMACOL.
FIG. 5. Radioactivity associated with total pancreatic protein during chase incubation of pancreatic tissue pieces fro111 fed ( F D ) and fasted ( F T ) rats. The incubation was done in the presence of 10 " A4 cycloheximide. The results are expressed as piconaoles of L-leucine associated with TCA-precipitable protein per milligram DNA. The data are from one representative experiment.
no L-[lHHqleucine was added to the medium and the concentration of L-["Hlleucine was 2.5 pCi/ml (specific activity of L-["Hqleucine, 64 Ci/rnmol). After 5 min the tissue pieces were washed with an excess incubation medium containing % mM L-['Hlleucine. The samples were then incubated for 4-8 h at 37 "C in fresh incubation medium containing 1 m M L-[~M]leucine. At the desired time the incubation was stopped by homogenizing the tissue in the incubation medium and the samples were treated as described in Materials and Methods to measure the radioactivity associated with TCAprecipitable ('total') protein and immunoprecipitable amylase, Cycloheximide ( 1 0 - W ) was used in some experiments to prevent reuse of any radioactive amino acid liberated during the incubation. As can be seen in Figs. 5 and 6, no detectable breakdown of newly synthesized protein or amylase occurred during incubation of tissue from fed or fasted rats during the time of incubation. In preliminary experiments it was found that gel filtration of the 105 000 X g supernatant from rat pancreas through a Sephadex (3-50 column separated the soluble pancreatic proteins into four fractions. Chymotrypsin and trypsin eluted together in the first protein peak, whereas amylase eluted in the third protein peak. Based on this observation an experiment was designed to determine the distribution of radioactivelly BabeIled proteins of the pancreas
VOL. 55, 1977
FIG. 6. Radioactivity associated with immunoprecipitable amylase during chase incubation of pancreatic tissue pieces from fed ( F D ) and fasted ( F T ) rats. The results are expressed as picsmoles of L-leucine associated with immunoprecipitable amylase per milligram DNA. The data are from one representative experiment.
and whether this distribution was altered by starvation. Pancreas pieces from both fcd and fasted rats were incubated as described in Materials and Methods, except that the tissue lroan fasted animals was incubated with L - [ ~ ~ C ] leucine (0.5 pCi/mI) and that from fed animals with L-[W]leucine ( 5 pCi/ml). The final leucine concentration in both incubation media was 1.8 mM. After 1 11 of incubation the tissues (and incubation media) from fed and fasted rats were pooled and homogenized. After 8 h of dialysis against 13 mi44 Tris-HC1, pH 8.3, and 3 mM CaCl.,, the homogenate was centrifuged at 105 000 X g for 2 h and the supernatant concentrated by ultrafihration using an Amicon ultrafiltration cell with a PM 10 membrane. Two inillilitres of the concentrated supernatant, containing 54 rng of protein, was applied to a 50 )< 2.8 cm Sephadex G-50 column which had been equilibrated with the Tris-HC1 buffer and the column eluted with the same buffer at a flow rate of 22.5 ml/h; 3-ml fractions were collected. The radioactivity in a 0.5-ml aliquot from each fraction eluted from the column was measured. The elution pattern of protein (optical density at 280 nm) and radioactivity (dpm) from one of these experiments is shown in Fig. 7. T o plot W and 14C radioactivity on the same scale, and thus facilitate comparison, the l 4 C values obtained for each fraction were divided by the 14C-3H ratio originally present in the sample applied to the column. The total recovery of radioactivity (both 3M and 14C) from
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VIELWA-MATOS AND TENENHOUSE
AMY T RY CHY
++ 4-4
++
---
F s g l r c t i an
Number
FIG.7. SepI-sadex 6 - 5 0 column chromatography of prelabelled 105 000 x g pancreatic supernatant from fed and fasted rats. The presence (++) or abscnce ( - - ) of amylase, trypsiw, and chyanotrypsin activities in the two main peaks are indicated. The incomqsoration of the radioactive amino acid is expressed as dpmiml of the sample. ( x 1, Optical clerasity at 280 wm; ( 0 ), 'H from fed rats, expressed as dpm; (e)),"C from fasted rats, expressed as dpm.
the co%umn was 95-98%. The radioactivity i n ~ c ~ r p c ~ r ainto t e d protein of pancreas from both fed and fasted rats consistently distributed between two fractions, the first, containing most of the radioactivity and all the trypsin and chyrnotrypsin activity and the third peak, cont a i n i n ~all the amylase activity, 30% of the total *'El counts, and about 15% of the I4C counts which eluted from the column. Thus, the same decrease (about 50% ) sf leucine incorporation into amylase by pancreas from fasted rats was found using the immunochemical method and column chromatography.
