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Ethanol withdrawal stimulates protein synthesis in rat pancreatic lobules
Biochimica et Biophysica Acta, 1036 (1990) 107-112
107
Elsevier BBAGEN 23397
Ethanol withdrawal stimulates protein synthesis in rat pancreatic lobules Biddanda C. Ponnappa, Jan B. Hoek, Leslie Jubinski and Emanuel Rubin Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA (U.S.A.)
(Received20 February 1990) (Revised manuscript received30 April 1990)
Key words: Ethanol withdrawal; Pancreaticlobule; Protein synthesis
Recent studies from our laboratory (Ponnappa et al. (1988) Biochim. Biophys. Acta 966, 390-402), indicate that in the pancreas of rats fed ethanol chronically, an overnight withdrawal of ethanol stimulated the rate of protein synthesis in vivo, whereas, during continuous ethanol ingestion, the rate of protein synthesis was the same as in the control group which did not receive ethanol. However, a stimulation of protein synthesis was also observed when isolated acini were prepared from the pancreas of continuously ethanol-fed rats. In the present studies, preparations of pancreatic lobules were used to further characterize the stimulatory effect observed in the ethanol-fed group. The rate of protein synthesis was studied in vitro by determining the rate of incorporation of [3H]leucine into proteins. Similar to in vivo observations, chronic ethanol feeding did not alter the rate of protein synthesis, but an overnight withdrawal of ethanol stimulated the rate of protein synthesis by 84%. The stimulation of protein synthesis reflected a general enhancement in the rate of synthesis of most of the digestive enzymes ranging from 60 to 110%. The maximal stimulation of protein synthesis occurred within 24 h of ethanol withdrawal and the rates rapidly decreased to control levels within 3 days. During the overnight ethanol withdrawal there was also a 30-40% decrease in the activities of most of the pancreatic digestive enzymes. This observation indicates that ethanol withdrawal also initiated the secretion and/or degradation of pancreatic digestive enzymes in vivo. The observation that the enhanced rate of protein synthesis can be observed in isolated acini but not in vivo or in lobular preparations from the continuously ethanol-fed rats indicates that the pancreas contains factors which supress this stimulatory effect of ethanol intake. The stimulation of protein synthesis, brought about either by ethanol withdrawal or by collagenase digestion of the tissue, may reflect the removal of such factors.
Introduction Excessive intake of alcohol often leads to the development of pancreatitis in man [1,2]. However, the biochemical events that precede the development of the disease are poorly understood. In experimental animals such as the rat, long-term oral administration of ethanol, provided in a liquid diet, did not induce pancreatitis [3]. By contrast, in the intragastric rat model developed by Tsukamoto et al. [4], sustained infusion of ethanol was reported to induce pancreatitis in about 20% of the rats in less than 5 months. Differences in the physiologic
Abbreviations: HRL, Hepes-buffered Ringer's solution containing 0.1% bovine serum albumin; CCK, cholecystokinin. Correspondence: B.C. Ponnappa, Department of Pathology and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, U.S.A.
response of the animals to oral and intragastric ethanol intoxication may have contributed to the development of pancreatitis in the intragastric model. In a recent report [5], we observed that in the pancreas of rats fed ethanol chronically, an overnight withdrawal of ethanol stimulated the rate of protein synthesis in vivo by 70%. By contrast, in the absence of ethanol withdrawal, that is, in the continuously ethanolfed rats, there was no change in the in vivo rate of protein synthesis when compared to the control group. However, in the same study [5], when isolated pancreatic acini were prepared, a stimulation (2-3-fold) of protein synthesis was also observed in vitro, in acini, from the continuously ethanol-fed rats. These observations suggested that withdrawal of ethanol overnight or collagenase digestion of the pancreas of chronically ethanol-fed rats might remove certain inhibitory factors, leading to stimulation of protein synthesis. We undertook the present studies to investigate further, the cir-
0304-4165/90/$03.50 © 1990 ElsevierScience Publishers B.V. (BiomedicalDivision)
108 cumstances which trigger the stimulation of protein synthesis during ethanol withdrawal. We used pancreatic lobules to monitor protein synthesis, since they showed properties similar to those observed in vivo during ethanol feeding and withdrawal [5]. The results indicate that an induction of digestive enzyme secretion precedes the stimulation of protein synthesis upon withdrawal of ethanol, and that intrapancreatic factors may play a role in the control of these processes. Materials and Methods
Animals Maximum Barrier Maintained male littermate Sprague-Dawley rats (Zivic-Miller Laboratories, Zelienopole, PA), in the weight range of 120-130 g, were pair-fed the control and the ethanol-containing liquid diets [6] (Bioserv, Frenchtown, N J) as described elsewhere [5]. The relative distribution of total calories in the ethanol-containing diet was, 18% protein, 35% fat, 11% carbohydrate and 36% ethanol. The control group had a similar composition except that carbohydrate replaced ethanol. Rats were maintained on the respective diets for periods ranging from 6-8 weeks, during which time the intake of ethanol averaged 12-14 g / k g body weight per day. During the first week of ethanol ingestion the animals maintained their body weights. Subsequently, the weight gain averaged 4-6 g per day, and at any time the total body weights of the ethanol-treated animals were within 90-95% of their pair-fed controls. Routinely, the animals were fed between 2-3 p.m. and were killed between 9-9:30 a.m. Determination of protein synthesis in pancreatic lobules Pancreatic lobules were prepared according to the procedure of Scheele and Palade [7]. Briefly, after the rat was killed, the pancreas was quickly removed and placed in ice-cold isotonic saline. After the removal of fat and ductal tissue, the pancreas was rinsed several times in saline and injected with an oxygenated, Hepesbuffered, Ringer's solution described previously [5]. The content of bovine serum albumin (BSA) was reduced to 0.1%. Hereafter, this buffer will be referred to as HRL. Small pieces of pancreas (1-2 mg protein) consisting of lobules, were cut out and rinsed in HRL. Lobules (10-20 mg protein per 10 ml of HRL) were preincubated for 45 min at 37 o C in a shaking (80 cycles/rain) waterbath, rinsed and resuspended in a similar volume of HRL. After a brief warm-up period, [3H]leucine (5 /,Ci/ml) was added and the contents incubated for predetermined intervals of time. The incorporation of the label was stopped by diluting the medium with three times the volume of ice-cold HRL, followed by immediate removal of the medium. The lobules were further washed three times with ice-cold H R L and twice more with ice-cold isotonic saline. Subsequently, the
tissue was homogenized in Tris buffer (25 mM, pH 8.9) containing 0.1% Triton X-100, such that the final concentration of protein in the homogenate was 4-5 mg/ml. After taking aliquots for the determination of protein, DNA and TCA-precipitable radioactivity, the rest of the homogenates were kept frozen at - 7 0 ° C . For the determination of the rate of total protein synthesis, the homogenates were diluted with water to a concentration of approx. 0.5 mg protein/ml, briefly sonicated and processed as described previously for acini [5]. However, for studies involving the determination of the synthetic rates of individual digestive enzymes, proteinase inhibitors [5] were included in the homogenizing medium. The homogenates were then centrifuged at 100 000 × g for 30 min to remove the particulate matter. The extent of incorporation of the label into individual digestive enzymes was determined in the soluble (supernatant) fraction, following separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described earlier [5]. Greater than 80% of the radioactivity associated with the TCAprecipitable component of the 'soluble fraction' was recovered among the digestive enzymes in the range of 12-55 kDa molecular mass. The extent of recovery was similar for both control and ethanol-fed groups.
