Pancreatic and intestinal carbohydrases are matched to dietary starch level in wild passerine birds

Evolutionary shifts in diet composition are presumably accompanied by simultaneous changes in digestive physiology. The adaptive modulation hypothesis predicts that activities of digestive enzymes should match the relative levels of their substrates in an animal's diet so that available membrane space and synthetic energy are not wasted on enzymes in excess of need. However, previous studies on captive passerine birds showed high intraspecific phenotypic flexibility only in proteases but not in carbohydrases in response to varying diet composition. In this study, we measured the activities of pancreatic, intestinal, and hepatic enzymes in six wild-caught passerine species. We predicted that if the adaptive modulation hypothesis holds during evolutionary shifts in diet composition in birds, then mass-specific activities of digestive enzymes should be correlated positively with the content of their relevant substrates in species' diets. Whereas mass-specific activities of proteases (aminopeptidase-N, trypsin, chymotrypsin, alanine aminotransferase) were not correlated with estimated dietary protein content, mass-specific activities of all studied carbohydrases (amylase, maltase, sucrase) were positively correlated with estimated dietary starch content. We conclude that activities of carbohydrases but not proteases are evolutionarily matched to diet composition in passerine birds. We hypothesize that the need for nitrogen and essential amino acids can prevent the evolution of a low activity of proteases, even in species feeding on a low-protein diet.     

Developmental changes in digestive physiology of nestling house sparrows, Passer domesticus

Six decades of studies have speculated that digestive capacity might limit avian growth rate or that developmental changes in the gut might determine developmental changes in digestive efficiency. However, there are no studies on digestive enzymes during avian development, except for studies on mainly domestic birds that exhibit the precocial mode of development. We studied alimentary organ masses, intestinal enzyme activities (sucrase, maltase, isomaltase, aminopeptidase-N), and pancreatic enzyme activities (amylase, trypsin, chymotrypsin) during development of a wild passerine bird exhibiting the altricial mode of development. Wild nestling house sparrows were studied immediately after removal from the nest (days 0, 3, 6 of age; day 0=hatch), whereas captives were raised in the laboratory beginning day 3 on a formulated casein/starch-based diet until fledging age (after day 12). Digestive biochemistry was dynamic. Tissue-specific activities of some digestive enzymes continued to increase through fledging, by >10 times in some cases (e.g., sucrase and maltase in midintestine). Total pancreatic amylase activity increased 100 times between hatch and day 12 through a combination of increases in tissue-specific activity and pancreas mass. House sparrows differ from poultry, in whom after about 2 wk of age the specific activity of intestinal and pancreatic digestive enzymes is generally constant or declines during development. The data on intestinal and pancreatic enzymes help explain why digestive efficiency of nestling house sparrows improves with age, and the data seem consistent with the idea that digestive capacity might limit feeding rate and hence growth rate.     

Dietary modulation of intestinal enzymes of the house sparrow (Passer domesticus): testing an adaptive hypothesis

Insectivorous/frugivorous passerine species studied so far lack the ability to modulate intestinal maltase activity, in contrast to galliformes. We tested for dietary modulation of small intestine (SI) enzymes including maltase in house sparrows to understand whether the difference between the galliformes on the one hand, and the passerines on the other, reflects a phylogenetic pattern (maltase modulated in galliformes but not passerines), a dietary pattern (maltase modulated in granivores but not insectivore/frugivores), some other pattern, or chance. We also tested the prediction that intestinal peptidase activity would be increased on a high protein (HP) diet. Birds were fed three diets high in starch, protein, or lipid for 10 days. For birds on the HP diet (60.3% protein) we observed the predicted upward modulation of aminopeptidase-N activity, as compared with the lower-protein, high starch (HS) (12.8% protein) diet. In contrast, birds eating the HS diet had similar maltase and sucrase activities, and only slightly higher isomaltase activity, compared with birds eating the high protein (HP), starch-free diet. Birds eating high lipid (HL) diet had low activities of both carbohydrases and peptidase. Considering that the statistical power of our tests was adequate, we conclude that house sparrows show little or no increase in carbohydrases in response to elevated dietary carbohydrate. We cannot reject the hypothesis that maltase lability among avian species has a phylogenetic component, or that high dietary fat has a depressing effect on both carbohydrase and peptidase activities.     