Discussion After a 72-h fast the rate of incorporation of [3H]Ieucine into immunoprecipitable amylase was decreased 5 0 % . Since there was no increase in the rate of an~y'lasebreakdown, this probably represents a decrease in the rate of amylase synthesis. Associated with this decrease in syant%nesiswas an equal ( 5 8 % ) decrease in total amylase activity within the tissue. In a series of preliminary experiments (not reported here), it was found that this decreased rate s f amylase synthesis was de-
tectable after 24-36 h of fasting and without any detectable decrease in total amylase activity; i.e., the change in rate of synthesis preceded and was the cause of the decrease in enzyme activity. The decrease in amylase synthesis occurred in the absence of any detectable change in the rate of synthesis of total pancreatic protein and witboaat any measurable decrease in the total activity of the other two pancreatic enzymes studied. There is no doubt that the assays used to measure trypsin and chymotrypsin were suiTiciently sensitive to detect a 25-50% decrease in activity. The fact that no significant decrease was found in a total of nine separate experiments indicates tlmat such a decrease did not occur. The effect of starvation on total protein synthesis is more diflicarlt to evaluate. In 'fed' rats, amylase accounts for about 30% of the total pancreatic protein synthesized in the in vitro system used in our studies. If only amylase synthesis was decreased, and if no other protein was being synthesized at an increased rate, then one would expect to find a decrease in total protein symthesis sf 155% after 72 h of starvation. It is possible that this change in total [3H]Beucine
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CAN. J. PHYSIOL. PHARMACOL. VQL. 55. I977
incorporation into total protein was not detected because of the lack of sensitivity of the method used. To improve our ability to detect small changes in the rate of synthesis of proteins other than amylase, we separated the soluble proteins of the rat pancreas homogenate into four fractions using Sephadex (3-50 column chron~atography and measured the ininto each fraction. corporation of ["]leucine All the amylase was recovered in a single protein peak separate from the peak which contained the trypsin and chymotrypsin activity. The 58% decrease in amino acid incorporation into amylase by the pancreas from starved rats was confirmed using this method; however, there was no consistent change in the radioactivity recovered in any of the other peaks, and no new radioactive peak was found. Since the recovery of radioactivity from these columns was 95-98%, it can be concluded that the incorporation of leucine into total protein (the sum of that incorporated into each peak) was decreased 15% by starvation, due entirely to the decrease in amylase synthesis. There are other factors which could contribute to the decrease in amylase activity in the pancreas after 72 h of starvation, e.g., increased rate of release of the enzyme, or an increased rate of destruction. This latter was demonstrated not to exist, at least when destruction is measured in vitro. We have not ruled out the possibility that during starvation amylase release from the gland is increased, but this seems highly unlikely. It is commonly believed that the exocrine pancreas contains a single population of zymogen granules, each containing a complement of all the secretory enzymes, and that during secretion the entire contents of the granule are discharged. If the rate of amylase release is increased such that the amylase content of the gland is significantly reduced, comparable decrease in gland content of the other secretory enzymes or an increased rate of synthesis of these proteins would be expected. We could find no evidence for either. Also, it seems unlikely that the depletion of an enzyme would result in a decrease in its synthesis; an increased rate of synthesis is a much more likely result. It must be concluded, therefore, that starvation for up to 72 h leads to a selective decrease in amylase synthesis in the rat exocrine pancreas. Although the mechanism of this effect is
not understood, it is clear from these results that, at least during starvation, the total amylase content of the tissue does not completely (if at all) control the rate of synthesis of the enzyme. AKIKUSA, Y. 1971. Effect of starvation on synthesis and release of growth hormone and prolactin in the rat anterior pituitary. Endocrinol. Jpn. 18, 41 1-414. ALI'FREY.V., DALY,M. M.. and M~RSKY, A. E. 1954. Synthesis of protein in the pancreas. II. The rolc of ribonucleoprotein in protein synthesis. J. Gen. Physiol. 37, 157-175. BERNFIELD, P. 1955. Amylase, a and p. Meth. Enzymol. 1, 149-150. BURTON,K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. CAMPBELL, D. H., JARREY, J. S., CREMER, N. E., and SUSSDORF, D. H. 1970. In Methods in immunology. W. A. Benjamin, Inc.. Melo Pk, CA. CHRIS'TOPHE, J., CAMUS,J., DISCHODT-LANCRMAN, M., RATHE,J., ROBBERECHT, P., VANDFRMEERS-PIKET, M. C., and VANDERMEERS, A. 1971. Factors regulating biosynthesis, intracellular transport and secretion of amylase and lipase in the rat exocrine pancreas. Horm. Metab. Res. 3, 3 9 3 4 0 % . DANIELSSOW, A., MARKLUND, S., and STPC~BRAND, T. 1974. Effects of starvation and islet hormones on the synthesis of amylase in isolated exocrine pancreas of the mouse. Acta Hepato-Gastroenterol. 21, 289297. DISCHE,Z. 1955. Color reaction of nucleic acid components. In The nucleic acids. Vol. 1. Edited by E. Chargaff and J. N. Davidson. Academic Press Inc., New York, N.Y. pp. 285-305. EAGLE,H. 1959. Amino acid metabolism in rnammalian cell cultures. Science, 130,432-437. EVANS,R. M., and SCHOLZ,W. W. 1971. Metabolic responses of chicks during adaptation to a high protein, 'bcarbohydrate-free" diet. J. Nutr. 181, 1127-1 136. HUMMEL,B. C. W. 1959. A modified spectrophotometric determination of chyrnotrypsin. trypsin, and thrombin. Can. J. Biochem. Physiol. 37, 1393-1399. LEE, P. C., and FISHER,J. R. 1971. Regulation of xanthine dehydrogenase levels in liver and pancreas of the chick. Biochim. Biophys. Acta, 237, 14-20. LOWRY,Q. H., ROSEBROUGH, N. J., FAWW, A. L., and RANDALL, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MELDOLESI, J. 1970. Effect of caerulein on protein synthesis and secretion in the guinea-pig pancreas. BP.J. Pharmacol. 40,721-73 1. MORISSET, J. A., and WEBSTER,P. B. 1971. In vitrcl and in vivo effects of pancreozymin, urecholine, and cyclic AMP on rat pancreas. Am. J. PfiysioI. 270, 202-208. 1972. Effects of fasting and feeding on protein synthesis by the rat pancreas. J. CLin. Invest. 51, 1-8.