DNA assay Aliquots of the pancreatic homogenates, containing 20-40 /~g DNA, were taken for extraction [8] and determination [9]. Standard DNA (calf thymus, DNA, Sigma Chemicals) in the concentration range of 0-60 /,g was similarly processed. Enzyme assays Amylase was assayed by the method of Rinderknecht [10] and lipase by the method of Tietez [11], using Sigma Kit 8500 with modifications as described elsewhere [12]. In these studies, a unit of amylase is defined as that activity of the enzyme at 25 °C and at pH 6.9 that liberates 1 mg of maltose from starch in 3 min. A unit of lipase is equivalent to the liberation of 1 /~mol of free fatty acids per min per mg protein. Trypsinogen and chymotrypsinogen, after activation with enterokinase and trypsin, respectively, were assayed by the method of Hummel [13]. Trypsin activity was expressed as T A M E (p-toluenesulphonyl-L-arginine methyl ester) units; 1 unit catalyzed the hydrolysis of 1 /,mol of TAME per min at 25 ° C. Similarly, chymotrypsin activity was expressed in BTEE (N-benzoyl-Ltyrosine ethyl ester) units, where 1 unit catalyzed the hydrolysis of 1 ~tmol of BTEE per min at 25 o C. Determination of protein Protein was assayed according to the procedure of Lowry et al. [14], using bovine serum albumin as the standard.
109
Statistical analysis Statistical analysis was carried out by the paired t-test. Materials L-[2,3,4,5-3H]Leucine, spec. act. 110 Ci/mmol, was purchased from ICN Radiochemicals, Irvine, CA. Electrophoresis chemicals were purchased from the following companies: Hoeffer Scientific, San Francisco; Fisher Scientific; and Eastman Kodak. All biochemicals were generally obtained from Sigma Chemicals. All other chemicals used were of reagent grade and obtained from Sigma (St. Louis, MO) or Fisher Scientific (King of Prussia, PA).
T A B L E II
Effect of ethanol feeding and withdrawal on pancreatic protein content Rats were maintained on control and ethanol-containing diets for 6 - 8 weeks. Pancreatic protein and D N A contents were determined before (0 days) and after ethanol withdrawal. All values are the m e a n s + S.E. of (n) pairs of animals. Duration of ethanol withdrawal (days)
Control
0(4) 1(5) 2(4) 3(3) 5(3)
34.14 + 35.35 + 38.45 + 35.86 + 40.85 +
Ethanol
(protein/DNA; mg/mg) 2.34 1.21 2.04 2.7 1.29
33.98 + 1.00 34.26 + 0.28 36.39 + 0.91 35.87 + 2.34 38.50+0.97
Results
Ethanol withdrawal and protein synthesis In pancreatic lobules from animals fed ethanol chronically, the rate of protein synthesis was comparable to preparations from pair-fed control animals (Table I). However, an overnight withdrawal of ethanol, induced by substituting the control diet for the ethanolcontaining one, increased the rate of protein synthesis (Table I). After more prolonged withdrawal this stimulatory effect rapidly decreased, and the rate of protein synthesis declined to control values by the third day (Table I). Since protein synthesis was measured by the rate of incorporation of [3H]leucine per mg protein, it was also necessary to determine the total protein content per cell in order to normalize the rates between the two groups. There was no significant difference in total protein content per cell (DNA) between the control and ethanol-fed groups under all conditions of treatment
(Table II). Regarding the specific activity of leucine pools, in the present studies a constant extracellular specific activity of [3H]leucine was maintained. In our previous study [5], we had already established that under conditions where stimulation of protein synthesis was observed in the ethanol-fed group, either in vivo or in vitro, there was no difference in the intracellular specific activity of [3H]leucine between the control and the ethanol-fed groups. Based on these observations, it can be concluded that the stimulation of protein synthesis observed in the present studies is also not due to a difference in the specific activity of leucine pools.
250 o 4,--
§ 200
150 c 0
g
TABLE I
Effect of ethanol feeding and duration of withdrawal on pancreatic protein synthesis Littermate rats were maintained on control and ethanol-containing diets for 6 - 8 weeks. The rate of incorporation of [3H]leucine (5 /~Ci/ml) into proteins was determined in the pancreatic lobules of animals before (0 days) or after 1 (18 h) 2, 3 and 5 days of ethanol withdrawal. Ethanol withdrawal was initiated by providing control diet to the ethanol-fed group. Values are the means_+ S.E. of (n) pairs of animals.
IO0
~' 5o if)
-~
0
a.
'~ -50
-I00
Duration of ethanol with-
Incorporation of [3 H]leucine (cpm × 1 0 - 3 / m g protein per 20 min)
drawal (days)
control
ethanol
% change
significance
0 (5) 1 (7) 2 (4) 3 (4) 5 (4)
11.68 + 1.41 12.31+0.85 11.20 + 1.73 11.31 +0.99 11.04+0.66
12.22 -t- 1.01 22.67+1.67 14.72 + 2.12 12.06+0.46 10.55+2.00
5 84 31 7 -4
N.S. P <0.01 P < 0.05 N.S. N.S.
N.S. = not significant.
N
I
i
I
I
I
I
0
I
2
3
4
5
Ethanol Withdrawal (days) Fig. 1. The effect of chronic ethanol feeding and withdrawal on the rate of pancreatic digestive enzyme synthesis. Littermate rats were maintained on control and ethanol-containing diets for 6 - 8 weeks. The rate of synthesis of individual digestive enzymes was determined in pancreatic lobules from the control and the ethanol-fed groups before (day 0) and after ethanol withdrawal as described in Materials and Methods. O, amylase; zx, chymotrypsinogen; O, lipase; and o, trypsinogen. *P < 0.005-0.02.
