hr) and a rapid single injection of

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1 J. Phyasol. (1976), 260, pp With 6 text-figurem Printed in Great Britain PROGRESSIVE ENHANCEMENT IN THE SECRETORY FUNCTIONS OF THE DIGESTIVE SYSTEM OF THE RAT IN THE COURSE OF COLD ACCLIMATION BY E. HARADA AND T. KANNO From the Department of Physiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, 060, Japan (Received 2 February 1976) SUMMARY 1. The secretary function of the exocrine pancreas and the stomach have been studied in the course of cold acclimation of rats that had been fed at an ambient temperature of 1 C in a climatic room. 2. The secretary responses of pancreatic enzymes evoked by continuous hr) and a rapid single injection of infusion of pancreozymin (PZ, 2-5 u./kg. PZ (1-7 u./kg) reached a maximum in the group of rats fed at 1 C for 4 weeks, and fell to the control levels after 8 weeks. The increase in the flow of pancreatic juice evoked by single injection of PZ was maximal at 4 weeks and slightly decreased after 8 weeks. 3. The insulin (3.0 i.u./kg) evoked secretion of pancreatic enzymes gradually increased after cold exposure, reached a maximum at 4 weeks and fell to the control levels after 8 weeks. The flow of pancreatic juice after insulin injection was almost the same in every group throughout the course of cold exposure. 4. The ratio of amylase to the total amount of the protein in the pancreatic juice decreased abruptly, in contrast to an increase in the ratio of protease in the process of cold acclimation. The change in the ratio of enzyme activity in the pancreatic juice may reflect parallel changes in enzyme activity in the exocrine pancreas. 5. The gastric secretion in response to insulin and bile secretion in the group fed at 1 C for 7 weeks was significantly higher than that in the control group. 6. It was thus concluded that the secretary activities of digestive system were enhanced by prolonged cold exposure and then returned to control level, and that the activities of the pancreatic enzymes were altered in the process of cold acclimation in rats.

2 630 E. HARADA AND T. KANNO INTRODUCTION Homoeotherms, i.e., birds and mammals, maintain their body temperature within a narrow range in spite of wide fluctuations in the ambient temperature. They conserve heat at low ambient temperatures and dissipate it at high ambient temperatures. Vhen a homoeotherm has been maintained in the cold for several weeks, characteristic signs of acclimation appear. In a rabbit, cold acclimation may represent almost exclusively a change in over-all insulation and in a wider thermal neutral zone (Heroux, 1967; Kockova' & Jansky, 1968; Harada & Kanno, 1975). In a rat, however, the principal mechanism of cold acclimation may be attributed to an increase in heat production by gradual development of non-shivering thermogenesis (Cottle & Carlson, 1954; Hart, Heroux & Depocas, 1956). This increase in heat production should be compensated by an increase in food consumption which may necessitate an enhanced digestive system. In fact, there is indirect evidence which suggests enhanced digestive functions during cold acclimation of homoeotherms: hypertrophy of the pancreas in the rat (Heroux & Gridgeman, 1958; Heroux & Campbell, 1959) and the enhanced gastric emptying and secretion in acute cold exposure of the dog (Sleeth & Van Liere, 1937). Kim, Kim, Kim and Hong (1970) observed that biliary-pancreatic secretion increased in the rat after 3 weeks of cold exposure. The present study has been carried out to get more direct evidence of enhancement of the digestive functions during cold acclimation of homoeotherms. The results showed that the secretary activities of the exocrine pancreas and the stomach were significantly enhanced, and that the activity of the pancreatic enzymes were significantly altered in the rat during cold acclimation. Preliminary accounts of the work described in this paper have already been published in an abstract form (Harada & Kanno, 1974). METHODS One hundred and twenty male Wistar rats, with an average weight of 220 g at the beginning of the study, were used. The rats were divided at random into two groups of sixty each, after about 2 weeks had been allowed for their adjustment to laboratory conditions. Then, one group was fed at an ambient temperature of 1 C for a maximum 8 weeks in an artificial climatic room having the characteristics as described in a previous paper (Harada & Kanno, 1975). The other group was used as a control. They were fed for the same period in a neighbouring room at about 20 C. Each animal was housed in wire mesh cages and fed a commercial rat diet (carbohydrate, 51X8%; protein, 25 3%; fat, 4-9 %; salt, 6 8%; cellulose, 4.2%, by weight) and water ad libitum. Body weight and food consumption for each animal was checked twice a week. In order to minimize the influence of diurnal variations all experiments were performed from about 10 a.m. to about 4 p.m.

