Effect of postnatal malnutrition on pancreatic zymogen enzymes in the rat1

Transcription

1 Effect of postnatal malnutrition on pancreatic zymogen enzymes in the rat1 T. F. Hatch,2 M.D., E. Lebenthal,3 M.D., J. Krasner,4 Ph.D., and D. Branski,5 M.D. ABSTRACT In order to characterize the response of the pancreas to undernutrition during the critical neonatal growth phase, acquired postnatal malnutrition was induced in the rat, using the expanded litter. An experimental nursing litter of 16 rats and control litters of 7 to 8 rats were formed. At 19 days of age, the pups were killed. Mean pancreatic wet weight was decreased in the malnourished rat to a greater extent than the decrease in total body weight (49 versus 60%). Decreased organ weight was predominantly the result of a decrease in DNA content and cell number. Enzyme activities expressed per total organ were all diminished; hipase to the greatest extent; trypsin and amylase to an intermediate extent; followed by chymotrypsin and the carboxypeptidases. The specific activities of hipase and trypsin were decreased with hipase, the most severely effected. The low trypsin levels can be attributed to trypsin inhibitor. It is possible therefore, that only the specific activity of lipase is significantly decreased. The decrease in enzyme activities, expressed both as specific activities and as total organ activities were decreased in a nonparallel fashion. Am. J. Clin. Nuir. 32: , Malnutrition, experienced during the postnatal preweaning period in the rat, results in dramatic stunting in the growth of many organs. Diminished organ weight is related to inhibition of normal cellular hyperplasia. DNA content and cell number are reduced; phenomena that are not reversed by adequate nutrition in the older rat (1). During the neonatal period, the pancreas of normal rats undergoes significant growth and functional maturation, primarily as the result of an increase in cell number, with little change in cell size (2). Pancreatic weight increases little during the 1st postnatal week, but during the ensuing 2 weeks, a steady increment in DNA content and organ weight results in a 10-fold enlargement of the organ. The functional development of pancreatic enzyme activities parallels structural enlargement. Total pancreatic contents of chymotrypsinogen, lipase, trypsinogen, and amylase increase by factors of 20, 30, 40, and 1000, respectively (3). Thus, the immediate neonatal period represents a critical phase in the structural and functional maturation of the pancreas. The response of the rat pancreas to a reduction of intake during a major part of the immediate postnatal period has not been studied. Because this period is critical for the normal development of exocrine pancreatic function, the changes induced might differ in degree or type from those seen with prolonged nutritional deprivation imposed during the major part of the prenatal or postwearnng periods. Our study was designed to assess several aspects of the response to underfeeding: 1) pancreatic weight, cell number and protein content; 2) the specific activities of pancreatic enzymes; and 3) the total organ activities of zymogen enzymes. Methods Ani,nals and deprivation technique Multiparous, 3 to 4 times pregnant female Wistar rats (Charles River), weighing approximately 350 g, were selected from an ongoing breeding colony. Normal litters delivering within 12 to 16 hr, were redistributed so that control rats belonged to optimally sized litters of 7 to 8 rats, while the experimental litter consisted of 16 rat pups. Nursing dams and pups were returned to the From the Division of Gastroenterology, Department of Pediatrics, School of Medicine, State University of New York and the The Children s Hospital of Buffalo, Buffalo, New York Fellow, Division of Gastroenterology. 3Chief of Division of Gastroenterology, Associate Professor of Pediatrics. 4Associate Research Professor of Pediatrics. Fellow, Division of Gastroenterology The American Journal of Clinical Nutrition 32: JUNE 1979, pp Printed in U.S.A.