110 T A B L E III
Effect of ethanol feeding on the enzyme activities and the in vitro rate of synthesis of pancreatic digestive enzymes Littermate rats were maintained on control and ethanol-containing liquid diets for 6 - 8 weeks. Tissue levels of digestive enzymes and the in vitro rate of incorporation of [3H]leucine into individual digestive enzymes and total proteins were determined in pancreatic lobules as described in Materials and Methods. Digestive enzyme
Amylase Lipase Chymotrypsinogen Trypsinogen Carboxypeptidase Proelastase
Enzyme activity ( U / m g protein)
[3 H]leucine incorporation (cpm) a
control
ethanol
% change
control
104.02 + 13.91 20.21+ 2.31 1.99_+ 0.19 4.37± 0.52 -
12.32 ± 1.26 31.14±2.30 3.53_+0.36 5.43_+0.45 -
- 88 54 77 24 -
1 955 505 581 869 1053 306
Protein synthesis (total)
ethanol + 333 _+ 48 _+ 60 _+ 98 ± 123 ± 17
634 728 991 1025 1 358 442
% change ± 37 ± 34 ± 93 ±118 ± 117 _+ 63
- 68 44 71 17 29 44
(cpm × 1 0 - 3/rag protein per 20 min) 11.68± 1.40 12.22± 1.01
a Indicates the amount of label (cpm) associated with individual digestive enzymes when 80 #g particulate-free protein were separated by SDS-PAGE. All values are the m e a n ± S.E. of (4-6) pairs of determinations.
In order to relate the increase in total protein synthesis to the synthesis of digestive enzymes, the synthetic rates of individual digestive enzymes were determined in pancreatic lobules at various stages of ethanol withdrawal. As shown in Table III, the rates of synthesis of all the proteolytic enzymes tested were higher in the ethanol-fed group during active ingestion of ethanol (see also Fig. 1, day 0), with chymotrypsinogen exibiting the highest rate (+ 77%). The rate of synthesis of lipase was also higher, while that of amylase was much lower (-68%) than in the control group. By contrast, after 1 day of ethanol withdrawal, there was a general increase in the synthesis of all of the digestive enzymes (Fig. 1). At the end of 3 days, with the exception of amylase, the synthetic rates of individual enzymes had returned to control values.
Ethanol withdrawal and tissue levels of digestive enzymes In order to determine whether the synthetic rates of digestive enzymes reflected the tissue levels of these proteins, the activities of some of these enzymes were measured. During continuous ethanol ingestion (i.e., before ethanol withdrawal), the steady-state levels of all of the digestive enzymes correlated well with their rates of synthesis (Table III). By contrast, after 1 day of ethanol withdrawal, when the rate of protein synthesis was maximal, there was a 30-40% reduction in the tissue levels of almost all of the enzymes tested (Figs. 1 and 2), amylase being the exception (see Discussion). Again, as in the case of their synthesis, after 3 days of ethanol withdrawal the elevated levels of some of the digestive enzymes (chymotrypsinogen, lipase and trypsinogen) reverted to control values and, after 5 days, remained at or below the levels of the enzymes in the control group (Figs. 1 and 2).
In order to distinguish the stimulatory effect induced by ethanol withdrawal from that due to dietary change, the effects of ethanol withdrawal were also studied after inducing withdrawal by overnight starvation. As indicated in Table IV, the results were similar to those observed when ethanol withdrawal was initiated by providing the control diet (Table I and Figs. 1 and 2), although the extent of stimulation of protein synthesis was lower (46 vs. 84%). These results suggest that stimulation of protein synthesis is a function of ethanol I00
A O
§ so g c u
:
~ -50 N C W
-IO0
I
I
I
I
i
I
0
I
2
3
4.
5
Ethanol Withdrawal (days)
Fig. 2. The effect of chronic ethanol feeding and withdrawal on tissue levels of pancreatic digestive enzymes. Littermate rats were treated as detailed in legend to Fig. 1. Tissue levels of digestive enzymes were determined before and after ethanol withdrawal as detailed in Materials and Methods. Symbols per Fig. 1; *P < 0.005-0.05.
111 T A B L E IV
Effect of ethanol withdrawal by starvation on the activities and rate of synthesis of pancreatic digestive enzymes Experimental details are similar to that described in legend to Table III except that animals from both control and ethanol-fed groups were fasted overnight, water being provided. All values are the m e a n s + S.E. of four pairs of animals. Digestive enzyme
Amylase Lipase Chymotrypsinogen Trypsinogen Carboxypeptidase Proelastase
Protein Synthesis (Total)
Enzyme activity ( U / m g protein)
[3 H]leucine incorporation (cpm) a
control
ethanol
%change
control
117.32+7.56 25.32_+2.93 2.24 + 0.21 4.32 + 0.41 -
21.98+4.34 24.90+1.52 3.41 + 0.21 4.25 ± 0.67
-81 -2 52 - 2 -
1983 404 747 992 1169 317
-
-
-
ethanol +226 ± 31 _+ 60 ± 160 ± 121 _+ 19
972 710 1860 1 405 1936 494
% change +127 5:120 ± 237 ± 210 ± 256 ± 51
(cpm × 1 0 - 3 / m g protein per 20 rain) 10.11_+ 1.01 14.80_+ 2.83
-51 76 149 42 76 56
46
a Indicates the amount of label (cpm) associated with individual digestive enzymes when 80 /~g particulate-free protein were separated by SDS-PAGE.
withdrawal per se and not simply the consequence of a dietary change. Discussion
Under physiological conditions the turnover of individual digestive enzymes in the pancreas is likely to be influenced by specific gastrointestinal hormones, such as cholecystokinin (CCK) [15-17], secretin [18], insulin [19,20] or a combination of these [16,21]. Cholinergic mechanisms may also be involved [22]. Chronic ethanol administration has been reported to alter the tissue levels of some of the pancreatic digestive enzymes [3,12,23-25]. It is, therefore, possible that ethanol in the diet might interfere with the regulatory mechanisms that control the levels of some of the digestive enzymes, thereby leading to altered tissue levels of these proteins. There is indeed evidence to show that ethanol administration stimulates the release of CCK [26]. Treatment with low levels (0.25 /~g/kg per h) of caerulein, an analogue of CCK, causes alterations in the synthesis of digestives enzymes [15], while a sustained infusion of high levels (5/~g/kg per h) has been reported to induce pancreatitis [27]. Whether alcohol-related pancreatitis is associated with sustained high circulating levels of CCK has not yet been determined. Against the background of a lack of tissue injury during long-term ethanol ingestion, the stimulation of pancreatic protein synthesis during ethanol withdrawal is of interest. We have recently reported [5] an enhanced rate of protein synthesis in pancreatic acini isolated from animals which had continuously ingested ethanol, even though no changes were observed in vivo. By contrast, after an overnight withdrawal of ethanol, a stimulation of protein synthesis occurred in vivo. In the pancreatic lobules of this study, maximal stimulation of
