T. Corring

Development of Digestive Enzymes in the Piglet from Birth to 8 Weeks

The changes with age of intestinal mucosa, protein, lactase, maltase and sucrase were followed in the piglet between day 105 of gestation and 8 weeks after birth. Lactase and maltase activities appeared during fetal life in the whole of the small intestine. Activity of sucrase was recorded after the 1st postnatal week. Lactase activity was high at birth and reached a maximum at 1 week (X 2.5); maltase activity which was low at birth increased to the 8th week (X 143). Activities of all enzymes were low in the duodenum; lactase was most active in the jejumum. Similar activities of maltase and sucrase were found in the two distal parts of the small intestine. Specific activity (related to protein content) of lactase reached a maximum at the end of the 1st week after birth and decreased afterwards. Specific maltase and sucrase activities were higher in the 2nd week, decreased between the 2nd and 4th week and increased afterwards (maltase) or decreased to the 6th--8th week (sucrase).

Effects of gastric digestive products from casein on CCK release by intestinal cells in rat

We investigated the ability of gastric digestive products from casein to stimulate cholecystokinin release by intestinal cells using the isolated vascularly perfused rat duodenojejunum. Casein digests were prepared with an in vitro system simulating gastric digestion and emptying. The luminal infusion of the digesta emptied from the artificial stomach for the first 10 minutes produced a sharp rise of portal cholecystokinin-like immunoreactivity to 300% of basal, followed by a well-sustained plateau secretion until the end of the infusion. The residual casein fraction of this digest brought about a modest cholecystokinin secretion, while the peptide component was as strong a stimulant as total digest. The peptide responsible for this effect was the glycomacropeptide that is a glycosylated fragment (106–169) of κ-casein. Only the slightly glycosylated forms of the peptide originating from variant A of κ-casein were active. The carbohydrate-free peptide did not alter basal cholecystokinin. The highly glycosylated forms of the peptide and the slightly glycosylated peptide from κ-casein variant B induced only a transient and low rise of portal cholecystokinin. The removal of N-acetylneuraminic acid from the active peptide suppressed its effect, while the infusion of an N-acetylneuraminic acid solution induced only a very low response. It is concluded that the glycomacropeptide released from dietary casein during gastric digestion can stimulate cholecystokinin release by intestinal cells in the rat. A well-defined structure is required for the peptide activity. A part of the peptide chain and some glycosidic chains containing N-acetylneuraminic acid, especially those bound to the amino acid residue threonyl 31 of caseinomacropeptide variant A, would be involved in this structure.

The influence of diet on the exocrine pancreatic secretion of growing pigs

1. Pancreatic juice was collected from six pigs of 48 kg initial weight fitted with a collection catheter in the pancreatic duct and a return catheter in the duodenum. 2. Measurements of flow and composition of the juice were made during 24 h periods after adaptation to isonitrogenous diets based on barley, wheatings and fish meal (diet BWF) or starch, sucrose, casein, maize oil and cellulose (diet SSC), given in a change-over design. Measurements were also made during the periods of adaptation to a change from one diet to the other. 3. Mean flow-rates for pigs adapted to diets showed a highly significant four-fold difference between diets; values were 4962 ml/d for diet BWF and 1273 ml/d for diet SSC. The hourly volumes of juice were very variable and showed no clear response to feeding and no consistent diurnal pattern for either diet. 4. There were no significant differences between diets in the specific activities of the proteases. Average values were (units/mg protein) trypsin (EC 3.4.21.4) 29.6, chymotrypsin (EC 3.4.21.1) 7.7, carboxypeptidase A diet BWF than with diet SSC. The specific activities and total outputs of alpha-amylase (EC 3.2.1.1) and lipase (EC 3.1.1.3) were significantly higher for diet BWF than for diet SSC; specific activities for the two diets respectively were: (units/mg protein) alpha-amylase 95-6 and 42.3, lipase 59.0 and 14.5. 5. The higher daily volume of juice with diet BWF was associated with significantly (but only slightly) higher levels of both sodium and potassium, compared with diet SSC. 6. The results are discussed in relation to previous studies on digestion at this Institute, in which pigs with intestinal cannulas were given the same diets.