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REID, I. M., VERNEY,E., and SIDRANSKY, N. 1970. Influence of acute nutritional stress on poIyribosomes and protein synthesis in brain and liver of young rats. J . Nutr. 100, 1149-1 156. SCHIMKE,R. T., and DOYLE, D. 1970. Control of enzyme levels in animal tissues. Annu. Rev. Biochem. 39,929-976. SIEKEVITZ.P. 1952. Uptake of radioactive alanine in vitro into the proteins o n rat liver fractions. J. Riol. Chem. 195,549-565. VANDERMEERS, A., and CHRISTOPHE,J. 1968. aAmylase et lipase du pancreas de rat. Purification chromatographique, recherche du poids moleculaire e t composition en acides amines. Biochim. Biophys. Acta, 154, 110-129.
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VEGBEI,YL,P. V., and KEMENY,T. T. 2962. Protein metabolism and pancreatic function. In Ciba Foundation Symposium on the exocrine pancreas. Edited by A. V. S. Reuck and M. P. Cameron. A. and J. Churchill, London. pp. 329-352. WEBSTER,P. D . 1969. Hormonal control of pancreatic acinar cell metabolism. I n The exocrine glands. Edited b y S. Y . Rotclh, F. P. Brooks, and W. B. Shelley. University of Pennsylvania Press, Philadelphia, PA. pp. 153-168. WEBSTER,P. D., SINGH, M., TUCKER,P. C., and BLACK,0. 1972. Effects of fasting and feeding on the pancreas. Gastroenterology, 62, 600-605.
The effect of fasting on the in vitro synthesis of amylase in rat exocrine pancreas E)epartnzent of Plzcmrmacology and Tlaerapeutics, McGilH &Tnitversidy, Montreal, P.Q., Ctxnada $136 1 Y6 Received July 14, 1976 VIEWA-MATOS, A. N., and TENENHOUSE, A. 1977. The effect of fasting on the in vitro synthesis of amylase in rat exocrine pancreas. Can. 5. Physiol. Pharmacol. 55,90-97. Immunological methods were used to study the effect of fasting on the in vitro synthesis of amylase (EC 3.2.1.1) in rat exocrine pancreas. After 72 h of fasting, the amylase enzyme activity of the pancreas and the rate of amylase synthesis were reduced 58%. No significant change in the activities of trypsin or chymotrypsin were detected. The decrease in leucine incorporation in total pancreas protein was accounted for by the decreased amylase synthesis. No change in the rate of amylase breakdown was detected. These results indicate that the rate of synthesis of amylase is controlled by food intake and is not directly related to the tissue content of enzyme. VIEIRA-M~ass, A. $I. et TENENHOUSE, A. 1979. The effect of fasting on the in vitro synthesis of amylase in rat exocrine pancreas. Can. 3. Physisl. Pharmacol. 55, 90-97. Des mkthodes immunologiques sont utiliskes pour Ctudier l'cffet du jeQne sur la synthkse de l'amylase, (EC 3.2.1.1 ) dans le pancrCas exocrine du rat in vitro. Apsks un jeQne de 72 h, B'activitC enzymatique de l'amylase et la vitesss de synthkse sont rkduites de 50%. Aucun changement significatif des activitks de la trypsin ou de la chy~notrypsinen'est relevk. La diminution d'incorporation de la leucine dans les protkines pancrkatiques pent Ctre expliquke par la diminution de synthkse de l'amylase. Abncun changemelat du taux de dkgradation de l'amylase n'est observk. Ces rksultats indiquent que la vitesse de synthkse de l'amylase est contr6lke par l'alimcntation et n'est gas directement like au eontenu d'enzyme tissulaire. [Traduit par le journal]
InQroduc~on The effect of starvation on the synthesis of the digestive enzymes of the exocrine pancreas has been studied by several investigators, with contradictory results. When enzyme activity was used as a measure of synthesis, some workers reported a decreased rate of synthesis of all enzymes (Veghelyl and Kerneny 1962) while others reported an increased rate of synthesis of certain enzymes (Christophe et a&. 1971) . When amino acid incorporation into total pancreatic protein was used as a measure sf protein synthesis, it was found that starvation either had no effect on the rate of synthesis (Allfrey et a&. 1954; Lee and Fisher 1971 ) or decreased the rate of synthesis (Webster 1969; Meldolesi 1970; Morisset and Webster 1971, 1972; Webster et al. 1972; ABBREVIATHONS: TCA, trichloroacetic acid; TAME, p-toluene sulfonyl-L-arginine methyl ester; BTEE, benzoyl-L-tyrosine ethyl ether. 'Present address: Bept. de ciencias fisiologicas, Escuela de Medicha U.B.O., Cd. Bolivar, Venezuela.