protein synthesis occurred within 24 h of ethanol withdrawal, followed by a rapid decline to control values during the next 48 h. Thus, the lobular preparation mimicked the responses observed in vivo. It is unlikely that the mere removal of ethanol from the tissue could have initiated the stimulatory process, since lobules prepared from ethanol-fed animals before withdrawal were washed several times with the incubation medium to remove ethanol, and did not show any stimulation of protein synthesis. Moreover, in our earlier studies [5] on isolated acini, ethanol (100 mM) added in vitro had little effect on the rate of protein synthesis in preparations from the ethanol-fed group. It is also unlikely that a change from the ethanol-containing diet to the control diet could have triggered increased protein synthesis, since ethanol withdrawal by starvation led to a similar increase (Table IV). These observations suggest that it is the physiological removal of ethanol that alters the regulation of protein synthesis in the exocrine pancreas. The stimulation of total protein synthesis by ethanol withdrawal was associated with a parallel increase in the rate of synthesis of digestive enzymes. This observation is in keeping with earlier reports that pancreatic protein synthesis generally reflects the synthesis of digestive enzymes [5,28]. However, with the exception of amylase, the maximal stimulatory effect on protein synthesis coincided with a 30-40% reduction in the tissue levels of digestive enzymes (Figs. 1 and 2). Short-term loss of large amounts of digestive enzymes from the pancreas could be due either to an increased secretion or degradation. In our previous report [5], in isolated pancreatic acini from ethanol-fed animals, stimulation of protein synthesis was not associated with a parallel increase in secretion or intracellular degradation of digestive enzymes. It is, therefore, likely that the reduction in the tissue levels of digestive enzymes occurred as
112 a result of increased pancreatic secretion in vivo during ethanol withdrawal. There are several reasons for the lack of change in the level of amylase during the first day of ethanol withdrawal. Firstly, the a m o u n t of carbohydrate, a determining factor for the induction of amylase synthesis [29], is low in the ethanol-containing diet (11% compared to 47% in the control group). During ethanol withdrawal, the provision of high levels of carbohydrate is likely to enhance the signal for the synthesis of amylase. Secondly, the addition of ethanol to the diet, particularly at low levels of carbohydrate, further depresses the levels of amylase in the pancreas [12]. This leads to the low steady-state levels of amylase during ethanol feeding. Withdrawal of ethanol removes the suppressive effects of ethanol ingestion on amylase levels [12], thereby leading to increased synthesis. Finally, the factors responsible for inducing the general increase in the synthesis of other digestive enzymes p r o b a b l y also contribute to the enhanced synthesis of amylase. Since the initial level of amylase is low (unlike that of other digestive enzymes), even if there was an initial loss of amylase during ethanol withdrawal, the a c c o m p a n y i n g signals for an increased synthesis more than compensate for the loss. The present study demonstrates that chronic ethanol ingestion modifies the regulation of pancreatic protein synthesis and secretion. However, such modifications, for most part, remain latent until perturbed by processes such as ethanol withdrawal (in lobules) or collagenase digestion of the tissue (in isolated acini). The biochemical mechanism by which the stimulation of protein synthesis occurs under various conditions in the ethanol-fed animals is not clear. Whereas the isolated acini from the animals continuously fed ethanol showed an enhanced rate of protein synthesis [5], there was no parallel increase in the rate of protein synthesis in the unprocessed tissue of the same group (Table I). One of the differences between isolated acini and the intact tissue, represented by the lobules, is that the acini do not contain the islet cells and hence are not under the active influence of these cells. The islet cells secrete peptide hormones, such as pancreatic polypeptide, glucagon and somatostatin, which are k n o w n to inhibit the effects of C C K and secretin [30]. The stimulation of protein synthesis observed upon isolation of the acini or withdrawal of the animal from ethanol, m a y reflect the removal of the suppressive effects of these inhibitors, which would otherwise compensate for an excessive release of an agonist like C C K in the ethanol-fed animals.
Acknowledgements This work has been supported in part by grants AA07571, AA07186 and AA07215 from the National Institute of Alcoholism and Alcohol Abuse.
References 1 Durbec, J.P. and Sarles, H. (1978) Digestion 18, 337-350. 2 Singh, M. (1986) in The Exocrine Pancreas: Biology, Pathology and Diseases (Go, V.L.W. et. al., eds.), pp. 423-442, Raven Press, New York. 3 Singh, M., LaSure, M.M. and Bockman, D.D. (1982) Gastroenterology 82, 425-434. 4 Tsukamoto, H., Towner, S.J., Yu, G.S.M. and French, S.W. (1988) Am. J. Pathol. 131, 246-257. 5 Ponnappa, B.C., Hoek, J.B., Jubinski, L. and Rubin, E. (1988) Biochim. Biophys. Acta 966, 390-402. 6 DeCarli, L.M. and Lieber, C.S. (1967) J. Nutr. 91, 331-336. 7 Scheele, G.A. and Palade, G.E. (1975) J. Biol. Chem. 250, 26602670. 8 Munro, H.N. and Fleck, A. (1966) Analyst 91, 78-88. 9 Burton, K. (1956) Biochem. J. 62, 315-323. 10 Rinderknecht, J., Wilding, P. and Haverback, B.J. (1967) Experientia 23, 805. 11 Tietz, N.W. and Fiereck, E.A. (1966) Clin. Chim. Acta 13, 352-53. 12 Ponnappa, B,C., Hoek, J.B., Sarchet, K. and Rubin, E. (1986) J. Lab. Clin. Med. 107, 556-562. 13 Hummel, B.C.W. (1959) Can. J. Biochem. Physiol. 37, 1393-1399. 14 Lowry, O.H., Roseborough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 15 Schick, J., Versphol, R., Kern, H. and Scheele, G.A. (1984) Am. J. Physiol. 138, 676-682. 16 Solomon, T.E., Peterson, H., Elashoff, J. and Grossman, M.I. (1978) Am. J. Physiol. 235, E714-E719. 17 Otsuki, M., Okabayashi, Y., Ohki, A., Hootman, S.R., Baba, S. and Williams, J.A. (1984) Am. J. Physiol. 246, G419-G425. 18 Rauch, U., Vasiloudes, P., Rudiger, K. and Kern, H.F. (1985) Cell Tissue Res. 242, 641-644. 19 Okabayashi, Y., Mossner, J., Logsdon, C.A., Goldfine, I.A. and Williams, J.A. (1987) Diabetes 36, 1054-60. 20 Korc, M., Owerback, D., Quinto, C. and Rutter, W.S. (1981) Science 213, 351-353. 21 Dagorn, J.C. and Mongeau, R. (1977) Biochim. Biophys. Acta 498, 76-82. 22 Scheele, G.A. (1981) Methods Cell Biol. 23, 345-358. 23 Chariot, J., LeBourhis, B., Rose, C. and Potet, F. (1980) Dig. Dis. Sci. 25, 656-659. 24 Sarles, H., Figarella, C. and Clemente, F. (1971) Digestion 4. 13-22. 25 Tsukamoto, H., Delgado, G., Reidelberger, R.D. and Largman, C. (1986) Biochem. Pharmacol. 35, 3623-3629. 26 Liddle, R.A., Goldfine, I.D. and Williams, J.A. (1984) Gastroenterology 87, 542-549. 27 Steer, M.L., Meldolesi, J. and Figarella, C. (1984) Dig. Dis. Sci. 29, 934-938. 28 Case, R.M. (1978) Biol. Rev. 53, 211 354. 29 Grossman, M.I., Greengard, H. and Ivy, A.C. (1943) Am. J. Physiol. 138, 676-682. 30 Henderson, J.R., Daniel, P.M. and Fraser, P.A. (1981) Gut 22, 158-167.