NUTRITIONAL REGULATION OF PANCREATIC AND BILIARY SECRETIONS

MECHANISMS OF PANCREATIC ADAPTATION , MOLECULAR REGULATION OF PANCREATIC ADAPTATION . Dietary changes and molecular adaptation . Peptides and molecular adaptation . BILIARY SECRETION A N D DIETARY FAT . BILE RESPONSE TO DIETARY FAT . Bile salts . Biliary phospholipids . Biliary cholesterol. . Relative proportions of biliary lipids : saturation of bile with cholesterol MECHANISMS OF BILE RESPONSE TO DIETARY FAT . . BILIARY SECRETION A N D DIETARY FIBRE . BILE RESPONSE TO DIETARY FIBRE . Choledocal secretion . Bile acid pool. . Bile acid metabolism . MECHANISMS OF BILE RESPONSE TO DIETARY FIBRE . CONCLUSIONS , REFERENCES . . 161 . 162 . 162 . 163 . 165 . 165 . 167 . 168 . 168 . 168 . 169 . 169 . 170 . 170 . 171 . 171 . 171 . 172 . 173 173 . 175 175

Ion-pair high-performance liquid chromatography of bile salt conjugates: application to pig bile.

The high-performance liquid chromatographic separation and quantitation of conjugated bile salts from pig bile is reported. Synthetic standards and bile samples were chromatographed on a C18 reversed phase column using acetonitrile/water/tetrabutyl ammonium phosphate as an isocratic mobile phase at a flow rate of 1 mL/min. Detection of the ion-pairs was at 214 nm. The method permits efficient separation of all conjugated pig biliary bile salts without prior modification or treatment of the samples. Analysis of 12 pig biles showed that 85% of the bile salts are conjugated to glycine. The three main conjugated bile salts were glyco-3α,6α,7α-trihydroxy-5β-cholanoic acid (GHC), glyco-3α,7α-dihydroxy-5β-cholanoic acid (GCDC), and glyco-3α,6α-dihydroxy-5β-cholanoic acid (GHDC). Glyco-3α-hydroxy-6-oxo-5β-cholanoic acid (G3α6oxo), tauro-3α,7α-dihydroxy-5β-cholanoic acid (TCDC), tauro-3α,6α,7α-trihydroxy-5β-cholanoic acid (THC), and tauro-3α,6α-dihydroxy-5β-cholanoic acid (THDC) were found to contribute each for 4 to 5% ot the total. An excellent correlation was found between the sum of conjugated bile salts quantitated by high-performance liquid chromatography (HPLC) and values obtained by conventional enzymatic assay. Simplicity, efficiency and relative rapidity of the method render it suitable for routine analyses.

Effects of diets containing casein and rapeseed on enzyme secretion from the exocrine pancreas in the pig

The effect of dietary protein on enzyme activity of pancreatic juice was studied in ten growing, castrated, Large White male pigs. Animals, fitted with permanent cannulas in the pancreatic duct and in the duodenum, were divided into two groups receiving either casein or rapeseed concentrate as a protein source. After a 15 d adaptation period to the experimental diet, the volume of pancreatic secretion was significantly higher, whereas the protein concentration was lower in the casein group compared with the rapeseed group. No statistical difference was observed in the daily protein output between groups. Total secreted activities of carboxypeptidase A (EC 3.4.17.1), and elastase (EC 3.4.21.36) were higher in the casein group during the nocturnal period, whereas total activities of trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), carboxypeptidase B (EC 3.4.17.2) and amylase (EC 3.2.1.1) in pancreatic secretions during the post-prandial periods were increased by the ingestion of the rapeseed diet. It is concluded that the pancreatic enzyme secretion is sensitive to the nature of the protein ingested.