3 DIGESTIVE SYSTEM IN COLD ACCLIMATION 631 Preparation for exocrine pancreatic 8ecretion Rats were subjected to 24 hr fasting before experimentation but were allowed to take water. Then, animals were removed from the climatic room after anaesthetized with urethane (1 1 gfkg body wt., i.p.). The abdomen was opened and a polyethylene tube (0.5 mm o.d.) was inserted into the bile duct toward the hilum of the liver for collection of bile. The bile duct was ligated below this point before entry into the pancreatic tissue. A polyethylene tube (0.5 mm o.d.) was also inserted into the duodenal end of the common duct and was used for collection of pure pancreatic juice. In all experiments, the pyrolus was ligated. The abdominal wall was closed with interrupted sutures. For i.v. infusions, a cannula was inserted into the femoral vein. The animal was placed on the cork board and maintained at a temperature of about 370 C (rectal) by an electric lamp. Preparation for ga8tric 8ecretion The gastric secretion in response to insulin was measured over a 3 hr period after the ligation of the pyloric region (Shay, Sun & Gruenstein, 1954). Before pyloric ligation, rats were subjected to 48 hr fasting, but water was available during this period. After anaesthetization with urethane (1 1 gfkg), the abdomen was shaved and a mid-line incision of about 2 cm in length was made extending from the xiphoid. Silk thread was placed around the pyloric region and a polyethylene catheter (3 mm o.d.) was passed into stomach by the oral route. Physiological saline (4 ml.) was injected and withdrawn immediately by gentle suction with a syringe, after which the catheter was withdrawn. Three hr later, insulin (3.0 i.u.lkg body wt.) was injected peritoneally, the stomach was removed and the gastric contents were drained into centrifuge tubes. The same procedure was repeated in the control group after injection of saline instead of insulin. After a 10 min period of centrifugation (3000 g), the volume of supernatant was measured, and the acid concentration was determined by titration with 0-02 NaOH to a ph 7-0 using a ph meter (Horiba Co., Japan). Dry gastric weights were measured after drying the stomach for 24 hr in an oven at 1000 C. Administration of drug Pancreozymin (PZ, Boots) was rapidly injected through the cannula in the femoral vein by a syringe or infused slowly for 90 min by a peristaltic pump (Tokyo- Rika Co., Japan). The dose of PZ was expressed in Crick, Harper & Raper (1950) units per 100 g body weight. Insulin (Novo) was given intraperitoneally in a dose of 3-0 i.u./kg body wt. Edtimation of digedtive enzymes After anaesthetization with urethane (1 1 g/kg), the pancreas was rapidly excised, trimmed of all excess fat, and frozen for a 24 hr period after weighing. The pancreatic tissue was homogenized in 5 ml. saline in a blender in the presence of 0-1 % Triton X-100 at 40 C for 5 min. The fluid was diluted 1000 to 5000 times by saline and protein, amylase and protease were assayed by the following methods. Total protein in the pancreatic juice and homogenized fluid were assayed by the method of Lowry, Rosebrough, Farr & Randall (1951). Amylase activity was assayed by a modified method of Bernfeld (1955). Protease activity was assayed by the method of Kunitz (1947).

