International Journal of Pancreatology, 5 (1989) 035-045 $02.20 Copyright ~ 1989 by The Hurnana Press Inc. All rights of any nature whatsoever reserved.

Time-Course of Changes in Pancreatic and Enzyme Composition in Rats

During Starvation

Size

Istvdn Nagy, * z4kos Pap, and Vince l/arr6

First Department of Medicine, Albert Szent-GyOrgyi Medical University, Szeged, PO Box 469, H-6701, Hungary

Received May 2, 1988; Revised July 27, t988; Accepted August, 1988

Summary

The effect of starvation for 3, 5, or 7 d on body weight, fat stores, pancre- atic weight, and enzyme composition was studied in 300 g rats and was compared with a 3-d fast in 200 g rats~ In the 300 g animals, fasting led to a gradual hypotrophy of the pancreas with a marked, continuous decrease in amylase content. Pancreatic lipase, trypsinogen, chymotrypsinogen, proelastase, and secretory trypsin inhibitor contents increased temporarily, but by d 7, they declined to about the initial values. This decline in enzyme levels coincided with the exhaustion of fat stores. The decrease in amylase content could be related to decreases in circulating insulin levels, whereas the temporary increase in lipase content may be owing to changes in plasma free fatty acid concentrations. In 200 g rats, starvation for 3 d led to ex- haustion of fat stores that was accompanied by greater losses of pancreatic weight, protein, and amylase contents. In addition, the levels of trypsino- gen and chymotrypsinogen decreased and lipase was unchanged. These findings indicate that during starvation, changes in pancreatic secretory enzymes are time-dependent and vary with the age, body weight, and/or adipose tissue mass of the rats.

Key Words: Amylase; lipase; secretory proteinases; pancreatic secretory trypsin inhibitor; insulin; fat metabolism.

I N T R O D U C T I O N

Prolonged food deprivation has been shown to exert anti trophic effects on the pancreas in experimental animals (1-3) and also in humans (4). In addi- tion, typical changes in secretory enzymes have also been described: in the rat

*Author to whom all correspondence and reprint requests should be addressed.

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36 Nagy, Pap, and Varr6

pancreas, fasting decreased amylase concentrations but increased lipase con- centrations (5-8)~ Insulin, glucose, and lipid metabolites were proposed to be involved in the inverse regulation of amylase and lipase synthesis during star- vation (6, 8). However, there is conflicting data about the changes in secretory proteinases during starvation: increased (5), unchanged (6, 7), or decreased (8,9) pancreatic trypsinogen and chymotrypsinogen concentrations have been reported. Moreover, little is known about the time-course of changes in secretory enzymes during starvation, and only a few reports provide data on total pancreatic enzyme contents (6,9).

Therefore, we have carried out experiments in 300 g rats fasting for in- creasing periods of time; the changes in body weight, adipose tissue mass, pancreatic size, and enzyme composition together with the circulating levels of insulin, glucose, and fatty acids were studied. Since metabolic responses to starvation have been shown to be dependent on the age and body weight of animals (10,11), the effect of fasting on pancreatic enzymes in younger rats, weighing about 200 g, was also examined for comparison.

MATERIAL AND METHODS

Animals

Male Wistar rats were kept at 24~ with a 12 h light cycle from 6.00 to 18.00 and fed a commercial laboratory chow containing 69% carbohydrate, 20% protein, and 4.7% fat (LATI, GiSdiSllb, Hungary).

Fasting Experiments

In the first part of the study, rats weighing about 300 g were used. Twenty- four animals were divided into groups of six and placed in wire-bottomed cages to prevent coprophagy and fasted for 1, 3, 5, or 7 d with only access to tap water. For enzyme studies, rats fasted for 1 d served as controls since pancreatic enzyme content of fed animals varies greatly owing to variable discharge of zymogen granules. Food deprivation for 24 h in rats does not significantly affect pancreatic protein synthesis and enzyme composition (12).

For other parameters (plasma glucose, insulin, and free fatty acid levels and epididymal fat pad weight) an additional six rats of identical body weight, fed ad libitum, were used as controls (0 d fasting animals).