Effects of dietary proteins on some pancreatic mRNAs encoding digestive enzymes in the pig

Abstract The response of pancreatic protease syntehsis to fish meal-enriched diets was investigated in the pig, which is generally held to be a suitable model for human digestive physiology. In three sets of experiments, pigs were fed either 7, 17, 48, or 68% protein diets for 8 days (1st set), or 17 or 48% protein diets for 3, 6, or 7 days (2nd set), or 7 or 48% protein diets for 4 days (3rd set). At the end of each experiment, the pancreata were removed for biochemical and gene expression assays. The specific activities of amylase, lipase, chymotrypsin, trypsin, and elastase decreased as the result of the 7% protein diet after an 8-day feeding period (1st set). In the same experiment, all the enzyme-specific activities (those of amylase, lipase, chymotrypsin, elastase as well as carboxypeptidases A and B) increased in response to the 48% protein diet, the most strongly affected enzyme being chymotrypsin. Only chymotrypsin and carboxy-peptidase A-specific activities were further increased after feeding with the 68% protein diet for 8 days as compared with the 48% protein diet. The amylase, lipase, and trypsinogen mRNA levels remained unchanged throughout the experiments, but the mRNA encoding procarboxypeptidase A2 decreased, and that coding for chymotrypsinogen was enhanced after the animals had been fed the experimental diets for 3 days, but showed no change thereafter. Procarboxypeptidase B mRNA increased slightly only after a 6-day feeding period. When pigs were fed the 7 and 48% protein diets for 4 days, the enzymes synthesized in vitro in pancreatic lobules were correlated with the relative levels of the corresponding mRNAs, as measured by means of an in vitro cell-free reticulocyte-lysate translation system: both amylase and carboxypeptidases specific activities and their mRNA levels decreased slightly, while those of serine proteases increased. It was concluded that the biosynthesis of each serine protease was regulated separately and transiently at the pre-translational level. On the other hand, it seems very likely that amylase may be regulated at the translational level, while a multiple-level control process may take place in the case of procarboxypeptidase A2.

Adaptation of exocrine pancreas to dietary proteins: Effect of the nature of protein and rat strain on enzyme activities and messenger RNA levels

Abstract We compared the effect of high protein diets enriched with casein, fish, or soybean on enzyme content and mRNA levels in pancreata of postweaning and adult rats of two different strains. In the first experiment, 72 male Fischer rats (age 5 weeks) were divided into six groups and fed with one of six diets containing 20% or 50% protein as fish meal, casein, or soybean for 1 or 3 weeks. In a second experiment, 36 Fischer and 36 Wistar rats were divided into six groups and fed with the same diets for 1 week. In both experiments, rats were sacrificed at the end of the experimental feeding period and pancreata were excised and prepared for biochemical assay and mRNA extraction. Activities and mRNA levels were determined for each enzyme (amylase, lipase, chymotrypsin, trypsin, and elastases). Pancreas weight and its total protein content were modulated by the amount of dietary protein and duration of diet. These parameters were significantly different in Fischer and Wistar rats. In the latter strain, the nature of dietary protein also influenced pancreas weight. In Fischer rats, amylase specific activity was decreased after feeding 50% casein diet for 1 or 3 weeks and 50% fish or soybean diets for 3 weeks. The decrease of specific mRNA was more pronounced after a 3-week than after a 1-week feeding, suggesting that transcriptional regulation replaced progressively a translational one. In Wistar rats, amylase specific activity was not modified, but mRNAs were decreased after feeding high-protein diets. Lipase specific activity and mRNAs were not modified by any diet in any group. Chymotrypsin specific activity was increased after feeding 50% casein and soybean diets for 1 week and any 50% protein diet for 3 weeks. This effect was more pronounced in adult rats fed high protein diets for 1 week. In young Fischer rats, chymotrypsinogen mRNAs were increased after feeding 50% casein diets: in adult rats of both strains this parameter was increased by all diets except when the 50% soybean diet was provided to Wistar. Trypsin specific activity was increased after feeding 50% casein diet for 1 week and 50% fish or soybean diet for 3 weeks in Fischer rats but not altered in Wistar. The expression of trypsinogen mRNA was only increased after feeding 50% casein diet to both strains and 50% soybean diet to Wistar rats, suggesting that the regulation of its expression is different with the nature of protein. Elastase specific activities were increased by high-protein diets in both strains, but this effect was more pronounced in Wistar rats: these enzyme mRNAs were not altered, suggesting that the regulation was translational. In conclusion, it appears that the kinetics of adaptation of enzymes is different depending on the nature of dietary protein and the strain of rats used in the experiment. Amylase biosynthesis is regulated at the transcriptional level. Chymotrypsinogen and trypsinogen mRNA level showed that casein-induced adaptation was modulated via transcription, while other diets induced adaptation via postranscriptional events. Elastases adapt differently depending on the nature of the protein and the regulation of their expression is mostly posttranscriptional.