4 632 E. HARADA AND T. KANNO Weighing of organs After anaesthetization with urethane (1.1 gfkg), the heart, liver, kidneys, pancreas, adrenals, stomach, small intestine, and testes were rapidly excised and trimmed of all excess fat and weighed immediately. The small intestine was cut open, the contents removed with a glass stick and weighed after drying for 24 hr in an oven at 1000 C. RESULTS Signs of cold acclimation The following measurements were compared between the control group fed at a mean ambient temperature of C and the group fed at an ambient temperature of C (Fig. 1A). The body weight of rats exposed to the cold for 1 week fell by 9 % and then rose by 8 % per week in the following 5 weeks and by about 3 % per week in the last 2 weeks. A similar rate of increase in body weight was also observed in the control group except for the initial falling phase (Fig. 1 B). The food consumption of the cold-exposed group rapidly increased in the initial phase of cold exposure, reached a maximum at about the second week, and maintained the high level, which was about 150 % of the control group. The food consumption of the control group was relatively constant throughout the whole period of feeding (Fig. 1C). TARix 1. Organ weights (mg/100 g body wt.) in rats fed at about 20 C or at 10 C for 6 weeks P for differences between Organ Control group Cold exposed group two groups Heart (6) ± 8'23 (6) <0.001 Liver (6) 3201P60± (6) < Kidneys ± (6) ±25-90 (6) <0-001 Pancreas (7) ±9-14 (6) <0-001 Adrenal glands ± 0-67 (6) ± 0 49 (6) < Stomach* (6) (6) < Small intestine* (4) ± (4) <0 05 Testes _ (6) '13 (6) <0)02 Body weight (g) (6) (6) <0-01 Note: figure in parentheses indicates number of animals, values are indicated by wet weight except those of the stomach and small intestine which are indicated by dry weight (*). Weights of various organs were compared after feeding for 6 weeks between the control group and the cold-exposed group (Table 1). The weights of pancreas, adrenal glands, stomach, heart, liver, kidneys, small intestine and testes of the cold-exposed group were significantly higher

5 DIGESTIVE SYSTEM IN COLD ACCLIMATION 633 than the corresponding values of the control group. The weight of the adrenal glands significantly increased after 2 weeks of cold exposure and reached a maximum at 4 weeks. The increasing ratios of these organs are comparable to corresponding values reported in previous papers He 300 B C 4.2* E c20 T T T U Feeding period (weeks) Fig. 1. Time courses of the changes in ambient temperature (A), body weight (B), and food consumption (C) in the control group shown by open circles (-O-), and those in the cold exposed group shown by filled circles (-@-). Each value for body weight and food consumption represents the mean value (± s.e..) of nine rats.

6 634 E. HARADA AND T. KANNO (Heroux & Gridgeman, 1958; Hale, Mefferd, Vawter, Foerster & Criscuolo, 1959; Jansk' & Hart, 1968). These measurements indicate that the rats may be acclimatized after 3-4 weeks of cold exposure, which corresponds well to the reported periods required for cold acclimation of thermogenesis in rats estimated by oxygen consumption (Gelineo, 1934). In the following experiments, the secretary functions of the digestive system were investigated in the course of cold acclimation. The secretary responses of the exocrine pancreas Physiological stimuli to which the pancreatic acinar cells respond by releasing their contents of zymogen granules are endogenous pancreozymin (PZ) and the vagus nerve. The secretary responses of the exocrine pancreas were examined for each group of rats that had been fed at 1 C for different periods. The responses were evoked by three different types of stimuli: a continuous i.v. infusion of PZ, a single, rapid I.v. injection of PZ, and a single i.p. injection of insulin. The secretary response topancreozymin. It has been shown that the secretory response varied markedly with the concentration of exogenous PZ: high concentrations of PZ (repeated injection of 8 u./kg body wt.) increased amylase output into the portal vein in contrast to a small increase in the output into the common duct (Saito & Kanno, 1973). A similar phenomenon has been observed (Debray, Vaille, DeLa Tour, Roze8& Souchard, 1963; Leroi, Morisset & Webster, 1971; Folsch & Wormsley, 1973). A physiological range of concentrations of PZ in the portal or the peripheral blood of the rat remains to be fully defined, but it seems to be in a range of several m-u./ml. (Kanno, Saito & Imai, 1973; Kanno & Imai, 1976). Folsch & Wormsley (1973) have reported that the maximal secretion of pancreatic enzymes was observed with a continuous infusion of 60 u. PZ/kg. hr combined with continuous infusion of 0 5 u. secretin/kg. hr into the anaesthetized rat. In the present study, we adopted a continuous hr as a weak stimulation, which was followed by a infusion of 2-5 u. PZ/kg. single, rapid injection of 1-7 u. PZ/kg to evoke the intermediate secretary response (represented by protein output) in the rat pancreas. Fig. 2 shows the changes in the release of pancreatic enzymes evoked by PZ in different groups of rats that had been fed at 1 C for different periods. The protein output evoked by the continuous stimulation with 2 5 u. PZ/kg. hr was enhanced with increasing time of cold exposure, reached a maximum at 4 weeks and gradually fell to the control levels after 8 weeks. The total amount of protein output in the pancreatic juice evoked by continuous stimulation with PZ was ug/90 min (mean of six rats and