Digestive tract development in rabbit according to the dietary energetic source: correlation between whole tract digestion,pancreatic and intestinal enzymatic activities

The developmental changes of both pancreatic and intestinal enzymes and the influence of dietary composition on enzyme activities were followed in suckling and weaning rabbits. In addition, whole tract digestibility of nutrients was recorded in response to two dietary energetic sources. Rabbits were fed ad libitum either a low fat and high starch diet (group LF), or a high fat and high fibre diet (group HF) between d 32 and d 42, with both groups receiving a growing finishing diet thereafter. Before weaning (d 32) nutrient digestion was high (>75% for organic matter, protein or fat), and then decreased sharply, except for fat. Between d 32 and d 42, digestion in the HF group was 7.5 and 4.6% lower, respectively, for organic matter and protein, while fibre and fat digestion was higher (+14.0 and +5.0%, respectively). Between d 25 and d 42 of age, pancreatic-specific activities of trypsin and chymotrypsin did not change while those of amylase and lipase increased by 1.5- and 76- fold (P<0.05), respectively. However, total activities and relative activities expressed on a LW basis were increased after weaning as a main consequence of a specific increased organ weight and pancreatic protein content. Relative activities of trypsin and chymotrypsin increased by 63 and 56% (P<0.01) after weaning, respectively. Total activities of pancreatic enzymes measured in the total small intestinal contents increased during the same period, but the range of variations was lower than those measured in the pancreatic gland. Total activities of lipase, trypsin and chymotrypsin measured in the small intestine contents were significantly correlated with pancreas enzyme potentialities. Total small intestine activity of lipase was 58% higher (P<0.001) in HF than in LF group while the other pancreatic and intestinal enzyme activities measured were not influenced by the energetic sources of the diet. Decreased digestibility of organic matter and protein observed with the HF diet could not be related to changes in pancreatic or intestinal enzymatic profiles and may be more dependent on quality of dietary ingredients.     

Are levels of digestive enzyme activity related to the natural diet in passerine birds?

Digestive capabilities, such as the rates nutrient hydrolysis and absorption, may affect energy intake and ultimately feeding behavior. In birds, a high diversity in gut biochemical capabilities seems to support the existence of a correlation between the morphology and physiology of the intestinal tract and chemical features of the natural diet. However, studies correlating the activity of digestive enzymes and the feeding habits at an evolutionary scale are scarce. We investigated the effect of dietary habits on the digestive physiological characteristics of eight species of passerine birds from Central Chile. The Order Passeriformes is a speciose group with a broad dietary spectrum that includes omnivorous, granivorous and insectivorous species. We measured the activity of three enzymes: maltase, sucrase and aminopeptidase-N. Using an autocorrelation analysis to remove the phylogenetic effect, we found that dietary habits had no effect on enzymatic activity. However, we found that granivorous and omnivorous species had higher levels of disaccharidase activities and insectivores had the lowest. The major difference in enzymatic activity found at the inter-specific level, compared to the reported lower magnitude of enzyme modulation owing to dietary acclimation, suggests that these differences to some extent have a genetic basis. However, the lack of a clear association between diet categories and gut physiology suggested us that dietary categorizations do not always reflect the chemical composition of the ingested food.     

Effects of diets rich in triglycerides and phospholipids on lipase and phospholipase A activities in rat pancreatic juice and pancreas]

Lipase and phospholipase A activities were increased in the pancreas and pancreatic juice of rats ingesting during two months 20% phospholipids- or triglycerides-rich diets. The activity of lipase is higher with the triglycerides-rich diet and the phospholipase A activity is higher with the phospholipids-rich diet. After seven days of the experiment, these two enzymes activities were less increased with diet containing 40% lipids.     