Danielsson et ak. 1974). A major reason for these conflicting results is that only in the studies of Morisset and Webster ( 1 972) and Danielsson et al. ( 1974) was degradation of a specific enzyme measured. It has been established in studies with several organs (Akikusa 1971; Reid et al. 1978; Evans and Scholtz 1971) that starvation effects the metabolism of various proteins differently. It is clear, therefore, that the behaviour of a specific protein cannot be inferred from changes in metabolism of total protein. Nor can the results for one protein be extrapolated to any other in the same organ. The experiments described in this report were designed to measure the effects sf starvation on the metabolism of pancreatic amylase using immunological techniques which have proven useful in the study of specific proteins in a number of organs (Shimke and Doyle 1970).
Materials and Methods Female Wistar rats weighing 250-386 g were used.
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VIEIRA-MATOS AND TENENHOUSE
One group of rats ( F D ) had free access to water and a commercial balanced diet (22% proteins, 69% carbohydrates, 4% fat. salts and vitamin supplement). The fasted rats (FT) were kept in metabolic cages with free access to watcr only, and were fasted for 72 h unless otherwise indicated. The rats were killed by decapitation, the dorsal part of the pancreas was excised, and placed in a petri dish containing cold ( 4 " 6 ) incubation medium. The incubation medium was KrebsRinger phosphate solution, supplemented with 10 mill glucose and Lamino acids at the concentrations described by Eagle (1959). The tissue was cleaned free of fat and connective tissue, and cut with scissors into 5- to 10-mg pieces. All the operations were done at 4 "C. The tissue (200 mg wet weight) was added to Erlenmeyer flasks containing 3 ml of the incubation medium (which had been bubbled with 100% 0, for 10 min) and preincubated for 10 min at 37 "C, before the addition of I.-["Haleucine. The final specific activity of the L-[3H]leucine was 10 pCi/mol. The incubation was terminated by homogenizing the total contents of the incubation flasks at 4 "C. The homogenate was filtered through gauze, and an aliquot of the filtrate (0.5 ml) precipitated with TCA (final concentration, 5 % ) containing 20 m M 'nonradioactive' ['Hlleucine. The precipitate was treated as decribed by Siekevitz (1952) for measurement of incorporation of radioactive amino acid into total protein. The remainder of the homogenate was dialysed at 4 " C for 8-12 h against phosphate saline buffer (0.01 M NaH,PO,-Na2HP04, 0.85% NaCl, pH 7.4). After dialysis the homogenate was centrifuged at 105 000 x g for 30 min, and the supernatant ('crude pancreatic extract') assayed for enzyme activities and [3H]leucine incorporation into amylase using the immunological techniques described below. Amylase was assayed using the method of Bernfield (1955), trypsin and chymotrypsin using the method of Hummel (1959). Both enzymes were activated by incubating homogenate supernatant with enterokinase ( E C 3.4.21.9) (20 mg/ml) at 37 " C for 30 min. Specific synthetic substrates were used: TAME for the trypsin assay and BTEE for the chymotrypsin assay. Purified bovine trypsin (217 U/mg) and chymotrypsin (47 U/mg) (Worthington Biochemical Corp.) were used as standards, and the enzyme activities are expressed as microgram equivalents of the bovine standards. In preliminary experiments, this dialysis procedure was found to be as effective as treatment with 0.5% Lubrol in releasing amylase activity and radioactive protein from particulate fractions of the rat pancreas homogenate. Rat pancreatic amylase was purified by the chromatographic method described by Vandermeers and Christophe (1968). The specific activity of the purified amylase varied between 500 and 700 U/mg protein (unit as defined by Bernfield 1955). Antibodies against the eleetrophoretically pure enzyme were produced in New Zealand rabbits by direct intrasplenic injections of 200-250 pg of the enzyme protein, mixed with complete Freund's adjuvant (1: 1, v/v). Booster injections were given intramuscularly. The y-globulin present in the serum of the immunized rabbits was
91
partially purified by repeated (three times) precipitation with arnnlonium sulphate ( 3 3 % saturation), King test, immunodiffusion, and immranoelcctrophoresis were performed as described by Campbell et al. (1978). Each antibody preparation was tested by immunodiffusion and immunoclectrophorcsis. and was used only if a single precipitin band was found by both methods. Optimal quantities of antibody preparation and of amylase contained in the crude pancreatic extract were mixed in I ml of phosphate saline buffer. The samples were incubated for 30 min at 37 " C and then overnight at 4 " 6 . The precipitate formed was iscslated by centrifugation 10 000 x I: for 20 min at 4 " 6 , washed three times with phosphate saline buffer containing 10 n1M L-flH]leucine, and used to measure the radioactivity incorporated into amylase. The supernatant was saved to test for antigen excess by ring test or measr~rement of amylase activity. The samples in which antigen was found in the supernatant were discarded. Nucleic acids were extracted from the TCA precipitate of the tissue homogenate by heating the samples which had been resuspended in 2 ml of TCA for 20 min at 90 " C . RNA was measured by the orcinol method described by Dische (1955) using purified yeast MNA (Worthington Co.) as standard, DNA as described by Burton (1956) using purified rat thymus DNA (Worthington Co.) as standard, and protein according to Lowry et al. ( 1951 ) .