7 DIGESTIVE SYSTEM IN COLD ACCLIMATION days (n=6) 1 week (n=7) 2 weeks (n=5) 4 weeks (n=6) 8 weeks (n=6) 0 -~ j200 I CL 0I 100 *.i, f.4...iji~i1..lij**i~lj~ : : t to l l l i l.l.l.l p >1500 l > >150 Time (min) Fig. 2. Time courses of changes in protein output into the pancreatic juice evoked by PZ in rats that had been fed at 1 C for different time periods. Each value represents the mean ( ± S.E.) for 10 min output (number of rats sampled is in the bracket). The stippled columns indicate the response of rats fed at about 20 C (number of rats for 4 weeks is ten and 8 weeks five). The striped horizontal bar indicates the period ofpz (2-5 u./kg. hr i.v.) infusion. Each arrow indicates the time of a single PZ injection (1.7 u.lkg. I.v.) days (n=6) 1 week (n=7) 2 weeks (n=5) 4 weeks (n=6) 8 weeks (n=6) 0-0 bo 10 I~~~~~I CD ~ ~ ou" X11, i ii A I > ~150So > Time (min) Fig. 3. Time courses of changes in the flow of the pancreatic juice evoked by PZ in the process of cold acclimation. Symbols as in Fig. 2.

8 636 E. HARADA AND T. KANNO + S.E.) in the group fed at 1 C for 4 weeks. The corresponding value in the control group fed at about 20 'C was ,ug/90 min (mean of ten rats and + s.e.). Analysis of these two values using a Student's t test showed that the former was significantly larger than the latter (P < 0 001). The secretary response induced by the rapid injection of 17 u. PZ/kg reached a maximum also at 4 weeks of cold exposure. The total protein output in 30 min in the group fed at 1 C for 4 weeks was ,ug/ 30 min (mean of six rats) and the corresponding value of the control group was 222x4 + 24x6 #ug 30 min (mean of ten rats). The difference between these two values was also significant (P < 0 001). The resting level of enzyme release before the continuous stimulation was almost constant in all groups of rats which were fed at 1 C for different periods of time. Fig. 3 shows the time course of changes in the flow of the pancreatic juice evoked by PZ in the process of cold acclimation of rats. Each result was simultaneously obtained from the corresponding group of rats depicted in Fig. 2. The increase in the rate of flow may be mainly due to PZ, although a part of it may be evoked by secretin admixed in the preparation of PZ. In the rat pancreas, it has been shown that PZ or caerulein strongly stimulates both the flow of pancreatic juice and enzyme release (Heatley, 1968; Dockray, 1972; Kanno, Suga & Yamamoto, 1976). Impurities in the PZ preparation may be a minor factor in the stimulation of juice flow (Dockray, 1972). In fact, it was recently confirmed in the isolated rat pancreas that pure natural PZ (3400 u./mg: GIH Research Unit, Karolinska Institutet, Stockholm) and synthesized desulphonated form of PZ evoke both responses (Yajima, Mori, Kiso, Koyama, Tobe, Setoyama, Adachi, Kanno & Saito, 1976). The juice flow evoked by continuous infusion of PZ (2-5 u./kg. hr) was almost the same in the whole course of cold acclimation (P < 0.05) except that measured after 1 week (P > 0-05). The juice flow induced by rapid injection of PZ (1.7 u. kg) significantly increased after 3 days of cold exposure (P < 0-001), reached a maximum at 4 weeks (P < 0 001) and then fell slightly after 8 weeks. The secretary responses to insulin. To study the effects of the vagus nerve on the pancreas, we attempted to use insulin, since it is known that insulin causes the activation of the vagus nerve possibly through a reflex via hypoglyeaemia (see Thomas, 1967). The insulin-evoked response of the pancreas was observed for each group ofrats following the preceding experiments and thus the time of insulin injection (3 0 i.u./kg) was at 150 min which corresponds to the final time of the preceding experiment. The protein output and the flow of pancreatic juice after a single insulin injection were measured for 10 min at intervals of 20 min. The protein output progressively increased after cold exposure, reached a maximum in the group of rats fed at 1 C for 4 weeks and gradually fell to the control levels after