107
Elsevier BBAGEN 23397
Ethanol withdrawal stimulates protein synthesis in rat pancreatic lobules Biddanda C. Ponnappa, Jan B. Hoek, Leslie Jubinski and Emanuel Rubin Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA (U.S.A.)
(Received20 February 1990) (Revised manuscript received30 April 1990)
Key words: Ethanol withdrawal; Pancreaticlobule; Protein synthesis
Recent studies from our laboratory (Ponnappa et al. (1988) Biochim. Biophys. Acta 966, 390-402), indicate that in the pancreas of rats fed ethanol chronically, an overnight withdrawal of ethanol stimulated the rate of protein synthesis in vivo, whereas, during continuous ethanol ingestion, the rate of protein synthesis was the same as in the control group which did not receive ethanol. However, a stimulation of protein synthesis was also observed when isolated acini were prepared from the pancreas of continuously ethanol-fed rats. In the present studies, preparations of pancreatic lobules were used to further characterize the stimulatory effect observed in the ethanol-fed group. The rate of protein synthesis was studied in vitro by determining the rate of incorporation of [3H]leucine into proteins. Similar to in vivo observations, chronic ethanol feeding did not alter the rate of protein synthesis, but an overnight withdrawal of ethanol stimulated the rate of protein synthesis by 84%. The stimulation of protein synthesis reflected a general enhancement in the rate of synthesis of most of the digestive enzymes ranging from 60 to 110%. The maximal stimulation of protein synthesis occurred within 24 h of ethanol withdrawal and the rates rapidly decreased to control levels within 3 days. During the overnight ethanol withdrawal there was also a 30-40% decrease in the activities of most of the pancreatic digestive enzymes. This observation indicates that ethanol withdrawal also initiated the secretion and/or degradation of pancreatic digestive enzymes in vivo. The observation that the enhanced rate of protein synthesis can be observed in isolated acini but not in vivo or in lobular preparations from the continuously ethanol-fed rats indicates that the pancreas contains factors which supress this stimulatory effect of ethanol intake. The stimulation of protein synthesis, brought about either by ethanol withdrawal or by collagenase digestion of the tissue, may reflect the removal of such factors.
Introduction Excessive intake of alcohol often leads to the development of pancreatitis in man [1,2]. However, the biochemical events that precede the development of the disease are poorly understood. In experimental animals such as the rat, long-term oral administration of ethanol, provided in a liquid diet, did not induce pancreatitis [3]. By contrast, in the intragastric rat model developed by Tsukamoto et al. [4], sustained infusion of ethanol was reported to induce pancreatitis in about 20% of the rats in less than 5 months. Differences in the physiologic
Abbreviations: HRL, Hepes-buffered Ringer's solution containing 0.1% bovine serum albumin; CCK, cholecystokinin. Correspondence: B.C. Ponnappa, Department of Pathology and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, U.S.A.
response of the animals to oral and intragastric ethanol intoxication may have contributed to the development of pancreatitis in the intragastric model. In a recent report [5], we observed that in the pancreas of rats fed ethanol chronically, an overnight withdrawal of ethanol stimulated the rate of protein synthesis in vivo by 70%. By contrast, in the absence of ethanol withdrawal, that is, in the continuously ethanolfed rats, there was no change in the in vivo rate of protein synthesis when compared to the control group. However, in the same study [5], when isolated pancreatic acini were prepared, a stimulation (2-3-fold) of protein synthesis was also observed in vitro, in acini, from the continuously ethanol-fed rats. These observations suggested that withdrawal of ethanol overnight or collagenase digestion of the pancreas of chronically ethanol-fed rats might remove certain inhibitory factors, leading to stimulation of protein synthesis. We undertook the present studies to investigate further, the cir-
0304-4165/90/$03.50 © 1990 ElsevierScience Publishers B.V. (BiomedicalDivision)
108 cumstances which trigger the stimulation of protein synthesis during ethanol withdrawal. We used pancreatic lobules to monitor protein synthesis, since they showed properties similar to those observed in vivo during ethanol feeding and withdrawal [5]. The results indicate that an induction of digestive enzyme secretion precedes the stimulation of protein synthesis upon withdrawal of ethanol, and that intrapancreatic factors may play a role in the control of these processes. Materials and Methods
Animals Maximum Barrier Maintained male littermate Sprague-Dawley rats (Zivic-Miller Laboratories, Zelienopole, PA), in the weight range of 120-130 g, were pair-fed the control and the ethanol-containing liquid diets [6] (Bioserv, Frenchtown, N J) as described elsewhere [5]. The relative distribution of total calories in the ethanol-containing diet was, 18% protein, 35% fat, 11% carbohydrate and 36% ethanol. The control group had a similar composition except that carbohydrate replaced ethanol. Rats were maintained on the respective diets for periods ranging from 6-8 weeks, during which time the intake of ethanol averaged 12-14 g / k g body weight per day. During the first week of ethanol ingestion the animals maintained their body weights. Subsequently, the weight gain averaged 4-6 g per day, and at any time the total body weights of the ethanol-treated animals were within 90-95% of their pair-fed controls. Routinely, the animals were fed between 2-3 p.m. and were killed between 9-9:30 a.m. Determination of protein synthesis in pancreatic lobules Pancreatic lobules were prepared according to the procedure of Scheele and Palade [7]. Briefly, after the rat was killed, the pancreas was quickly removed and placed in ice-cold isotonic saline. After the removal of fat and ductal tissue, the pancreas was rinsed several times in saline and injected with an oxygenated, Hepesbuffered, Ringer's solution described previously [5]. The content of bovine serum albumin (BSA) was reduced to 0.1%. Hereafter, this buffer will be referred to as HRL. Small pieces of pancreas (1-2 mg protein) consisting of lobules, were cut out and rinsed in HRL. Lobules (10-20 mg protein per 10 ml of HRL) were preincubated for 45 min at 37 o C in a shaking (80 cycles/rain) waterbath, rinsed and resuspended in a similar volume of HRL. After a brief warm-up period, [3H]leucine (5 /,Ci/ml) was added and the contents incubated for predetermined intervals of time. The incorporation of the label was stopped by diluting the medium with three times the volume of ice-cold HRL, followed by immediate removal of the medium. The lobules were further washed three times with ice-cold H R L and twice more with ice-cold isotonic saline. Subsequently, the
tissue was homogenized in Tris buffer (25 mM, pH 8.9) containing 0.1% Triton X-100, such that the final concentration of protein in the homogenate was 4-5 mg/ml. After taking aliquots for the determination of protein, DNA and TCA-precipitable radioactivity, the rest of the homogenates were kept frozen at - 7 0 ° C . For the determination of the rate of total protein synthesis, the homogenates were diluted with water to a concentration of approx. 0.5 mg protein/ml, briefly sonicated and processed as described previously for acini [5]. However, for studies involving the determination of the synthetic rates of individual digestive enzymes, proteinase inhibitors [5] were included in the homogenizing medium. The homogenates were then centrifuged at 100 000 × g for 30 min to remove the particulate matter. The extent of incorporation of the label into individual digestive enzymes was determined in the soluble (supernatant) fraction, following separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described earlier [5]. Greater than 80% of the radioactivity associated with the TCAprecipitable component of the 'soluble fraction' was recovered among the digestive enzymes in the range of 12-55 kDa molecular mass. The extent of recovery was similar for both control and ethanol-fed groups.