2 POSTNATAL MALNUTRITION IN RATS 1225 colony and maintained in the same environment with stock feeding given ad libitum. At 19 days of age, pups from both littergroups were killed in a random order, without starvation, by exsanguination under anesthesia. Each pancreas was carefully resected, immediately weighed, and divided for histological assessment and biochemical determination. Preparation of samples After being weighed, each pancreas was sectioned for analysis. A small piece was placed in 10% buffered formahin for histological assessment. The remaining tissue was minced and homogenized in microtubes and stored at 0 C for 3 to 4 weeks. For enzyme determination, pancreatic tissue was weighed, diluted with ice cold saline (0.1 ml/mg of tissue), and homogenized in a Potter-Elvehjem glass homogenizer equipped with a Teflon plunger. It has been determined that these technics of tissue preparation and storage do not result in a loss of enzyme activity. Biochemical analysis In order to activate the pancreatic proenzymes, a trace amount of enterokinase was added to the homogenate and allowed to incubate before determination of enzyme activities. Trypsin (EC ) activity was measured from the hydrolysis of p-nitroaniline from the substrate benzoyl-dl-arginine-p-nitroaniline (BAPNA) at ph 8.2 and 25 C (4, 5). Units are expressed as nanomoles of p- nitroaniline hydrolyzed per minute per milligram of protein. Chymotrypsin (EC ) was determined from the rate of hydrolysis of N-benzoyl-DL-tyrosine ethyl acetate ester as measured by the change in absorbance at 256 nm with time (6). Units are micromoles of substrate hydrolyzed per minute per milligram of protein, a-amylase (EC ) was determined from the colored F Total Body Weights p<.0o1 Q product formed by the reduction of 3,5 dinitrosalicylic acid by maltose liberated per minute per milligram of protein. Lipase (EC ) activity was determined by the potentiometric titration (at a constant ph 8) of ionized fatty acids liberated from a triglyceride (olive oil) emulsion with normal NaOH (8). Units are expressed as micromoles of NaOH per minute per milligram of protein. Carboxypeptidase A (EC ) was determined by the rate of increase in the 154 nm absorbance of 25 C from the hydrolysis of hippuryl-l-phenylalanine (9). The determination of carboxypeptidase B (EC ) was performed using hippuryl-l-arginine as the substrate (10). Activities are expressed as microcmoles of substrate hydrolyzed per minute per milligram of protein. DNA was determined from the reaction with a diphenylamine chromogenic reagent after precipitation with cadmium (II) salts (11). Total proteins were done according to Sutherland s adaptation of the Lowry method using the Fohin phenol reagent (12, 13). Results Pancreatic Weights 2SOr The expanded litter model, used in this study, for the induction of acquired postnatal malnutrition in the nursing rat was effective in producing significant growth retardation (Fig. 1). The mean total body weight for malnourished rats was significantly diminished (P < 0.001), being approximately 60% of the mean for optimally nourished controls. Mean pancreatic wet weight was also significantly diminished (P < 0.001) in the malnourished rat pup. Organ weight in the malnourished rat was 49% of the mean for well- p<.0o1 0.lOi- p N.S. H FIG. 1. Effect of malnutrition on body and tissue weight. The bar graphs represent average values ± 1 SD for normal rats () on the left. Malnourished rats () on the right. Left, total body weight in grams; middle, pancreatic weight in milligrams; and right, the ratio of gram of protein per gram of tissue.

3 1226 HATCH ET AL. nourished controls. Stunting of both total body and pancreatic growth was present in our malnourished rat model, yet, the degree of pancreatic dystrophy was disproportionately greater than the reduction in total body weight. Pancreatic DNA in malnourished and control rats, expressed as total DNA (micrograms), DNA per gram of tissue and DNA per milligram of protein, is shown in Figure p< FIG. 2. Comparison of pancreatic DNA between normal () and malnourished () rats. Left, represents average values of micrograms DNA per gram of tissue; middle, microgram DNA per milligram of protein; and right, total DNA (micrograms). 45 pcos p<.005 a.trypsin b.lipose C.Amylase d.chymotrypsin acorboxypeptidasea tcorboxypeptidase8 15 T TT 0 T IP<05 ht i_il Measurement of tissue protein content, expressed as grams of protein per gram of tissue revealed no significant differences (P not significant) in the malnourished animals as compared to control. The mean total pancreatic DNA of malnourished rats approximated 60% of the mean for controls. Total organ DNA was significantly depressed in the malnourished rat (P < 0.005). The decrease in mean pancreatic DNA was II pttsl I / 4 2 [LI [III[FI 15 as W4 FIG. 3. Specific activities of pancreatic enzymes in normal and malnourished rats. Units as given in the methods per milligram of protein. a, Trypsin; b, amylase; c, lipase; d, chymotrypsin; e, carboxypeptidase A; and f, carboxypeptidase B.

