Changes in mRNA levels of rat pancreatic lipase in the early days of consumption of a high-lipid diet

The time-course response of rat pancreatic enzymes to a diet containing 25% sunflower oil was investigated. A 1.2-fold enhancement in lipase specific activity was observed as early as the first day of diet consumption and was further increased up to 1.9-fold on the 5th day. On the other hand, colipase activity was slightly decreased during the first two days of high-lipid diet intake and then increased. An immediate and direct effect was also exerted by the 25% lipid diet on lipase biosynthesis. Both fractional synthetic rate and specific activity of lipase were comparably induced. Due to a 1.6-fold increase in the overall protein synthesis following 5 days of lipid diet consumption, the absolute synthesis of lipase and amylase was increased by 3.5-fold and 0.98-fold, respectively, as compared to control animals. By contrast, the synthesis of procarboxypeptidases and serine proteases did not increase before day 5, probably as the result of a distinct adaptive mechanism. The pancreatic mRNA levels in control and adapted animals, which were determined by dot-blot hybridization with amylase and lipase cDNAs, were consistent with a biphasic induction of lipase synthesis since a first increase in the level of the enzyme-specific mRNA during the first two days of diet intake (4-fold on day 1) was followed by a second increase after the fourth day (6.5-fold on day 5). On the other hand, amylase mRNA level was unchanged during the dietary manipulation. Thus, hyperlipidic diets exerted an both lipase activity and synthesis but a delayed effect on procarboxypeptidase and serine protease synthesis. In a similar manner, the immediate induction of lipase mRNA level by dietary fat, followed by another increase a few days later, suggested that at least two different mechanisms are involved in lipase mRNA induction.     

Effects of inverse changes in dietary lipid and carbohydrate on the synthesis of some pancreatic secretory proteins

The effect of ingesting isocaloric and isonitrogenous diets with increasing amounts of lipid (0-30%) and consequently decreasing amounts of carbohydrates (68.7-1.25%) on the exocrine pancreas was studied in adult male Wistar rats. Pancreatic contents of chymotrypsin, lipase and colipase activity, as well as synthesis of amylase, lipase, procarboxypeptidases and individual serine proteases were examined. Lipid-free diets and diets containing 1% lipid were found to have little effect on pancreatic proteins as compared with lipid-rich diets where two distinct patterns of response were observed. Ingestion of diets containing 3-20% lipid resulted in a progressive increase in the activity of lipase, colipase and chymotrypsin up to 2-fold in the first case and 1.6-fold in the two other cases when animals were fed the 20% fat diet. Under the latter conditions, the relative synthesis of secretory proteins, as expressed as percentage of the radioactivity incorporated into individual proteins compared to that incorporated into the total mixture of exocrine proteins, was unchanged for procarboxypeptidases, whereas it was stimulated for lipase (2-fold) and serine proteases (1.6-fold). Amylase relative synthesis progressively decreased as the lipid content of diets increased. Consumption of hyperlipidic diets containing 25% and 30% fat resulted in a further enhancement in the activity of lipase and colipase in the gland in contrast with chymotrypsin activity which was unchanged as compared to the control diet (3% lipid). As far as biosynthesis was concerned, a plateau in the relative synthesis of lipase and serine protease was reached. Amylase relative synthesis further decreased down to 2.2-fold when rats were fed the 30% fat-rich diet whereas that of procarboxypeptidases was markedly increased (about 1.7-fold). Absolute rates of synthesis of total pancreatic secretory proteins, as expressed with regard to the DNA content of the tissue, indicated that biosynthesis of all secretory pancreatic proteins was stimulated by hyperlipidic diets (at least 2-fold with the 30% lipid diet). Consequently, when such an increase was taken into consideration, the absolute synthesis of amylase was found to be unchanged throughout the dietary manipulations, whereas that of lipase, procarboxypeptidases and serine proteases were stimulated by 4.0-fold, 3.4-fold and 3.2-fold, respectively.     

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.     

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.     

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.     

Attenuation of skeletal muscle atrophy via protease inhibition.