Induction of pancreatic trypsin by dietary amino acids in rats: four trypsinogen isozymes and cholecystokinin messenger RNA

We previously demonstrated that feeding a diet containing a high level of amino acid mixture simulating casein (AA) induced an increase in pancreatic protease activities in rats. In the present study, this effect of dietary AA was further characterized with three separate experiments. These experiments (1) examined periodic changes in pancreatic and small intestinal trypsin activities after switching from a 20% (a normal nitrogen level) AA diet to a 60% AA (a high nitrogen level) diet; (2) measured the abundance of mRNA for four trypsinogen isozymes and for intestinal cholecystokinin (CCK) and secretin in rats fed 20% and 60% AA diets for 10 days compared with rats fed 20% and 60% casein diets; and (3) measured the abundance of mRNA for four trypsinogen isozymes after chronic administration of CCK. Trypsin activities were gradually increased in both the pancreas and the small intestinal lumen and reached maximum at 5 days after the switch to the 60% AA diet (Exp. 1). This result is evidence that the increase in the protease activity in the pancreas is due to enhancement of pancreatic trypsin production. In experiment 2, pancreatic trypsinogen isozymes I, II, III, and IV mRNA abundance were evaluated by the Northern blotting method using cDNA probes specific for each isozyme mRNA. Abundance of trypsinogen mRNA without trypsinogen I tended to increase in the rats fed the 60% casein diet but tended to decrease in the rats fed the 60% AA diet compared with the respective normal nitrogen level diet groups without significant difference. CCK mRNA abundance in the jejunal mucosa increased as a result of feeding the 60% casein diet, but not the 60% AA diet. Subcutaneous CCK injections (3.5 nmole/kg body weight/day, twice daily, at 8:30 am and 7:30 pm) for 10 days resulted in increased pancreatic trypsin activity, whereas the changes in mRNA of the four trypsinogen isozymes was similar between the 20% and 60% casein groups but differed between the 20% and 60% AA groups (Exp. 3). These results suggest that CCK is not involved in the induction of pancreatic trypsin that occurs with feeding of a high AA diet and that the mechanism of protease induction by dietary AA is different from that in the case of dietary protein.     

Variation of basal EROD activities in ten passerine bird species--relationships with diet and migration status

Inter-specific differences in animal defence mechanisms against toxic substances are currently poorly understood. The ethoxyresorufin-O-deethylase (EROD) enzyme plays an important role in defence against toxic chemicals in a wide variety of animals, and it is an important biomarker for environmental contamination. We compared basal hepatic EROD activity levels among ten passerine species to see if there is inter-specific variation in enzyme activity, especially in relation to their diet and migration status. Migratory insectivores showed higher EROD activity compared to granivores. We hypothesize that the variable invertebrate diet of migratory insectivores contains a wider range of natural toxins than the narrower diet of granivores. This may have affected the evolution of mixed function oxidases (MFO) system and enzyme activities. We further tested whether metabolic rates or relative liver size were associated with the variation in detoxification capacity. We found no association between EROD activity and relative (per mass unit) basal metabolic rate (BMR). Instead, EROD activity and relative liver mass (% of body mass) correlated positively, suggesting that a proportionally large liver also functions efficiently. Our results suggest that granivores and non-migratory birds may be more vulnerable to environmental contaminants than insectivores and migratory birds. The diet and migration status, however, are phylogenetically strongly connected to each other, and their roles cannot be fully separated in our analysis with only ten passerine species.     

Effects of whole wheat feeding on the development of the digestive tract of broiler chickens

The objective of the current study was to investigate the impact of including whole wheat in broiler diets on the development of the digestive tract. Chickens were fed a standard feed containing 400 g ground wheat/kg or the same diet with a part of the wheat given separately as whole grains that increased progressively from 200 g/kg at 8 d to 400 g/kg at 22 d. Every week, from 16 to 44 d, growth performance, modifications of the size of the digestive tract organs and intestinal enzyme activities were investigated. Morphology of villi and crypts in the small intestinal segments (duodenum, jejunum, ileum) were analyzed at 23 and 44 d. Microbacterial counts were performed in jejunal, ileal and caecal contents weekly from 16 to 44 d.During the adaptation period from 8 to 15 d, the birds fed the whole wheat diet had lower feed intake and lower weight gain. Thereafter, they showed improved growth performance so that by the end of the experiment, they had higher body weight compared to the standard-fed birds, 2430 ± 29 versus 2331 ± 36 g.Higher relative weights of gizzard (+26%) and pancreas (+12%) were observed from 16 to 44 d for whole wheat-fed birds compared to standard-fed birds. No differences in relative size of the different intestinal segments were observed, except that the jejunum was shorter. Increased villus to crypt length and surface ratios were observed at 23 d in the duodenum of whole wheat-fed birds, with no differences in morphometry between groups thereafter. Alkaline phosphatase activity was higher from 16 to 44 d in the duodenum and jejunum of whole wheat-fed birds. However, the activities of the digestive enzymes, leucine aminopeptidase and maltase, were similar between the two diets in the measured intestinal segments.A lower number of facultative anaerobic bacteria was found in the ileum of the whole wheat-fed birds, with no differences between treatments for Escherichia coli and for Lactobacillus counts. In the jejunum and the caeca, no differences in microflora counts were observed.The present results showed that feeding whole grains to broilers led mainly to modifications in the upper part of the digestive tract (gizzard, pancreas) and had little influence on the small and large intestine.     