Results Preliminary experiments were done to determine optimal conditions for immunoprecipitation of amylase and to assess the specificity of the immunochemical method. The crude pancreatic extract sf tissue which had been incubated in the presence of L-[~P%]leucinc was prepared as described in Materials and Methods. Aliquots containing increasing amounts of amylase activity were incubated in the presence of a fixed volume of the antibody preparation (1 0 p l ) , as described in Materials and Methods for quantitative immunoprecipitation. The radioactivity associated with the antigen-antibody complex was measured. The results of a typical experiment are shown in Fig. I . The increasing amount of radioactivity associated with the antigen-antibody complex indicated that the newly synthesized amylase reacted to form an insoluble complex with the antibody. A point was obtained where neither antigen nor antibody excess was found (equivalence point). At the equivalence point and in thc zone of slight antibody excess the precipitation of amylase was quantitative. In all the following experiments the antigen-anti-
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CAN. J. FHYSBOE. P H A M A W L . VQL. 55, 1977
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TABLE 1. Radioactivity specifical%yand nonspecifically associated with the antigen-antibody complex
Incubation of L- [3H]leucine, min
TCAprecipitable radioactivity from crude homogenate, dpnlm
I st antibody treatment Antibodyprecipitable radioactivity, dl?m
2nd antibody treatment
Antibody TCA,
c a p /dpm
Antibodyprecipitable radioactivity,
An tibody TCA, dpm /dpm
ase was then added to the supernatant after removal of the antigen-antibody precipitate and the procedure repeated. Less than 2 % of the original total TCA-precipitable radioactivity was recovered with the antigen-antibody precipitate.
8 3 Ag Excess -
25
I5
38
60
90
AMY.
Units
- - _+ + +' + If Ab Excess + FIG.1 . Determination of optimal antigen-antibody proportions for quantitative precipitation of amylase synthesized by rat pancreas pieces incubated in vitro. Radioactivity (dprn) associated with the antigen-antibody complex is plotted against the Bog of the units of amylase atsed in the reaction mixture. One amylase unit was defined as the amount of enzyme that hydrol y z d H rng sf maltose in 3 min at 24 "C. Details of the procedure are given in the text.
body proportion which gave slight antibody excess was used to determine the rate of incorporation of [aH]leucine into amylase. The specificity of the immunoprecipitation method was tested by deternaining whether any amylaseantibody-precipitable radioactivity remained after quantitative precipitation of amylase enzymatic activity. These results are shown in Table 1. In these experiments pancreas pieces were incubated in the presence of L-["Ileucine for 10, 20, or 40 min. The tissue was then extracted and precipitated with amylase antibody as described in Materials and Methods. AS can be seen from Table 1, about 30% of the total TCA-precipitable radioactivity was precipitated by the antibody. Urnlabelled amyl-
Effect of Starvation on RNA and Protein Content and Digestive Enzyme Activities in the Rat Pancreas Table 2 summarizes the results of experiments in which the pancreatic content of RNA and protein, as well as the tissue chymotrypsin, trypsin, and amylolytic activities were measured in fed and fasted rats. Although a slight decrease in all parameters was observed, only amylase was significantly changed after fasting for 72 h. The enzyme activity was decreased about 50%. The possibility that this decrease was due to inhibition of the enzyme activity rather than to a decrease in the enzyme protein content was considered. That this was unlikely was shown by measuring the equivalence point of a preparation of amylase antibody using the tissue homogenate from both fasted and fed rats as the source of amylase (Fig. 2 ) . Twenty microlitres of amylase antibody preparation was incubated with 20-90 U of amylase from crude pancreatic extract derived from fasted and fed rats. T o provide the same amount of amylolytic activity in samples from fasted and fed animaIs, the volume of 'fasted' extract was twice that of 'fed' extract. After centrifugation of the antigen-antibody complex the amylolytic activity in the supernatant was measured and plotted against the antigen concentration added to the incubation mixture. Amylase activity appeared in the supernatant from both samples when the amount of amylase activity added exceeded 60 U; i.e., the equivalence point was the same using amylase f r ~ mthe pancreas of
VIEIRA-MATBS AND TENENHOUSE
TABLE 2. RNA and protein content, amylase, trypsin, and chymotrypsin activities in
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pancreas from fed and fasted rats
Fed Fasted
yochange
RNA, mg /rng DNA
Protein, rng /mg DNA
Amylase, U /mg DNA
Trypsin, , ~ /mg g DNA
Chymotrypsin, pg /mg DNA
2.53 + 0.32 (13) 2.02 f 0.28 (1 3) -20 NS
21.25 f 2 . 7 (10) 17.33 f 2 . 4 (109 -20 NS
1596.2 f 150 (30) 709.0 f 57 (30) -55 p < 0.01
145 f 28 (9) 109 f IS) (9) -25 NS
483 f 55 (9) 395 _+ 46 (9) -18 NS
NOTE:The values given are mean values -b SE; the number of determinations is given in parentheses. NS, not significant.