9 DIGESTIVE SYSTEM IN COLD ACCLIMATION 350 r 3 days (n=6) 1 week (n=6) 2 weeks (n=5) 4 weeks (n=4) 8 weeks (n=6) : "o C 200 I IF F- 637 'a I a- 02 VP 50 0' S Time (min) Fig. 4. Time courses of changes in protein output (shown by open columns) in the pancreatic juice evoked by insulin in each group of rats fed at 1 C. Each value represents the mean ( ± S.E.) for a 10 min output collected at intervals of 20 min (number of rat sampled is in the bracket). The stippled columns indicate the response of rats fed at about 20 C (number of rats for 4 weeks is ten and for 8 weeks five). Each arrow indicates the time of insulin (3.0 i.u./kg i.r.) injection. The time of insulin injection was represented by 150 min, which corresponded to the final time of the preceding experiments. 10 F 3 days (n=6) I week (n=6) 2 weeks (n=5) 4 weeks (n=6) 8 weeks (n=6) 0 la ;0.0 bo 8a6 V_ C or v I I- I II r r1r ! Time (min) Fig. 5. Time courses of changes in the flow of the pancreatic juice evoked by insulin in the process of cold acclimation. Symbols as in Fig. 4.

10 638 E. HARADA AND T. KANNO 8 weeks (Fig. 4). The total amount of seven measurements of the group fed at 10 C for 4 weeks (depicted in Fig. 4) was compared with measurements made on the control group. The difference was significant (P < 0*01). In contrast, the flow of pancreatic juice after insulin injection was almost the same in every group subjected to cold exposure, although the total amount of flow measured after cold exposure for 1 week was larger than that of the control (P < 0.05) (Fig. 5). Comparison of enzyme activity in the pancreatic juice and pancreas. In the preceding experiments, we measured the output of the pancreatic protein E 9 8 T T E7 0 6 / C 7 &J ~T E 700 C a, 600,,500_ E " PI T~~~ 400t\o Feeding period (weeks) Fig. 6. Time courses of the changes in ratios of protease (upper line) and of amylase (lower line) to the total amount of protein in pancreatic juice, estimated in rats fed at 10 C for different periods of time. Each value represents the mean ratio ( ± S.E.) of six rats. Each open circle indicates the value for rats fed at about 200 C.

11 DIGESTIVE SYSTEM IN COLD ACCLIMATION 639 as an index of the output of pancreatic enzymes. But there was a possibility that enzyme activity may also be altered in the course of cold acclimation. This possibility was examined in the following experiments. Fig. 6 shows changes of amylase and protease activity in the insulinevoked juice of the pancreas. The ratio of amylase activity to total pancreatic protein (u. amylase/mg protein) decreased after cold exposure, reached a minimum in the group fed at 1 C for 2 weeks, and this minimum ratio remained constant in the groups fed longer than 2 weeks. This decrease in the ratio after cold exposure for 2 weeks was significant (P < 0-001). On the contrary, the ratio of protease activity to total pancreatic protein (u. protease/mg protein) fell slightly in the group fed at 1 C for 3 days, rose to a maximum in the group fed for 2 weeks and gradually fell to the initial ratio in the groups which were fed longer than 2 weeks. The increase in the ratio in the group fed for 2 weeks was significant (P < 0.02). TABLE 2. Amylase and protease ratios in rats fed at about 200 C or at 10C for 7 weeks Cold exposed P for differences between Ratio Control group group groups Pancreas (mg/l 00g ± 11F37 (6) (6) < body wt.) Total protein (mg/loo mg ± 0.92 (6) (6) >0 1 pancreas) Amylase (u./lomg pancreas) ± (5) ±56'69 (5) <001 Protease (u.1100 mg pancreas) ± (6) ± (6) <0 01 Amylase (u./mg protein) 94*53 ± 4.72 (5) 67*58 ± 3*32 (6) < 0*001 Protease (u./mg protein) (6) 1' *085 (6) < 0*001 Note: figure in parentheses indicates number of animals. The change in the ratio of enzyme activity in the pancreatic juice may reflect the parallel change in the enzyme activity in the pancreatic acinar cells: amylase contained in the pancreas (u. amylase 100 mg of pancreatic tissue) fed at 1 C for 7 weeks was significantly lower than that of the control group (P < 0-01). The amount of pancreatic protease (u. protease/i00 mg of pancreatic tissue) in the group subjected to low temperature was significantly higher than that of the control group (P < 0.01) (Table 2). Bile and gastric secretions. Gastric juice was collected for 3 hr after a single i.p. injection of insulin (3.0 i.u./kg). As shown in Table 3, the amount two