In the second part of the study, rats weighing about 200 g were used. Eight rats were fasted for 3 d and six animals, fasted for 24 h, were used as con- trols.

Each animal was weighed before and after the experiments. At the end of experiments, at 8-10 AM, rats were anesthetized with sodium pentobarbital (50 mg/kg body wt), and blood samples were taken from the aorta with a heparinized syringe. Plasma collected after centrifugation of the blood was stored at - 2 0 ~ The whole pancreas was dissected out, cleaned, weighed, frozen in liquid nitrogen, and stored at - 20 ~ until assayed. Epididymal fat

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Pancreatic Changes in Rats 3 7

pads were also removed, blotted, and weighed. The weight of epididymal fat pads has been shown to reflect that of total body adipose mass (13).

Biochemical Determinations

The pancreas was homogenized in nine volumes (w/v) of ice-cotd buffer containing 0.02M Tris-HC1, pH 7.8, 0.15M NaC1, and 0.1% Triton X-100. Samples for DNA determination were precipitated with ice-cold 0.8M per- chloric acid, washed in 5% trichloroacetic acid, and then hydrolyzed with 0.8M perchloric acid at 90~ for 10 min (14). DNA was estimated by the diphenytamine method (15). Pancreatic secretory trypsin inhibitor (PSTI) was extracted and measured as described by Fritz et al. (16). The homogenate (1 mL) was immediately precipitated with an equal volume of 0.4~14 perchloric acid. After incubation at 60~ for 3 min, the samples were centrifuged at 20,000g for 15 min. The supernatant fraction was neutralized with 2MK2CO3. Trypsin inhibitor capacity was estimated by incubating 0.15-0.3 mL of tissue extract with 7.5 ~g trypsin (from bovine pancreas, Type III-S, Sigma Chem. Co., St. Louis, MO) in 0.2M Tris-HC1 buffer, pH 8.0, at 37 ~ for 5 mino Residual trypsin activity was measured with benzoyl-D,L-Arg-p-nitro-anilide. Trypsin inhibition was linear up to 75% inhibition and measurements per- formed in this linear range.

Enzyme measurements were carried out from supernatant fractions of homogenates after centrifuging at 20,000g for 20 rain. Amylase was assayed by the Phadebas | Amylase test (17). Lipase activity was determined at 25 ~ pH 8.0, by a pH stat method (18) modified for the assay of rat pancreatic lipase. The reaction mixture contained 1 mL of Sigma Lipase Substrate (Sigma, St. Louis, MO), O.15MNaC1 and 1 m M CaClz in a final vol of 10 mL. Tryp- sinogen, chymotrypsinogen, and proelastase were activated after a 200-fold dilution of the homogenate in the assay buffer containing 80 mdvI Tris-HCt, pH 8.0, 25 mM CaC12, and 100/xg/mL bovine serum albumin. Trypsinogen was activated with enterokinase (Sigma Chem. Co., St. Louis, MO, 0.02 U//xg pancreatic protein) for 120 min at 37 ~ Chymotrypsinogen and proelastase were activated by bovine trypsin (Type III-S, Sigma Chem. Co., St. Louis, MO, 250 ~g/mg pancreatic protein) at 4 ~ for 2 h. These conditions yielded optimal activation of zymogens and are comparable to those described earlier (I9,20). Trypsin was measured with benzoyl-D,L-Arg-p-nitroanilide (21), chymotrypsin with acetyl-Tyr-p-nitroanilide (22), and elastase with succinyl- Ala3-p-nitroanilide (23) as substrates at 37 *C. Enzyme activities are expressed as total pancreatic contents and given in SI units (/xkat or nkat per pancreas; 1 /~kat (microkat)= 1/60 IU (international unit)). Protein concentration from the supernatant fraction of the homogenates was measured using the micro- biuret method of Goa U4).

Plasma insulin was determined with a radioimmunoassay kit (Institute of Isotopes, Budapest, Hungary) using human insulin as the standard. Plasma glucose was measured with o-toluidine (25). Free fatty acids were determined by a colorimetric method (26).