4 POSTNATAL MALNUTRITION IN RATS 1227 a. Trypsin b. Lipose c. Amylose 900 pcooi 750 p<.0o d.chymofryps 30[ JO5 ecorboxypeptidosea f.cboxypeptidoseb 80 p<005 2( C IC - FIG. 4. Total organ activities. The same enzymes as presented in Figure 3 except that activities are expressed as the total present in the pancreas. in proportion to the difference in mean organ weights. Since total DNA content is related to cell number, there was a significant decrease in the cellularity of the malnourished pancreas. The ratios DNA per gram of tissue (P < 0.1) and DNA per miffigram of protein (P < 0.05), tended to be increased in the malnourished litter mates. These parameters bear on cell size, therefore, there was a tendency to smaller cells in the pancreas of malnourished rats. Decreased total pancreatic wet weight in the malnourished rat relates predominantly to a reduction in cell number. Cell size tends to be decreased also, but would play only a minor role in effecting total organ weight. The effect of undernutrition on pancreatic enzymes, expressed as specific activities and total organ activities, is shown in Figures 3 and 4. Lipase (P < 0.05) and trypsin (P < 0.05), expressed as specific activities were significantly depressed in the malnourished rat compared to control, with lipase the most severely affected (Fig. 3). However, amylase, chymotrypsin, and carboxypeptidases A and B were not significantly different between the two groups. These latter enzyme activities appeared resistant to the effects of malnutrition. Expressed as total organ activities, the mean activities of all enzymes were significantly diminished (Fig. 4). Discussion The normal development of the pancreas has been studied in the rat and in other species with similar patterns of ontogeny (14, 17). Amylase may be detected by the 16th day of gestation in embryonic rat pancreas (18). During the last week of fetal development in the rat, zymogen enzymes in the pancreas accumulate to high levels. At term, the total pancreatic contents of zymogen enzymes range from 2.5% that of adult rats (27 days) for trypsinogen, to 15 to 20% for chymotrypsinogen, amylase, and lipase (3). Postnatal pancreatic development includes several distinct stages, apparently predetermined by a developmental clock in the healthy neonatal rat. Pancreatic growth lags during the 1st week, followed by an increase in cell division and organ weight (3). At 17 to 18 days of postnatal age, a phase of rapid cell division begins, with a parallel increase in organ weight, lasting until 34 to 35 days of age. After this period, weight and DNA content increase more gradually until 100 days of age. Cell size increases to a small extent from 17 to 35 days and remains constant thereafter (2). Pancreatic zymogen enzyme contents follow, to a certain degree, the postnatal changes in organ size. The zymogen contents in the healthy term rat are high, except for trypsin-

5 1228 HATCH ET AL. ogen. During the first 1 to 2 days of postnatal life, these activities fall to low levels, suggesting that enzyme depletion occurs in response to the initial feedings, perhaps due to a limited reserve capacity for new enzyme synthesis. Enzyme contents gradually increase in a nonparallel fashion, reaching birth levels at 10 to 20 days of age. During the 3rd postnatal week, proenzyme activities increase dramatically, reaching adult levels at 27 to 30 days of age (3). The malnourished rat, in our study, experiences a delay in pancreatic growth. Pancreatic wet weight in 19-day-old malnourished rats (49% of normal weight), is approximately that of the normal 11 to 12-day-old rat. Pancreatic DNA content is significantly diminished in the malnourished rat, being approximately 60% that of control animals. Since the DNA content of individual cells is presumed to be relatively constant, the similar decreases in pancreatic weight and DNA content suggest that the diminished organ weight in malnourished rats is explained by a diminished cell number. The measurements, DNA per gram of tissue and DNA per gram of protein, comment on cell size, since the DNA content per cell is constant and uniform. The differences between malnourished and control are significant, indicating a smaller cell size in the malnourished rat. Thus, the diminished pancreatic organ weight, seen in malnutrition is significant, with the degree of dystrophy exceeding that of total body weight changes. Loss of mass is predominantly due to a loss in cell number, with a significant but lesser role accorded to cellular hypotrophy. The effect of immediate postnatal malnutrition has been studied in the rat (1). In contrast to isolated prenatal or adolescent malnutrition, preweaning malnutrition is characterized by proportional decreases in weight, protein, RNA, and DNA, that reflect a decrease in cell number, thereby simulating the organ of a younger animal. Cell size is minimally affected if at all. Our study of the pancreas in postnatal malnutrition demonstrates changes similar to those previously described for other organs. Two implications may be drawn from this fmding. First, in developing animals, organ size is related to the degree of functional maturation (16, 17), suggesting that an immature pattern of pancreatic enzyme activity should be seen in the malnourished rat. Second, in previous studies of preweaning malnutrition, subsequent normalization of dietary intake did not correct the diminished cell content of the organs studied as compared to normally nourished adult controls. Such an insult during this critical period may result in permanent stunting in pancreatic size and impairment in zymogen enzyme reserve. Acquired postnatal malnutrition produces an alteration in pancreatic enzyme content. Expressed as specific activities, lipase and trypsin are significantly depressed, with mean values being approximately 50 and 70% of control. Amylase tended to be decreased, but not to a significant degree. The activities of chymotrypsin and the carboxypeptidases A + B, expressed as specific activities, were the most resistant to the effects of malnutrition. Total organ activities of all enzymes were significantly diminished in malnourished rats, yet, the degree of change was similar to the alterations in specific activities. Lipase content was the most severely depressed, being less than 20% of the control value. Trypsin and amylase were effected to an intermediate degree, with mean values being 30 to 40% of control. The low trypsin levels may be attributed to the probable presence of trypsin inhibitor(s) in pancreatic tissue. This inhibitor might complex with free trypsin, thus lowering the total measurable free trypsin. It is possible, therefore, that only the specific activity of lipase is significantly decreased in the undernourished animals. The remaining enzymes were the least diminished, being 50 to 70% that of control. Thus, the zymogen enzymes of the malnourished rat are all diminished in a nonparallel fashion. Lipase is the most sensitive enzyme, in its response to the effects of malnutrition. The uniform but nonparallel diminution in pancreatic zymogen enzymes in association with decreased organ size may relate to several factors that influence pancreatic growth and functional development. Starvation induced by litter expansion is uniform for all animals, relating to decreased intake. Less foodstuff is presented to the intestinal tract resulting in minimal or infrequent release of gastrointestinal hormones and neurotrans-