Inducing cholesterol precipitation from pig bile with β-cyclodextrin and cholesterol dietary supplementation

BACKGROUND/METHODS In this study, pigs fed for 3 weeks a well-balanced semi-purified diet enriched with 0.3% cholesterol and 0, 5 or 10% beta-cyclodextrin were proposed as new animal donors of gallbladder bile exhibiting different rates of cholesterol crystallization, in order to gain insight into the early mechanisms underlying cholesterol precipitation in vivo. The appearance and growth of cholesterol crystals were monitored in the incubated freshly collected gallbladder biles through light microscopy and concomitant time-sequential determination of crystallized cholesterol concentration, and interpreted in terms of the composition of the bile. RESULTS Although the concentration of total lipids and proteins and the relative proportions of bile acids, phospholipids, and cholesterol remained unchanged under beta-cyclodextrin, the cholesterol crystallization increased in the following order: 0<<10<5% beta-cyclodextrin. Concomitantly, the proportion of chenodeoxycholic acid in bile, and the hydrophobicity index of the biliary bile acid mixture increased in the following order: 0<5<10% beta-cyclodextrin (the same as reported elsewhere for the decrease in the antinucleating ApoA1), while sn-2 arachidonoyl biliary lecithins were specifically increased with 5% beta-cyclodextrin in the diet. CONCLUSIONS We hypothesized that lecithin molecular species may be the determinant factor in modulating high cholesterol crystallization rates in biles otherwise enriched with hydrophobic bile acids.

Influence of caecal microflora and of two dietary protein levels on the adaptation of the exocrine pancreas: comparative study in germ-free and conventional rats

Dietary proteins are degraded by both endogenous enzymes and the caecal microflora. In conventional rats the enzyme content of the pancreas depends on the amount of dietary protein. The influence of the caecal microflora on this process is unknown. We report here the effect of the caecal microflora on pancreatic enzymes (proteases, amylase (EC 3.2.1.1), lipase (EC 3.1.1.3)) and on colonic metabolites (NH3, urea, short-chain fatty acids). Germ-free and conventional male Fischer rats were fed for 3 weeks with a diet containing 220 or 450 g protein/kg provided as a mixture of fish concentrate and soyabean isolate. The excretion of NH3 and the pH were specifically increased by the high-protein diet in the germ-free rats. The higher production of isobutyrate, valerate and isovalerate in conventional rats fed on the high-protein diet reflected a high bacterial proteolytic activity since these short-chain fatty acids are specific indicators of this activity. The microflora hydrolysed urea to NH3 and maintained the pH at neutrality whatever the amount of protein in the diet since there were changes in germ-free rats but not in conventional ones. In germ-free rats, amylase, trypsin (EC 3.4.21.4), elastase (EC 3.4.21.36) and carboxypeptidase A (EC 3.4.17.1) specific activities were significantly lower than in conventional rats. The adaptation of the pancreas to the 450 g protein/kg diet was not impaired by the bacterial status except for the specific activity of chymotrypsin (EC 3.4.21.1) which was more increased by this diet in germ-free than in conventional rats. Moreover, the specific activity of lipase increased only in conventional rats fed on the 450 g protein/kg diet. In conclusion, we observed a relationship between the enzyme content of the pancreas and the presence or absence of the caecal microflora suggesting that bacterial fermentation influences pancreatic function.