-
A M Y units added Supern. v o l . FD
5
10
6Q 15
-1 28
I00 25
0
FIG. 2. Determination of the equivalence point of the amylase antibody. Crude pancreatic extract from fed ( 0 ) and fasted ( x ) rats was used as antigen. Details of the procedure are given in the text.
fasted or fed rats. Thus, the decrease in the amylase activity found in the extract of pancreas from fasted rats was paralleled by a decrease in the amount of immunoprecipitable protein. The rate of incorporation of L-[3H]leucine into TCA-precipitable protein and immunoprecipitable amylase of rat pancreas tissue was measured as described in Materials and Methods. There was no difference between the rate of incorporation of L-[3M]leucine into total protein of pancreas from fasted and fed rats (Fig. 3.) Incorporation into immunoprecipitable amylase, however, was decreased almost 50% (Fig. 4). An attempt was made to determine the in v i t r ~rate of breakdown of newly synthesized total protein and irnmunoprecipitable amylase by measuring the rate of disappearance of radioactivity incorporated during pulse labelling of pancreatic tissue pieces from both fed and fasted rats. The tissue was incubated as described in Materials and Methods, except that
10
30
40
120
Time (mimB
FIG.3. Rate of incorporation of L-['H]leucine into total protein by pancreatic tissue pieces from fed ( 0 ) and fasted ( 0 ) rats. Each point is the mean ? SE of five experiments. The results are expressed as wanomoles of L-leucine incorporated into TCA-precipitable protein per milligram DNA.
a
.p_
5
a
a
I
9
O
W
30
68
126
FIG.4. Rate of incorporation of [3H]leucine into immunoprecipitable amylase by pancreatic tissue pieces from fed ( 8 ) and fasted ( ) rats. Each point is the mean + SE of five experiments. The results are expressed as nanomoles of L-leucine associated with irnmunoprecipitable amylase per milligram DNA.
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CAN. B. PHYSIOL. PHARMACOL.
FIG. 5. Radioactivity associated with total pancreatic protein during chase incubation of pancreatic tissue pieces fro111 fed ( F D ) and fasted ( F T ) rats. The incubation was done in the presence of 10 " A4 cycloheximide. The results are expressed as piconaoles of L-leucine associated with TCA-precipitable protein per milligram DNA. The data are from one representative experiment.
no L-[lHHqleucine was added to the medium and the concentration of L-["Hlleucine was 2.5 pCi/ml (specific activity of L-["Hqleucine, 64 Ci/rnmol). After 5 min the tissue pieces were washed with an excess incubation medium containing % mM L-['Hlleucine. The samples were then incubated for 4-8 h at 37 "C in fresh incubation medium containing 1 m M L-[~M]leucine. At the desired time the incubation was stopped by homogenizing the tissue in the incubation medium and the samples were treated as described in Materials and Methods to measure the radioactivity associated with TCAprecipitable ('total') protein and immunoprecipitable amylase, Cycloheximide ( 1 0 - W ) was used in some experiments to prevent reuse of any radioactive amino acid liberated during the incubation. As can be seen in Figs. 5 and 6, no detectable breakdown of newly synthesized protein or amylase occurred during incubation of tissue from fed or fasted rats during the time of incubation. In preliminary experiments it was found that gel filtration of the 105 000 X g supernatant from rat pancreas through a Sephadex (3-50 column separated the soluble pancreatic proteins into four fractions. Chymotrypsin and trypsin eluted together in the first protein peak, whereas amylase eluted in the third protein peak. Based on this observation an experiment was designed to determine the distribution of radioactivelly BabeIled proteins of the pancreas
VOL. 55, 1977
FIG. 6. Radioactivity associated with immunoprecipitable amylase during chase incubation of pancreatic tissue pieces from fed ( F D ) and fasted ( F T ) rats. The results are expressed as picsmoles of L-leucine associated with immunoprecipitable amylase per milligram DNA. The data are from one representative experiment.