Effect of fasting on the structure and function of the gastrointestinal tract of house sparrows (Passer domesticus)

Starvation is a condition that often affects animals in nature. The gastrointestinal tract is the organ system displaying the most rapid and dramatic changes in response to nutrient deprivation. To date, little is known about starvation phases and effects on the organ morphology and digestive function in small passerine birds. In this study, we determined the phases of starvation and examined the effect of final stage of starvation in the organ morphology and, intestinal histology and enzymatic function in the small intestine. Our results show the three phases of the classical model of fasting in a shorter period of time. The mass of heart, pancreas, stomach, small intestine and liver of long-term fasted birds was reduced between 20 and 47%. The mass decrease in small intestine was correlated with reduction in small intestinal histology: perimeter, mucosal thickness, villus height and width. In contrast, the enzyme activity of sucrase-isomaltase and aminopeptidase-N in enterocytes, all expressed per μg of protein, was higher in long-term fasted birds than fed animals. This suggest that, while autophagy of digestive organs is induced by starvation, consistent with phenotypic plasticity, the activity of sucrase-isomaltase and aminopeptidase-N remains high, probably as an anticipatory strategy to optimize digestion at re-feeding time.     

Comparison of growth and development of the exocrine pancreas in pigs and rats during the immediate postnatal period

This study compared pancreatic tissue growth and functional changes during the first 3 postnatal days in piglets and rat pups. In piglets the absolute weight and the relative weight per unit body weight of the pancreas increased by 97 and 70%, respectively, while in rat pups the same parameters decreased by 33 and 48%, respectively, during this period. The specific activity of pancreatic amylase rose by 336% while that of trypsin, chymotrypsin and lipase remained at newborn level in piglets. In rat pups the specific activities of all enzymes measured declined by 61 to 92% during the first 3 postnatal days. The rate of postnatal pancreatic growth in the two species coincide with the levels of epidermal growth factor and insulin-like growth factors in maternal milk as reported in the literature, suggesting that milk-borne growth factors may stimulate pancreatic development in newborn animals.     

Pancreatic exocrine secretion in the pig following test meals of different composition and intra-duodenal loads of glucose and maltose.

Eleven pigs were fitted with pancreatic and duodenal fistulae, and pancreatic juice collected permanently. Amylase, chymotrypsin, lipase and total proteins were determined in juice collected within 2 and 6 hours after different test-meals or intraduodenal loads of glucose and maltose. In the pancreatic juice of pigs adapted to a high-lipid diet and submitted to a high-carbohydrate test-meal the activity of amylase was increased by 50%. When the consumption of the high-lipid meal was associated with an intraduodenal load of 100 g of glucose all the enzyme activities were stimulated when compared to the effect of meal alone, but only the activity of amylase was significantly increased (+ 82%). In the juice of pigs adapted to a balanced diet and submitted to intraduodenal loads of 150 ml of water, 50 g of glucose, 50 g of maltose and 150 g of maltose, the enzyme activities remained almost constant with the load of water and 50 g of maltose but with 50 g of glucose and 150 g of maltose loads, amylase activity was increased by 20% and 30% respectively. It is suggested, that the exocrine pancreas of the pig adapts itself rapidly to the changes in the size of the intestinal pool of starch hydrolysis products.     

Distinction between ''A''-esterases and arylesterases. Implications for esterase classification.