DNA assay Aliquots of the pancreatic homogenates, containing 20-40 /~g DNA, were taken for extraction [8] and determination [9]. Standard DNA (calf thymus, DNA, Sigma Chemicals) in the concentration range of 0-60 /,g was similarly processed. Enzyme assays Amylase was assayed by the method of Rinderknecht [10] and lipase by the method of Tietez [11], using Sigma Kit 8500 with modifications as described elsewhere [12]. In these studies, a unit of amylase is defined as that activity of the enzyme at 25 °C and at pH 6.9 that liberates 1 mg of maltose from starch in 3 min. A unit of lipase is equivalent to the liberation of 1 /~mol of free fatty acids per min per mg protein. Trypsinogen and chymotrypsinogen, after activation with enterokinase and trypsin, respectively, were assayed by the method of Hummel [13]. Trypsin activity was expressed as T A M E (p-toluenesulphonyl-L-arginine methyl ester) units; 1 unit catalyzed the hydrolysis of 1 /,mol of TAME per min at 25 ° C. Similarly, chymotrypsin activity was expressed in BTEE (N-benzoyl-Ltyrosine ethyl ester) units, where 1 unit catalyzed the hydrolysis of 1 ~tmol of BTEE per min at 25 o C. Determination of protein Protein was assayed according to the procedure of Lowry et al. [14], using bovine serum albumin as the standard.
109
Statistical analysis Statistical analysis was carried out by the paired t-test. Materials L-[2,3,4,5-3H]Leucine, spec. act. 110 Ci/mmol, was purchased from ICN Radiochemicals, Irvine, CA. Electrophoresis chemicals were purchased from the following companies: Hoeffer Scientific, San Francisco; Fisher Scientific; and Eastman Kodak. All biochemicals were generally obtained from Sigma Chemicals. All other chemicals used were of reagent grade and obtained from Sigma (St. Louis, MO) or Fisher Scientific (King of Prussia, PA).
T A B L E II
Effect of ethanol feeding and withdrawal on pancreatic protein content Rats were maintained on control and ethanol-containing diets for 6 - 8 weeks. Pancreatic protein and D N A contents were determined before (0 days) and after ethanol withdrawal. All values are the m e a n s + S.E. of (n) pairs of animals. Duration of ethanol withdrawal (days)
Control
0(4) 1(5) 2(4) 3(3) 5(3)
34.14 + 35.35 + 38.45 + 35.86 + 40.85 +
Ethanol
(protein/DNA; mg/mg) 2.34 1.21 2.04 2.7 1.29
33.98 + 1.00 34.26 + 0.28 36.39 + 0.91 35.87 + 2.34 38.50+0.97
Results
Ethanol withdrawal and protein synthesis In pancreatic lobules from animals fed ethanol chronically, the rate of protein synthesis was comparable to preparations from pair-fed control animals (Table I). However, an overnight withdrawal of ethanol, induced by substituting the control diet for the ethanolcontaining one, increased the rate of protein synthesis (Table I). After more prolonged withdrawal this stimulatory effect rapidly decreased, and the rate of protein synthesis declined to control values by the third day (Table I). Since protein synthesis was measured by the rate of incorporation of [3H]leucine per mg protein, it was also necessary to determine the total protein content per cell in order to normalize the rates between the two groups. There was no significant difference in total protein content per cell (DNA) between the control and ethanol-fed groups under all conditions of treatment
(Table II). Regarding the specific activity of leucine pools, in the present studies a constant extracellular specific activity of [3H]leucine was maintained. In our previous study [5], we had already established that under conditions where stimulation of protein synthesis was observed in the ethanol-fed group, either in vivo or in vitro, there was no difference in the intracellular specific activity of [3H]leucine between the control and the ethanol-fed groups. Based on these observations, it can be concluded that the stimulation of protein synthesis observed in the present studies is also not due to a difference in the specific activity of leucine pools.
250 o 4,--
§ 200
150 c 0
g
TABLE I
Effect of ethanol feeding and duration of withdrawal on pancreatic protein synthesis Littermate rats were maintained on control and ethanol-containing diets for 6 - 8 weeks. The rate of incorporation of [3H]leucine (5 /~Ci/ml) into proteins was determined in the pancreatic lobules of animals before (0 days) or after 1 (18 h) 2, 3 and 5 days of ethanol withdrawal. Ethanol withdrawal was initiated by providing control diet to the ethanol-fed group. Values are the means_+ S.E. of (n) pairs of animals.
IO0
~' 5o if)
-~
0
a.
'~ -50
-I00
Duration of ethanol with-
Incorporation of [3 H]leucine (cpm × 1 0 - 3 / m g protein per 20 min)
drawal (days)
control
ethanol
% change
significance
0 (5) 1 (7) 2 (4) 3 (4) 5 (4)
11.68 + 1.41 12.31+0.85 11.20 + 1.73 11.31 +0.99 11.04+0.66
12.22 -t- 1.01 22.67+1.67 14.72 + 2.12 12.06+0.46 10.55+2.00
5 84 31 7 -4
N.S. P <0.01 P < 0.05 N.S. N.S.
N.S. = not significant.
N
I
i
I
I
I
I
0
I
2
3
4
5
Ethanol Withdrawal (days) Fig. 1. The effect of chronic ethanol feeding and withdrawal on the rate of pancreatic digestive enzyme synthesis. Littermate rats were maintained on control and ethanol-containing diets for 6 - 8 weeks. The rate of synthesis of individual digestive enzymes was determined in pancreatic lobules from the control and the ethanol-fed groups before (day 0) and after ethanol withdrawal as described in Materials and Methods. O, amylase; zx, chymotrypsinogen; O, lipase; and o, trypsinogen. *P < 0.005-0.02.
110 T A B L E III
Effect of ethanol feeding on the enzyme activities and the in vitro rate of synthesis of pancreatic digestive enzymes Littermate rats were maintained on control and ethanol-containing liquid diets for 6 - 8 weeks. Tissue levels of digestive enzymes and the in vitro rate of incorporation of [3H]leucine into individual digestive enzymes and total proteins were determined in pancreatic lobules as described in Materials and Methods. Digestive enzyme
Amylase Lipase Chymotrypsinogen Trypsinogen Carboxypeptidase Proelastase
Enzyme activity ( U / m g protein)
[3 H]leucine incorporation (cpm) a
control
ethanol
% change
control
104.02 + 13.91 20.21+ 2.31 1.99_+ 0.19 4.37± 0.52 -
12.32 ± 1.26 31.14±2.30 3.53_+0.36 5.43_+0.45 -
- 88 54 77 24 -
1 955 505 581 869 1053 306
Protein synthesis (total)
ethanol + 333 _+ 48 _+ 60 _+ 98 ± 123 ± 17
634 728 991 1025 1 358 442
% change ± 37 ± 34 ± 93 ±118 ± 117 _+ 63
- 68 44 71 17 29 44
(cpm × 1 0 - 3/rag protein per 20 min) 11.68± 1.40 12.22± 1.01
a Indicates the amount of label (cpm) associated with individual digestive enzymes when 80 #g particulate-free protein were separated by SDS-PAGE. All values are the m e a n ± S.E. of (4-6) pairs of determinations.