and whether this distribution was altered by starvation. Pancreas pieces from both fcd and fasted rats were incubated as described in Materials and Methods, except that the tissue lroan fasted animals was incubated with L - [ ~ ~ C ] leucine (0.5 pCi/mI) and that from fed animals with L-[W]leucine ( 5 pCi/ml). The final leucine concentration in both incubation media was 1.8 mM. After 1 11 of incubation the tissues (and incubation media) from fed and fasted rats were pooled and homogenized. After 8 h of dialysis against 13 mi44 Tris-HC1, pH 8.3, and 3 mM CaCl.,, the homogenate was centrifuged at 105 000 X g for 2 h and the supernatant concentrated by ultrafihration using an Amicon ultrafiltration cell with a PM 10 membrane. Two inillilitres of the concentrated supernatant, containing 54 rng of protein, was applied to a 50 )< 2.8 cm Sephadex G-50 column which had been equilibrated with the Tris-HC1 buffer and the column eluted with the same buffer at a flow rate of 22.5 ml/h; 3-ml fractions were collected. The radioactivity in a 0.5-ml aliquot from each fraction eluted from the column was measured. The elution pattern of protein (optical density at 280 nm) and radioactivity (dpm) from one of these experiments is shown in Fig. 7. T o plot W and 14C radioactivity on the same scale, and thus facilitate comparison, the l 4 C values obtained for each fraction were divided by the 14C-3H ratio originally present in the sample applied to the column. The total recovery of radioactivity (both 3M and 14C) from
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VIELWA-MATOS AND TENENHOUSE
AMY T RY CHY
++ 4-4
++
---
F s g l r c t i an
Number
FIG.7. SepI-sadex 6 - 5 0 column chromatography of prelabelled 105 000 x g pancreatic supernatant from fed and fasted rats. The presence (++) or abscnce ( - - ) of amylase, trypsiw, and chyanotrypsin activities in the two main peaks are indicated. The incomqsoration of the radioactive amino acid is expressed as dpmiml of the sample. ( x 1, Optical clerasity at 280 wm; ( 0 ), 'H from fed rats, expressed as dpm; (e)),"C from fasted rats, expressed as dpm.
the co%umn was 95-98%. The radioactivity i n ~ c ~ r p c ~ r ainto t e d protein of pancreas from both fed and fasted rats consistently distributed between two fractions, the first, containing most of the radioactivity and all the trypsin and chyrnotrypsin activity and the third peak, cont a i n i n ~all the amylase activity, 30% of the total *'El counts, and about 15% of the I4C counts which eluted from the column. Thus, the same decrease (about 50% ) sf leucine incorporation into amylase by pancreas from fasted rats was found using the immunochemical method and column chromatography.
Discussion After a 72-h fast the rate of incorporation of [3H]Ieucine into immunoprecipitable amylase was decreased 5 0 % . Since there was no increase in the rate of an~y'lasebreakdown, this probably represents a decrease in the rate of amylase synthesis. Associated with this decrease in syant%nesiswas an equal ( 5 8 % ) decrease in total amylase activity within the tissue. In a series of preliminary experiments (not reported here), it was found that this decreased rate s f amylase synthesis was de-
tectable after 24-36 h of fasting and without any detectable decrease in total amylase activity; i.e., the change in rate of synthesis preceded and was the cause of the decrease in enzyme activity. The decrease in amylase synthesis occurred in the absence of any detectable change in the rate of synthesis of total pancreatic protein and witboaat any measurable decrease in the total activity of the other two pancreatic enzymes studied. There is no doubt that the assays used to measure trypsin and chymotrypsin were suiTiciently sensitive to detect a 25-50% decrease in activity. The fact that no significant decrease was found in a total of nine separate experiments indicates tlmat such a decrease did not occur. The effect of starvation on total protein synthesis is more diflicarlt to evaluate. In 'fed' rats, amylase accounts for about 30% of the total pancreatic protein synthesized in the in vitro system used in our studies. If only amylase synthesis was decreased, and if no other protein was being synthesized at an increased rate, then one would expect to find a decrease in total protein symthesis sf 155% after 72 h of starvation. It is possible that this change in total [3H]Beucine
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CAN. J. PHYSIOL. PHARMACOL. VQL. 55. I977
incorporation into total protein was not detected because of the lack of sensitivity of the method used. To improve our ability to detect small changes in the rate of synthesis of proteins other than amylase, we separated the soluble proteins of the rat pancreas homogenate into four fractions using Sephadex (3-50 column chron~atography and measured the ininto each fraction. corporation of ["]leucine All the amylase was recovered in a single protein peak separate from the peak which contained the trypsin and chymotrypsin activity. The 58% decrease in amino acid incorporation into amylase by the pancreas from starved rats was confirmed using this method; however, there was no consistent change in the radioactivity recovered in any of the other peaks, and no new radioactive peak was found. Since the recovery of radioactivity from these columns was 95-98%, it