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38 Nagy, Pap, and Varr6

Table 1 Effect of Starvation on Body Weight, Pancreatic Weight, Pancreatic Protein,

and DNA Contents in 300 g Rats a

Pancreatic Pancreatic wt. Protein, DNA, Days of Body weight, g weight, per body wt~ rng per mg per starvation (initial) (final) mg mg/g pancreas pancreas

1 302 278 804 2.89 t06.8 4.10 +6.4 _+8.1 _+61 _+0.10 +7.7 _+0.30

3 302 255 b 692 b 2.74 91.0 b 4.12 _+6.1 _+5.1 +_52 -+0.14 _+5.9 +0.86

( - 8 % ) (-14~ ( - 5 % ) ( - 1 5 % ) (+0.5%)

5 303 238 '~ 635 b 2.67 81.8 b 3.86 +7.3 _+9.6 +51 +0.28 _+6.9 -+0.34

(-1407o) (-21070) (-807o) (-23%o) ( - 6%o)

7 302 210 b 534 b 2.56 b 66.2 b 3.77 +7.4 _+13.0 _+44 +0.10 -+ 1 1 . 1 -+0,41

( - 2 5 % ) (-34070) ( - 11070) (-38~ (-8070)

~Values are means _+ SD, For all groups, n = 6. Percentage differences from d 1 values are given in parentheses. bp<0 .05 vs respective d 1 values.

Statistical Analysis

Results are expressed as means _+ SD for each group of animals. Data were statistically evaluated by analysis of variance followed by "least significant dif- ference" (LSD) tests (27) or Student 's t-test, as appropriate. P values of less than 0.05 were accepted as significant.

RESULTS

Effects of Fasting on 300 g Rats

After a 24-h fast, body weight decreased 8%, mostly owing to an emptying of the gastrointestinal tract, and thereafter it decreased with time (Table I). During the first 5 d of the fast, the decrease in pancreatic weight paralleled the body weight's, but the ratio of pancreatic weight to body weight had decreased by d 7. Total pancreatic content of soluble proteins decreased in a similar rate as did pancreatic weight. Fasting tended to diminish pancreatic DNA content, but the differences were not significant.

Changes in pancreatic secretory enzyme contents are shown in Figs. 1 and 2. Fasting for 3, 5, and 7 d was associated with 31, 58, and 78~ decreases in amylase content. At all stages of the fast, the percentage loss of amylase con- tent far exceeded that of the pancreatic protein content. On the contrary, fast- ing for 3 or 5 d increased the lipase content 70-75~ and, to a lesser degree, increased trypsinogen, chymotrypsinogen, PSTI, and proelestase. By d 7, the levels of these enzymes had declined again to about initial values. The changes in PSTI and trypsinogen content closely paralleled each other; the ratios of

International Journal o f Pancreatology Volume 5, 1989

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40 Nagy, Pap, and Varr6

PSTI capacity to trypsinogen activity (1.35, 1.36, 1.32, and 1.33% at days l, 3, 5, and 7, respectively) were not significantly modified during the fast. Analysis of the 24 pairs of data indicated a significant linear correlation between PSTI and trypsinogen concentrations (r = 0.95, p < 0.001).

Plasma insulin concentration decreased early in the fast (35% after 24 h and 67% after 3 d) and remained low thereafter (Fig. 3). The level of plasma glucose was relatively well maintained throughout the fast except for a 16% decrease on the first day. Fasting for 24 h did not significantly alter the weight of epididymal fat pads, but decreased continuously thereafter. By d 7, epididymal fat stores became almost completely exhausted. Plasma concen- trations of free fatty acids increased rapidly following food deprivation and then decreased, presumably owing to exhaustion of fat stores.

Effects of Starvation for 3 D in 200 g Rats

In these animals, starvation for 3 d decreased body weight, pancreatic weight, and protein content to a greater degree than in 300 g rats and led to an almost complete exhaustion of epididymat fat stores (Table 2). The per- centage loss of amylase content was greater than in the 300 g animals; the content of lipase remained unchanged, whereas those of trypsinogen and chymotrypsinogen showed a significant decrease. Pancreatic DNA content was not altered in this experiment either.