'A'-esterase activities (substrates paraoxon and pirimiphos-methyloxon) and arylesterase activities (substrate phenyl acetate) were assayed in the sera of 14 species of birds representing seven different orders and 11 species of mammal representing five different orders. Ten species of birds had no detectable 'A'-esterase, and the remaining four species only low activity, yet all birds showed considerable arylesterase activity (16.8-99.3 mumol/min per ml of serum). Ten species of mammal showed both 'A'- and 'aryl'-esterase activities. In humans, gel filtration of serum completely separated peaks representing paraoxonase and arylesterase activities. Thus, in both birds and humans, serum enzymes exist that express arylesterase activity but not 'A'-esterase activity. These findings suggest that a distinction should be made between these two types of esterase in future classifications.     

Prolonged feed deprivation does not permanently compromise digestive function in migrating European glass eels Anguilla anguilla

The effects of prolonged feed deprivation (40 days at 18° C) and re-feeding (30 days) on body mass, growth and the activity of selected pancreatic and intestinal enzymes were evaluated in migrating European glass eels Anguilla anguilla by comparison with a control group fed to satiation with hake Merluccius merluccius roe for the duration of the experiment. Feed deprivation resulted in mass loss and a reduction in digestive function, as revealed by a decrease in the total and specific activities of pancreatic (trypsin and α-amylase) and intestinal brush border (alkaline phosphatase and leucine aminopeptidase) enzymes. The total activity of intestinal brush border enzymes diminished after 5 days of feed deprivation, whereas that of pancreatic enzymes did not decrease until 10 days, indicating that the intestine is more sensitive to feed deprivation than the pancreas. Re-feeding A. anguilla that were starved for 40 days resulted in compensatory growth, with specific growth rates that were 2·6 times higher than the control group. This compensatory growth was associated with the recovery of trypsin and intestinal brush border enzyme activities, which were restored to control levels within 5 days of re-feeding. The ability to maintain pancreatic enzyme activity during 40 days of feed deprivation, and rapidly recover capacity for protein digestion upon re-feeding, would enable A. anguilla at this glass eel stage to withstand periods without food but rapidly provide amino acids for protein synthesis and growth when suitable food was available.     

Precocious alteration of digestive enzyme activities in small intestine and pancreas by chronic oral administration of protease inhibitor in suckling rats.

1. The role of endogenous CCK in the development of digestive enzyme activities in small intestine and pancreas was investigated in suckling rats. Synthetic protease inhibitor (camostat 100 micrograms/g bwt) was orally administered twice daily for 5 days from 11 days of age. 2. Pancreatic hypertrophy and hyperplasia, and alteration of pancreatic enzyme composition, especially decreases in amylase activity and increases in trypsin and chymotrypsin activities were produced by camostat treatment. These changes were completely suppressed by simultaneous administration of the potent CCK receptor antagonist L-364,718 (1 microgram/g bwt). 3. With camostat treatment, intestinal lactase activity decreased to 41%, while maltase and sucrase activities increased 3 and 2.5 times respectively. These changes in enzyme activities were not affected by the application of L-364,718. 4. The mucosal disaccharidase and pancreatic enzyme activities could not be modified by chronic subcutaneous injection of camostat. The precocious induction of maltase and sucrase activities by camostat treatment was also observed in the adrenalectomized pups. 5. These results indicate that pancreatic growth accompanied by alteration of digestive enzyme composition in the suckling rats is regulated by endogenous CCK, but the precocious induction of disaccharidase activities is not mediated by endogenous CCK released by camostat treatment.     

Activity, Composition, and Distribution of Pancreatic Serine Proteinases in Various Taxonomic Groups of Hydrobionts

Comparative analysis is presented of our own and literature data on activity, isoform composition, and molecular mass of enzymes of the trypsin and chymotrypsin types in pancreatic tissue of several species of marine echinodermates, molluscs, crustaceans, fish, and mammals. Specific activities of the enzymes vary widely inside classes, depend on the pancreatic tissue structure, and can be correlated with alimentary needs of individual species. Substrate specificity, isoform composition, and molecular mass are the most stable parameters in the studied taxonomic groups. The scheme is proposed of distribution of the species according to the enzyme properties and the pancreas structure.     

Intestinal cholesterol esterase: intracellular enzyme or contamination of cytosol by pancreatic enzymes?