12 640 E. HARADA AND T. KANNO of gastric juice secreted from the group fed at 1 C for 7 weeks was significantly larger than that from the control group (P < 0-001), whereas little difference was detected after a saline injection. Total acidity of the gastric juice of the cold exposed group was also significantly higher than that of the control group (P < 0.05) (Table 3). In the same group fed at 1' C for 7 weeks, the total amount of bile was collected for the whole course of 6 hr experiments. The total amount of bile secreted from the cold exposed group was significantly larger than that from the control group (P < 0.01) (Table 3). TABLE 3. Bile and gastric secretions in rats fed at about 200 C or at 1 C for 7 weeks P for differences between Control group Cold exposed group two groups Stomach (dryweightmg/100g body wt.) 100X01±4'57 (8) 118X13±1X65 (12) <0*001 Gastric juice (tl.(3 hr. 100 mg stomach) Control ± (4) (6) >0 1 Insulin (3.0 i.u./kg) administration ± (4) (5) < N-NaOH/ Acid output (il. 3 hr. 100 mg stomach) Control ±91-28 (4) (6) >0 1 Insulin (3.0 i.u.fkg) administration ± (4) ± (5) <0-05 Bile (#s.i6 hr. 100 mg body wt.) ± (7) (6) < 0 01 Note: figure in parentheses indicates number of animals. DISCUSSION Enhancement of the digestive secretory system in the process ofcold acclimation The principal conclusion of our study is that the digestive secretary system was enhanced in rats which were fed at a low ambient temperature; definite enhancements were demonstrated in the PZ- and insulin-evoked enzyme release and juice flow of the exocrine pancreas; the insulin-evoked gastric juice and acidity; the total amount of bile secretion. The enhancement in the pancreatic secretion reached a maximum after about 4 weeks of cold exposure. This corresponds well with a previous report that 3-4 weeks were needed to reach a state of cold acclimation of thermogenesis in the rat (Gelineo, 1934). These enhanced secretary functions returned to the control level after 8 weeks of cold exposure. This pattern in the time course of enhancement in the exocrine pancreas during cold acclimation is similar to that observed in various endocrine glands, such as the secretion rate