6 POSTNATAL MALNUTRITION IN RATS 1229 mitters such as cholecystokinin, gastrin, and acetylcholine. These agents are trophic to the pancreas; gastrin and cholinergic agents result in hypertrophy (18, 14), while cholecystokinin causes both hypertrophy and hyperplasia (20). Atrophy of the stomach and pancreas occurs in rats given complete total parenteral nutrition, pointing to the importance of the intestinal delivery of foodstuffs (21). In the older rat, prolonged fasting results in a dramatic and reversible decrease in DNA synthesis and content, glandular weight and enzyme and electrolyte output (14, 29). The influence of these trophic agents during the critical developmental period and the differential effects on the development of individual enzyme activities remain unexplored. A second area relating to pancreatic development involves the influence of endocrine hormones. In older rats hypophysectomy results in pancreatic atrophy, that may be prevented by the administration of thyroxine, cortisone, and growth hormone (24, 25). Thus, the development and maintenance of pancreatic mass and function appears to require endocrine hormone stimulation. In our malnourished rat model, abnormalities in endocrine function may be partially responsible for the deficiencies in pancreatic size and proenzyme content. Indeed, in studies of prenatal malnutrition induced by maternal deprivation abnormalities in body weight and bone maturation may be reversed by the administration of growth hormone and thyroid replacement (26). Decreased organ size in the growing animal correlates with delayed functional maturation. This is also true in our model rat, the 19-day malnourished rat pancreas being similar in weight and enzyme content to the 13- to 14-day-old rat. Thus, a developmental clock for normal pancreatic enzyme development may be reset by the effects of malnutrition, and the nonparallel decrease in enzyme activities might reflect the nonparallel course of enzyme development that is normally seen (4). Protein-calorie malnutrition in the human infant is a significant world-wide problem. Undernutrition is present in one-half of the world s population. Over 300 million children suffer from chronic malnutrition. Digestive disturbances are common in the malnourished child. Steatorrhea is common, but is not necessarily due just to inadequate pancreatic lipase function. Pancreatic enzyme output is diminished in the malnourished child as studied by pancreozymin-secretin stimulation (27). Decreased enzyme output is proportional to the decrease in serum albumin and the decreased availability of amino acid precursors (28). Exocrine pancreatic enzymes diminish in a characteristic order, first lipase, then trypsin and fmally amylase (30). Volume output and neutralization capacity are said to be retained. The enzyme deficiencies associated with malnutrition are reported to respond rapidly to improvement in the nutritional status. Though, the clinical situation presented by the protein-calorie malnourished child is most often the result of a complex array of factors, including a variable degree of prenatal malnutrition, intestinal infection and infestation, and the effects of acquired postnatal malnutrition, the latter is certainly an important facet in the altered digestive function. This study demonstrates the alterations in growth and enzyme content of the pancreas, produced by isolated postnatal acquired malnutrition in the rat. Depressed organ size related to a reduction in cell number with a tendency to decreased cell size. The enzyme activities were reduced in a nonparallel fashion. Amylase, chymotrypsin, and the carboxypeptidase activities were diminished in proportion to organ weight. Lipase, and to a lesser extent, trypsin were uniquely sensitive to the effects of malnutrition. The contents of these two latter enzymes were disproportionately diminished. Adequate nutrition in the postnatal period appears to be more important for the development of lipase and trypsin than for the other enzymes. Lipase, in particular, is uniquely sensitive to undernutrition. Significant and possibly permanent changes in pancreatic structure and function may result from undernutrition in the neonatal period. a The authors thank Ms. Bridget Krantz and Ms. Ok Kim for their valuable technical assistance. References 1. WINICK, M., AND A. NOBLE. Cellular response in rats during malnutrition at various ages. J. Nutr. 89: 300, 1966.

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