DISCUSSION

The results of this study show that pancreatic enzyme changes during star- vation in rats are dependent on the initial body weight and duration of the fast. In accordance with previous findings (1,2) in the 300 g rats, fasting for increased periods of time led to gradual losses of body weight, pancreatic mass, and protein content. However, pancreatic DNA content did not signifi- cantly diminish even after 7 d of starvation. These results indicate a pancre- atic hypoplasy without a significant decrease in cell number.

The nonparallel changes in pancreatic secretory enzyme contents observed during starvation cannot be explained by a general reduction in the rate of protein synthesis or secretory changes (6). They are presumably owing to nonparallel changes in the synthetic rates of individual enzymes. Similar to earlier findings (1,5-8), pancreatic amylase content dramatically decreased with the fast's duration. Since it has been found that the breakdown of amyl- ase is not increased in the pancreas of fasted animals (7), the selective loss of amylase content may reflect a slower synthetic rate of this enzyme (3, 7). Pan- creatic amylase synthesis has been shown to be regulated by the insulin and glucose supply of the pancreas (5,28-30).

Insulin, by interaction with its receptor, increases glucose transport and stimulates protein synthesis in acini (31). Insulin alone is not sufficient to in- crease amylase synthesis (12). It acts indirectly, via promoting glucose uptake and utilization by acinar cells (32). In accordance with previous observations (7,11,33), our results show a slight lowering in the plasma glucose concentra- tion with a progressive reduction in the plasma insulin level in fasted rats.

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Pancreatic Changes in Rats 41

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International Journal of Pancreatology Volume 5, 1989

42 Nagy, Pap, and Varr6

Table 2 Effect of Starvation on Body Weight, Eididymal Fat Stores,

and Pancreatic Composition in 200 g Rats a

Control Starved for 3 d

Body weight , g, init ial Body weight , g, f inal E p i d i d y m a l fat pads , g Pancrea t i c weight , mg Soluble pro te in , m g / p a n c r e a s D N A , m g / p a n c r e a s Amylase , i zka t /pancreas c L i p a s e , / x k a t / p a n c r e a s c T ryps inogen , n k a t / p a n c r e a s C h y m o t r y p s i n o g e n , n k a t / p a n c r e a s

195 _+4.8 195 +4.1 187+6.2 152_+2.2 ~ (-19%)

1.39+0.38 0.22+_0.12 b (-84%) 644+58 509+_19 b (-21%)

83.0+5.0 61.9+_3.4 b (-25%) 3.53+0.12 3.54_+0.11 (+0.3%) 242_+41 117_+18 b (-52%)

102.6+10.5 98.0_+25.2 (-4~ 253_+35 183_+15 b (-28%) 204 _+ 25 147 _+ 22 b ( - 28%)

~Values are means _+ SD. For control group, n = 6, for starved group, n = 8. ences are given in parentheses. ~p<0.05 vs controls. c 1/zkat = 1/60 !U (international unit).

Percentage differ-

Thus, the selective decrease in amylase content during starvation is most probably a consequence of the lowered circulating glucose and insulin levels. Indeed, it has been demonstrated that these two substances can reverse the effect of fasting on pancreatic amylase levels in mice (34). However, contra- dictory results have also been published for rats (6).