The location of cholesterol esterase in rabbit intestine was re-evaluated. In three different experiments that were designed to eliminate contaminating mucus and pancreatic enzymes from the lumen of the small intestine, it was observed that the activities of cholesterol esterase and amylase in intestinal cytosol and whole homogenate decreased in parallel fashion. After the mucus was carefully wiped from the intestinal mucosa prior to the preparation of cytosol, amylase and cholesterol esterase activities decreased sevenfold. The recovery of the total activity of both enzymes in the cytosol was approximately 15%. When the lumen of the small intestine was filled with phosphate buffer and incubated at 37 degrees C for 20 min, cholesterol esterase and amylase activities in the cytosol prepared from this segment were further decreased. Moreover, the activities of amylase and cholesterol esterase were completely recovered from the lumen. Amylase and cholesterol esterase activities in the cytosol were eliminated if dithiothreitol was used as a mucolytic agent to prepare intestinal mucosa for the isolation of intestinal cells. In whole homogenates prepared from these intestinal segments, approximately 10-15% of the total cholesterol esterase activity remained. This activity, which could not be accounted for by pancreatic contamination, was associated with intestinal nuclei and cellular debris. Progesterone, ethinyl estradiol, and 25-hydroxycholesterol regulated microsomal acyl CoA:cholesterol acyltransferase activity and caused similar directional changes in the rate of cholesteryl ester synthesis in isolated intestinal cells. These same sterols, however, failed to affect cytosolic cholesterol esterase activity in vitro.     

Morphological relationships of passerine birds from Australia and New Guinea in relation to their diets

Beak, wing, leg and intestinal lengths, and gizzard widths, were all significantly related to body mass in 51 honeyeater species from Australia, 48 honeyeater species from New Guinea and 31 purely insectivorous passerine bird species from Australia. The nectar-feeding honeyeaters had smaller gizzards and intestines than wholly insectivorous birds of comparable size, although their wing and leg lengths did not differ; New Guinean and Australian honeyeaters were similar in these respects. Overall, honeyeaters had longer beaks than pure insectivores. Among Australian honeyeaters, those genera consuming more nectar than insects had longer beaks than the less nectarivorous, more insectivorous genera. Indeed, the latter group had beaks comparable in length to wholly insectivorous birds. All morphological differences revealed were attributable to known differences in diet.     

Regulation of proteolytic enzyme activities and mRNA concentrations in rat pancreas by food content

Regulation by food content of the expression of genes encoding pancreatic proteases was studied in rats fed diets containing 15%, 25% or 70% protein (w/w) (diet I, II and III). Trypsin, chymotrypsin and elastase activities in pancreas were 1.4, 2.8 and 2 times higher in diet III than in diet I whereas carboxypeptidase A level was unchanged. As compared to diet I, the pancreatic concentration of mRNAs encoding trypsinogen I and chymotrypsinogen B, measured by filter hybridization to specific cDNA probes, were found respectively 3.6 and 3.9 times higher in diet III, and 1.9 and 2.6 times higher in diet II. Elastase I mRNA concentration was 1.8 times higher in diet III, but unchanged in diet II. Procarboxypeptidase A mRNA concentration was not affected. It is concluded to a coordinate pre-translational regulation of serine protease genes expression by the protein content of diet, differing however in amplitude and sensitivity among the three species of enzymes studied.     

Luminal dietary protein, not amino acids, induces pancreatic protease via CCK in pancreaticobiliary-diverted rats

We determined whether pancreatic adaptation to a high-protein diet depends on ingested protein in the intestinal lumen and whether such adaptation depends on a CCK or capsaicin-sensitive vagal afferent pathway in pancreaticobiliary-diverted (PBD) rats. Feeding a high-casein (60%) diet but not a high-amino acid diet to PBD rats increased pancreatic trypsin and chymotrypsin activities compared with those after feeding a 25% casein diet. In contrast, feeding both the high-nitrogen diets induced pancreatic hypertrophy in PBD rats. These pancreatic changes by the diets were abolished by treatment with devazepide, a CCK-A receptor antagonist. Protease zymogen mRNA abundance in the PBD rat was not increased by feeding the high-casein diet and was decreased by devazepide. Perivagal capsaicin treatment did not influence the values of any pancreatic variables in PBD rats fed the normal or high-casein diet. We concluded that luminal protein or peptides were responsible for the bile pancreatic juice-independent induction of pancreatic proteases on feeding a high-protein diet. The induction was found to be dependent on the direct action of CCK on the pancreas. Pancreatic growth induced by high-protein feeding in PBD rats may depend at least partly on absorbed amino acids.