In order to relate the increase in total protein synthesis to the synthesis of digestive enzymes, the synthetic rates of individual digestive enzymes were determined in pancreatic lobules at various stages of ethanol withdrawal. As shown in Table III, the rates of synthesis of all the proteolytic enzymes tested were higher in the ethanol-fed group during active ingestion of ethanol (see also Fig. 1, day 0), with chymotrypsinogen exibiting the highest rate (+ 77%). The rate of synthesis of lipase was also higher, while that of amylase was much lower (-68%) than in the control group. By contrast, after 1 day of ethanol withdrawal, there was a general increase in the synthesis of all of the digestive enzymes (Fig. 1). At the end of 3 days, with the exception of amylase, the synthetic rates of individual enzymes had returned to control values.
Ethanol withdrawal and tissue levels of digestive enzymes In order to determine whether the synthetic rates of digestive enzymes reflected the tissue levels of these proteins, the activities of some of these enzymes were measured. During continuous ethanol ingestion (i.e., before ethanol withdrawal), the steady-state levels of all of the digestive enzymes correlated well with their rates of synthesis (Table III). By contrast, after 1 day of ethanol withdrawal, when the rate of protein synthesis was maximal, there was a 30-40% reduction in the tissue levels of almost all of the enzymes tested (Figs. 1 and 2), amylase being the exception (see Discussion). Again, as in the case of their synthesis, after 3 days of ethanol withdrawal the elevated levels of some of the digestive enzymes (chymotrypsinogen, lipase and trypsinogen) reverted to control values and, after 5 days, remained at or below the levels of the enzymes in the control group (Figs. 1 and 2).
In order to distinguish the stimulatory effect induced by ethanol withdrawal from that due to dietary change, the effects of ethanol withdrawal were also studied after inducing withdrawal by overnight starvation. As indicated in Table IV, the results were similar to those observed when ethanol withdrawal was initiated by providing the control diet (Table I and Figs. 1 and 2), although the extent of stimulation of protein synthesis was lower (46 vs. 84%). These results suggest that stimulation of protein synthesis is a function of ethanol I00
A O
§ so g c u
:
~ -50 N C W
-IO0
I
I
I
I
i
I
0
I
2
3
4.
5
Ethanol Withdrawal (days)
Fig. 2. The effect of chronic ethanol feeding and withdrawal on tissue levels of pancreatic digestive enzymes. Littermate rats were treated as detailed in legend to Fig. 1. Tissue levels of digestive enzymes were determined before and after ethanol withdrawal as detailed in Materials and Methods. Symbols per Fig. 1; *P < 0.005-0.05.
111 T A B L E IV
Effect of ethanol withdrawal by starvation on the activities and rate of synthesis of pancreatic digestive enzymes Experimental details are similar to that described in legend to Table III except that animals from both control and ethanol-fed groups were fasted overnight, water being provided. All values are the m e a n s + S.E. of four pairs of animals. Digestive enzyme
Amylase Lipase Chymotrypsinogen Trypsinogen Carboxypeptidase Proelastase
Protein Synthesis (Total)
Enzyme activity ( U / m g protein)
[3 H]leucine incorporation (cpm) a
control
ethanol
%change
control
117.32+7.56 25.32_+2.93 2.24 + 0.21 4.32 + 0.41 -
21.98+4.34 24.90+1.52 3.41 + 0.21 4.25 ± 0.67
-81 -2 52 - 2 -
1983 404 747 992 1169 317
-
-
-
ethanol +226 ± 31 _+ 60 ± 160 ± 121 _+ 19
972 710 1860 1 405 1936 494
% change +127 5:120 ± 237 ± 210 ± 256 ± 51
(cpm × 1 0 - 3 / m g protein per 20 rain) 10.11_+ 1.01 14.80_+ 2.83
-51 76 149 42 76 56
46
a Indicates the amount of label (cpm) associated with individual digestive enzymes when 80 /~g particulate-free protein were separated by SDS-PAGE.
withdrawal per se and not simply the consequence of a dietary change. Discussion
Under physiological conditions the turnover of individual digestive enzymes in the pancreas is likely to be influenced by specific gastrointestinal hormones, such as cholecystokinin (CCK) [15-17], secretin [18], insulin [19,20] or a combination of these [16,21]. Cholinergic mechanisms may also be involved [22]. Chronic ethanol administration has been reported to alter the tissue levels of some of the pancreatic digestive enzymes [3,12,23-25]. It is, therefore, possible that ethanol in the diet might interfere with the regulatory mechanisms that control the levels of some of the digestive enzymes, thereby leading to altered tissue levels of these proteins. There is indeed evidence to show that ethanol administration stimulates the release of CCK [26]. Treatment with low levels (0.25 /~g/kg per h) of caerulein, an analogue of CCK, causes alterations in the synthesis of digestives enzymes [15], while a sustained infusion of high levels (5/~g/kg per h) has been reported to induce pancreatitis [27]. Whether alcohol-related pancreatitis is associated with sustained high circulating levels of CCK has not yet been determined. Against the background of a lack of tissue injury during long-term ethanol ingestion, the stimulation of pancreatic protein synthesis during ethanol withdrawal is of interest. We have recently reported [5] an enhanced rate of protein synthesis in pancreatic acini isolated from animals which had continuously ingested ethanol, even though no changes were observed in vivo. By contrast, after an overnight withdrawal of ethanol, a stimulation of protein synthesis occurred in vivo. In the pancreatic lobules of this study, maximal stimulation of