13 DIGESTIVE SYSTEM IN COLD ACCLIMATION 641 of the thyroid hormone (Bauman, Anderson & Turner, 1968), the activity of adrenal medulla (Le Blanc & Nadeau, 1961; Leduc, 1961), adrenal cortical function (Sch6mbaum, 1960) and release of thyrotropin (Itoh, Hiroshige, Koseki & Nakatsugawa, 1966). Hildebrandt (1967) has already proposed that in the process of cold acclimation, the endocrine function of mammals may be enhanced gradually in the initial phase, and then may return to the pre-exposed level. This view may be extended to include the process of cold acclimation of the exocrine system. Temporal sequence in the cold acclimation of the exocrine digestive system The question then raised is the temporal sequence in the process of cold acclimation of the digestive system. The cold acclimation of secretion in the digesive exocrine gland may be preceded by changes in the function of the gastrointestinal endocrine system and the autonomic nervous system that controls their secrteory functions. Enhancement of the secretory functions of PZ and secretin, and vagus nerve activity may precede enhancement of the secretary function of the exocrine pancreas in the temporal sequence of cold acclimation. The enhancement in the secretary function of PZ and secretin may be the consequence of an increase in food consumption. In fact, the present study shows that food consumption increased immediately after cold exposure and was maintained at a high level during the whole period of cold exposure. Enhanced intestinal absorption may also be associated with enhancement in the secretory function of the digestive system. Musacchia & Barr (1969) showed a significant rise in intestinal glucose absorption after 2 weeks of cold exposure in the hamster. Enhancement in the autonomic nervous system has been regarded as the preceding event in the sequence of the cold acclimation (see Hildebrandt, 1967), and should be taken into account as a factor controlling cold acclimation. Carlson (1966) has shown that enhancement in the sympathetic activity of cold exposed animals leads to increased metabolic sensitivity to catecholamines. The parasympathetic system is also known to be activated in cold acclimation (LeBlanc & Cote, 1967; Harri & Tirri, 1974). Functional changes in other endocrine glands seem to enhance the digestive exocrine secretion, although direct evidence supporting this assumption remains to be defined. Activity changes in pancreatic enzymes after cold acclimation Results of the present study showed that in the process of cold acclimation, the ratio of amylase activity to total pancreatic protein in the pancreatic juice decreased abruptly in contrast to an increase in the ratio of protease activity of total protein.

14 642 E. HARADA AND T. KANNO The mechanism of this change in enzyme activity remains unknown, but several possibilities may be considered. Firstly, the change in enzyme activity may be due to an increase in food consumption during cold acclimation. An adaptation in enzyme activity of the exocrine pancreas has been reported when the composition of the diet was changed (Grossman, Greengard & Ivy, 1943; Ben Abdeljlil, Visani & Desnuelle, 1963; Howard & Yudkin, 1963; Morisset & Dunnigan, 1971; Snook, 1971). A preponderant component in the diet induces an increase in activity in the pancreatic enzyme digesting that component. For example, an increase in dietary casein raises the protease activity and reduces the amylase activity in the pancreas (Howard & Yudkin, 1963; Budko & Kopec, 1968) and pancreatic juice of the rat (Ben Abdeljlil et al. 1963). These phenomena, however, may not be the main mechanism of enzyme activation in cold acclimation, since the proportion of the food components was the same throughout the present study, although the total amount of food consumed increased. Secondly, an increase in the endocrine functions may also induce a change in enzyme activity. However, Rothman & Wells (1967) demonstrated that when relatively large amounts of PZ (20 u./kg) were administered in vivo for 4 days, amylase, chymotrypsinogen, and trypsinogen increased similarly in the pancreatic tissue along with a moderate increase in the weight of the pancreas. Thus, the increased release of endogenous PZ may not be the main mechanism in the activity change in the pancreatic enzymes. Finally, a decrease in insulin secretion may be taken into consideration. Insulin deficiency in rats fed on whole egg protein was shown to induce both a decrease in amylase activity and an increase in the activity of trypsinogen and chymotrypsinogen (Snook, 1968). A decrease in insulin secretion during cold acclimation has been reported by Beck, Zaharko & Kalser (1967). The decrease in insulin secretion may further be accelerated by an increase in catecholamine secretion, since it is known that catecholamines suppress insulin secretion (Porte & Williams, 1966). The authors wish to acknowledge the valuable assistance afforded by Mr S. Imai and Mr K. Ishikawa in the course of the present study. This study was supported by research grants from the Mitsubishi Foundation, Japan, the Matsunaga Foundation, Japan, the Hokkaido Government Office, Japan, the Ministry for Education Culture and Science, Japan. REFERENCES BAuMANN, T. R., ANDERSON, R. R. & TupuNER, C. W. (1968). Thyroid hormone secretion rates and food consumption of the hamster (Me8ocricetua auratue) at C and 4.50 C. Gen. comp. Endocr. 10, BEcK, L. V., ZAHARKO, D. S. & KALSER, S. C. (1967). Variation in serum insulin and glucose of rats with chronic cold exposure. Life Sci. 6,

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