can be concluded that the incorporation of leucine into total protein (the sum of that incorporated into each peak) was decreased 15% by starvation, due entirely to the decrease in amylase synthesis. There are other factors which could contribute to the decrease in amylase activity in the pancreas after 72 h of starvation, e.g., increased rate of release of the enzyme, or an increased rate of destruction. This latter was demonstrated not to exist, at least when destruction is measured in vitro. We have not ruled out the possibility that during starvation amylase release from the gland is increased, but this seems highly unlikely. It is commonly believed that the exocrine pancreas contains a single population of zymogen granules, each containing a complement of all the secretory enzymes, and that during secretion the entire contents of the granule are discharged. If the rate of amylase release is increased such that the amylase content of the gland is significantly reduced, comparable decrease in gland content of the other secretory enzymes or an increased rate of synthesis of these proteins would be expected. We could find no evidence for either. Also, it seems unlikely that the depletion of an enzyme would result in a decrease in its synthesis; an increased rate of synthesis is a much more likely result. It must be concluded, therefore, that starvation for up to 72 h leads to a selective decrease in amylase synthesis in the rat exocrine pancreas. Although the mechanism of this effect is
not understood, it is clear from these results that, at least during starvation, the total amylase content of the tissue does not completely (if at all) control the rate of synthesis of the enzyme. AKIKUSA, Y. 1971. Effect of starvation on synthesis and release of growth hormone and prolactin in the rat anterior pituitary. Endocrinol. Jpn. 18, 41 1-414. ALI'FREY.V., DALY,M. M.. and M~RSKY, A. E. 1954. Synthesis of protein in the pancreas. II. The rolc of ribonucleoprotein in protein synthesis. J. Gen. Physiol. 37, 157-175. BERNFIELD, P. 1955. Amylase, a and p. Meth. Enzymol. 1, 149-150. BURTON,K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. CAMPBELL, D. H., JARREY, J. S., CREMER, N. E., and SUSSDORF, D. H. 1970. In Methods in immunology. W. A. Benjamin, Inc.. Melo Pk, CA. CHRIS'TOPHE, J., CAMUS,J., DISCHODT-LANCRMAN, M., RATHE,J., ROBBERECHT, P., VANDFRMEERS-PIKET, M. C., and VANDERMEERS, A. 1971. Factors regulating biosynthesis, intracellular transport and secretion of amylase and lipase in the rat exocrine pancreas. Horm. Metab. Res. 3, 3 9 3 4 0 % . DANIELSSOW, A., MARKLUND, S., and STPC~BRAND, T. 1974. Effects of starvation and islet hormones on the synthesis of amylase in isolated exocrine pancreas of the mouse. Acta Hepato-Gastroenterol. 21, 289297. DISCHE,Z. 1955. Color reaction of nucleic acid components. In The nucleic acids. Vol. 1. Edited by E. Chargaff and J. N. Davidson. Academic Press Inc., New York, N.Y. pp. 285-305. EAGLE,H. 1959. Amino acid metabolism in rnammalian cell cultures. Science, 130,432-437. EVANS,R. M., and SCHOLZ,W. W. 1971. Metabolic responses of chicks during adaptation to a high protein, 'bcarbohydrate-free" diet. J. Nutr. 181, 1127-1 136. HUMMEL,B. C. W. 1959. A modified spectrophotometric determination of chyrnotrypsin. trypsin, and thrombin. Can. J. Biochem. Physiol. 37, 1393-1399. LEE, P. C., and FISHER,J. R. 1971. Regulation of xanthine dehydrogenase levels in liver and pancreas of the chick. Biochim. Biophys. Acta, 237, 14-20. LOWRY,Q. H., ROSEBROUGH, N. J., FAWW, A. L., and RANDALL, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MELDOLESI, J. 1970. Effect of caerulein on protein synthesis and secretion in the guinea-pig pancreas. BP.J. Pharmacol. 40,721-73 1. MORISSET, J. A., and WEBSTER,P. B. 1971. In vitrcl and in vivo effects of pancreozymin, urecholine, and cyclic AMP on rat pancreas. Am. J. PfiysioI. 270, 202-208. 1972. Effects of fasting and feeding on protein synthesis by the rat pancreas. J. CLin. Invest. 51, 1-8.
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REID, I. M., VERNEY,E., and SIDRANSKY, N. 1970. Influence of acute nutritional stress on poIyribosomes and protein synthesis in brain and liver of young rats. J . Nutr. 100, 1149-1 156. SCHIMKE,R. T., and DOYLE, D. 1970. Control of enzyme levels in animal tissues. Annu. Rev. Biochem. 39,929-976. SIEKEVITZ.P. 1952. Uptake of radioactive alanine in vitro into the proteins o n rat liver fractions. J. Riol. Chem. 195,549-565. VANDERMEERS, A., and CHRISTOPHE,J. 1968. aAmylase et lipase du pancreas de rat. Purification chromatographique, recherche du poids moleculaire e t composition en acides amines. Biochim. Biophys. Acta, 154, 110-129.
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VEGBEI,YL,P. V., and KEMENY,T. T. 2962. Protein metabolism and pancreatic function. In Ciba Foundation Symposium on the exocrine pancreas. Edited by A. V. S. Reuck and M. P. Cameron. A. and J. Churchill, London. pp. 329-352. WEBSTER,P. D . 1969. Hormonal control of pancreatic acinar cell metabolism. I n The exocrine glands. Edited b y S. Y . Rotclh, F. P. Brooks, and W. B. Shelley. University of Pennsylvania Press, Philadelphia, PA. pp. 153-168. WEBSTER,P. D., SINGH, M., TUCKER,P. C., and BLACK,0. 1972. Effects of fasting and feeding on the pancreas. Gastroenterology, 62, 600-605.