protein synthesis occurred within 24 h of ethanol withdrawal, followed by a rapid decline to control values during the next 48 h. Thus, the lobular preparation mimicked the responses observed in vivo. It is unlikely that the mere removal of ethanol from the tissue could have initiated the stimulatory process, since lobules prepared from ethanol-fed animals before withdrawal were washed several times with the incubation medium to remove ethanol, and did not show any stimulation of protein synthesis. Moreover, in our earlier studies [5] on isolated acini, ethanol (100 mM) added in vitro had little effect on the rate of protein synthesis in preparations from the ethanol-fed group. It is also unlikely that a change from the ethanol-containing diet to the control diet could have triggered increased protein synthesis, since ethanol withdrawal by starvation led to a similar increase (Table IV). These observations suggest that it is the physiological removal of ethanol that alters the regulation of protein synthesis in the exocrine pancreas. The stimulation of total protein synthesis by ethanol withdrawal was associated with a parallel increase in the rate of synthesis of digestive enzymes. This observation is in keeping with earlier reports that pancreatic protein synthesis generally reflects the synthesis of digestive enzymes [5,28]. However, with the exception of amylase, the maximal stimulatory effect on protein synthesis coincided with a 30-40% reduction in the tissue levels of digestive enzymes (Figs. 1 and 2). Short-term loss of large amounts of digestive enzymes from the pancreas could be due either to an increased secretion or degradation. In our previous report [5], in isolated pancreatic acini from ethanol-fed animals, stimulation of protein synthesis was not associated with a parallel increase in secretion or intracellular degradation of digestive enzymes. It is, therefore, likely that the reduction in the tissue levels of digestive enzymes occurred as
112 a result of increased pancreatic secretion in vivo during ethanol withdrawal. There are several reasons for the lack of change in the level of amylase during the first day of ethanol withdrawal. Firstly, the a m o u n t of carbohydrate, a determining factor for the induction of amylase synthesis [29], is low in the ethanol-containing diet (11% compared to 47% in the control group). During ethanol withdrawal, the provision of high levels of carbohydrate is likely to enhance the signal for the synthesis of amylase. Secondly, the addition of ethanol to the diet, particularly at low levels of carbohydrate, further depresses the levels of amylase in the pancreas [12]. This leads to the low steady-state levels of amylase during ethanol feeding. Withdrawal of ethanol removes the suppressive effects of ethanol ingestion on amylase levels [12], thereby leading to increased synthesis. Finally, the factors responsible for inducing the general increase in the synthesis of other digestive enzymes p r o b a b l y also contribute to the enhanced synthesis of amylase. Since the initial level of amylase is low (unlike that of other digestive enzymes), even if there was an initial loss of amylase during ethanol withdrawal, the a c c o m p a n y i n g signals for an increased synthesis more than compensate for the loss. The present study demonstrates that chronic ethanol ingestion modifies the regulation of pancreatic protein synthesis and secretion. However, such modifications, for most part, remain latent until perturbed by processes such as ethanol withdrawal (in lobules) or collagenase digestion of the tissue (in isolated acini). The biochemical mechanism by which the stimulation of protein synthesis occurs under various conditions in the ethanol-fed animals is not clear. Whereas the isolated acini from the animals continuously fed ethanol showed an enhanced rate of protein synthesis [5], there was no parallel increase in the rate of protein synthesis in the unprocessed tissue of the same group (Table I). One of the differences between isolated acini and the intact tissue, represented by the lobules, is that the acini do not contain the islet cells and hence are not under the active influence of these cells. The islet cells secrete peptide hormones, such as pancreatic polypeptide, glucagon and somatostatin, which are k n o w n to inhibit the effects of C C K and secretin [30]. The stimulation of protein synthesis observed upon isolation of the acini or withdrawal of the animal from ethanol, m a y reflect the removal of the suppressive effects of these inhibitors, which would otherwise compensate for an excessive release of an agonist like C C K in the ethanol-fed animals.
Acknowledgements This work has been supported in part by grants AA07571, AA07186 and AA07215 from the National Institute of Alcoholism and Alcohol Abuse.
References 1 Durbec, J.P. and Sarles, H. (1978) Digestion 18, 337-350. 2 Singh, M. (1986) in The Exocrine Pancreas: Biology, Pathology and Diseases (Go, V.L.W. et. al., eds.), pp. 423-442, Raven Press, New York. 3 Singh, M., LaSure, M.M. and Bockman, D.D. (1982) Gastroenterology 82, 425-434. 4 Tsukamoto, H., Towner, S.J., Yu, G.S.M. and French, S.W. (1988) Am. J. Pathol. 131, 246-257. 5 Ponnappa, B.C., Hoek, J.B., Jubinski, L. and Rubin, E. (1988) Biochim. Biophys. Acta 966, 390-402. 6 DeCarli, L.M. and Lieber, C.S. (1967) J. Nutr. 91, 331-336. 7 Scheele, G.A. and Palade, G.E. (1975) J. Biol. Chem. 250, 26602670. 8 Munro, H.N. and Fleck, A. (1966) Analyst 91, 78-88. 9 Burton, K. (1956) Biochem. J. 62, 315-323. 10 Rinderknecht, J., Wilding, P. and Haverback, B.J. (1967) Experientia 23, 805. 11 Tietz, N.W. and Fiereck, E.A. (1966) Clin. Chim. Acta 13, 352-53. 12 Ponnappa, B,C., Hoek, J.B., Sarchet, K. and Rubin, E. (1986) J. Lab. Clin. Med. 107, 556-562. 13 Hummel, B.C.W. (1959) Can. J. Biochem. Physiol. 37, 1393-1399. 14 Lowry, O.H., Roseborough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 15 Schick, J., Versphol, R., Kern, H. and Scheele, G.A. (1984) Am. J. Physiol. 138, 676-682. 16 Solomon, T.E., Peterson, H., Elashoff, J. and Grossman, M.I. (1978) Am. J. Physiol. 235, E714-E719. 17 Otsuki, M., Okabayashi, Y., Ohki, A., Hootman, S.R., Baba, S. and Williams, J.A. (1984) Am. J. Physiol. 246, G419-G425. 18 Rauch, U., Vasiloudes, P., Rudiger, K. and Kern, H.F. (1985) Cell Tissue Res. 242, 641-644. 19 Okabayashi, Y., Mossner, J., Logsdon, C.A., Goldfine, I.A. and Williams, J.A. (1987) Diabetes 36, 1054-60. 20 Korc, M., Owerback, D., Quinto, C. and Rutter, W.S. (1981) Science 213, 351-353. 21 Dagorn, J.C. and Mongeau, R. (1977) Biochim. Biophys. Acta 498, 76-82. 22 Scheele, G.A. (1981) Methods Cell Biol. 23, 345-358. 23 Chariot, J., LeBourhis, B., Rose, C. and Potet, F. (1980) Dig. Dis. Sci. 25, 656-659. 24 Sarles, H., Figarella, C. and Clemente, F. (1971) Digestion 4. 13-22. 25 Tsukamoto, H., Delgado, G., Reidelberger, R.D. and Largman, C. (1986) Biochem. Pharmacol. 35, 3623-3629. 26 Liddle, R.A., Goldfine, I.D. and Williams, J.A. (1984) Gastroenterology 87, 542-549. 27 Steer, M.L., Meldolesi, J. and Figarella, C. (1984) Dig. Dis. Sci. 29, 934-938. 28 Case, R.M. (1978) Biol. Rev. 53, 211 354. 29 Grossman, M.I., Greengard, H. and Ivy, A.C. (1943) Am. J. Physiol. 138, 676-682. 30 Henderson, J.R., Daniel, P.M. and Fraser, P.A. (1981) Gut 22, 158-167.