Metabolic adaptations to starvation entail the increased use of lipid fuels (fatty acids, ketone bodies) in place of glucose by most of the tissues (11,35,36). In rats, fat is mobilized readily during starvation, and circulating levels of fatty acids and ketone bodies increase early in the fast and remain elevated as long as fat stores are abundant (10,11,36). Our results on the changes in plasma free fatty acids levels and fat pad weights are in accordance with these findings and suggest that the rise in pancreatic lipase content during the first few days of starvation is most probably related to the increase in circulating fatty acids or ketone bodies since it has been shown that parenteral infusion of lipids (29) or beta-hydroxybutyrate (8) can increase pancreatic lipase levels. The decline in lipase content, seen at d 7 of the fast, coincided with the ex- haustion of fat stores associated with a fall in plasma free fatty acids. These data support the view proposed by Bazin and Lavau (8,32) that pancreatic amylase and lipase syntheses are under control of the glucose and lipid utili- zation of the acinar cells; thus, the inverse changes in these enzyme levels during starvation might be regulated by inverse changes in intracellular glu- cose and fat metabolites. To check this hypothesis, experiments with specific regulators of mitochondriat fatty acid utilization are now in progress to ex- plore the role of fatty acid metabolism in the regulation of pancreatic lipase levels.

An unexpected finding from this study is that in 300 g rats, pancreatic trypsinogen, chymotrypsinogen, proelastase, and PSTI contents were increased

International Journal o f Pancreatotogy Volume 5, 1989

Pancreatic Changes in Rats 43

during the first 5 d of starvation. Increased specific activities of chymotrypsin- ogen and trypsinogen were also observed in the pancreas of fasting animals by Deschodt-Lanckman et al. (5). However, total pancreatic contents had not been reported. The reason for this increase in secretory proteinase levels, observed during the early phase of starvation, are not known. The rise in these enzyme levels is unlikely owing to CCK, a known inducer of these en- zymes (12,37), since fasting levels of this hormone are low in rats (38) and prolonged fasting is associated with low circulating CCK levels (4). Consis- tent with our findings are the reported increased synthesis of anionic, but not of cationic forms of trypsinogen, chymotrypsinogen, and proelastase together with decreased synthetic rates of all other enzymes in rats in response to a protein-free diet (39). Further experiments exploring the isoenzyme composi- tion should be performed to examine whether a similar redistribution of pan- creatic enzyme synthesis occurs in fasting animals~

In contrast to our findings in 300 g rats, no changes in the specific activities of chymotrypsinogen and trypsinogen have been observed in the pancreas of fasting 250-300 g Sprague-Dawley rats (6, 7), whereas in rats weighing about 200 g, these activities have been found to be decreased after a few days of starvation (8,9). Confirming these latter data, we have found that pancreatic enzyme responses to starvation for 3 d in 200 g rats differ from those seen in 300 g animals. In these younger rats, fasting led to an early exhaustion of fat stores with accelerated losses of body weight and pancreatic protein content. Thus, the lack of rise in pancreatic protease and lipase contents was most probably owing to a general defect in pancreatic protein synthesis. Similarly, a profound fall in all pancreatic enzymes was found in the 300 g rats at d 7 of the fast as fat stores became exhausted. These results are consistent with the findings of Goodman et al. (10,11), who have shown that metabolic adapta- tions to starvation in the rat are dependent on the age, body weight, and size of fat stores. Thus, older rats having considerable fat stores are able to con- serve significant amounts of tissue proteins for several days of starvation by using lipid fuels instead of glucose and amino acids, whereas in younger rats, the early depletion of fat stores leads to an accelerated loss of tissue proteins.

Therefore, the present results indicate that increases in pancreatic lipase and proteinase levels are characteristic for the early phase of starvation in the rat, but these responses are depressed in the absence of abundant fat stores sufficient for protein sparing.

The concentration of PSTI in the pancreas of control rats (4.4 nkat/g tissue) and the percentage ratio of PSTI capacity to trypsinogen activity (about 1.3%) found in this study are comparable to those reported by others (16,40). A new observation in this study is that during starvation, pancreatic trypsin inhibitor content varies in parallel with trypsinogen content. This results sug- gests that some common factors may be involved in the regulation of these activities.

In conclusion, the data presented here indicate that starvation in the rat results in nonparallel changes in pancreatic enzyme composition, which are dependent on the body weight and adipose tissue mass, and are regulated by metabolic alterations affecting the enzyme synthesis of acinar ceils.

international Journal o f Pancreatotogy Volume 5, 1989

44 Nagy, Pap, and Varr6

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Pancreatic Changes in Rats 45

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