Skeletal muscle atrophy in response to a number of muscle wasting conditions, including disuse, involves the induction of increased protein breakdown, decreased protein synthesis, and likely a variable component of apoptosis. The increased activation of specific proteases in the atrophy process presents a number of potential therapeutic targets to reduce muscle atrophy via protease inhibition. In this study, mice were provided with food supplemented with the Bowman-Birk inhibitor (BBI), a serine protease inhibitor known to reduce the proteolytic activity of a number of proteases, such as chymotrypsin, trypsin, elastase, cathepsin G, and chymase. Mice fed the BBI diet were suspended for 3-14 days, and the muscle mass and function were then compared with those of the suspended mice on a normal diet. The results indicate that dietary supplementation with BBI significantly attenuates the normal loss of muscle mass and strength following unloading. Furthermore, the data reveal the existence of yet uncharacterized serine proteases that are important contributors to the evolution of disuse atrophy, since BBI inhibited serine protease activity that was elevated following hindlimb unloading and also slowed the loss of muscle fiber size. These results demonstrate that targeted reduction of protein degradation can limit the severity of muscle mass loss following hindlimb unloading. Thus BBI is a candidate therapeutic agent to minimize skeletal muscle atrophy and loss of strength associated with disuse, cachexia, sepsis, weightlessness, or the combination of age and inactivity.     

Ecotin: lessons on survival in a protease-filled world.

Ecotin, an Escherichia coli periplasmic protein of 142 amino acids, has been shown to be a potent inhibitor of a group of homologous serine proteases with widely differing substrate recognition. It is highly effective against a number of enzymes, including both pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. Recent structural and functional studies on ecotin and its interactions with different serine proteases have clarified these initial observations and revealed the remarkable features of this protein in inhibiting a strikingly large subset of the chymotrypsin family of serine proteases. The structures of the ecotin:serine protease complexes provide the first examples of protein-protein recognition where the concept of specificity of interactions needs to be reexamined. The binding sites show a fluidity of protein contacts derived from ecotin's innate flexibility in fitting itself to proteases while strongly interfering with their function.     

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.     

Prey-mediated effects of the protease inhibitor aprotinin on the predatory carabid beetle Nebria brevicollis

To investigate the potential non-target impacts of transgenic pest-resistant plants, prey-mediated impacts of a protease inhibitor (PI) on the predatory carabid, Nebria brevicollis, were investigated. The PI used was aprotinin, a serine PI of mammalian origin with insecticidal properties when incorporated in artificial diet or expressed in transgenic plants. Field-collected N. brevicollis adults, kept at 23 °C, 16:8 L:D, were fed, over their pre-aestivation activity period of 24 days, with Helicoverpa armigera larvae reared on an artificial diet containing 0.5% (w:w, fresh mass) aprotinin. These larvae contained 22.62 μg aprotinin/g insect. Control prey was reared on diet without aprotinin. Beetle survival and body mass were unaffected by prey type. Beetles consuming PI-fed prey lost significantly more mass than the control beetles during two periods of mass loss, but gained significantly more mass during the final period of mass gain. This was not due to differences in amounts of prey supplied or consumed. The final mass gain coincided with increased consumption of PI-prey. Female beetles were significantly heavier than males, but we found no consistent gender-based differences in response to PI-prey. At the end of the experiment, body mass of all beetles was similar to field-collected ones (approximately 55 mg). All experimental beetles had significantly lower activities of digestive cysteine proteases and the serine proteases chymotrypsin and trypsin than field-collected ones. Beetles consuming PI-fed prey had significantly lower levels of trypsin and higher levels of chymotrypsin and elastase than the control beetles.     

Irreversible inhibition of serine proteases by peptide derivatives of (alpha-aminoalkyl)phosphonate diphenyl esters

Peptidyl derivatives of diphenyl (alpha-aminoalkyl)phosphonates have been synthesized and are effective and specific inhibitors of serine proteases at low concentrations. Z-PheP(OPh)2 irreversibly reacts with chymotrypsin (kobsd/[I] = 1200 M-1 s-1) and does not react with two elastases. The best inhibitor for most chymotrypsin-like enzymes including bovine chymotrypsin, cathepsin G, and rat mast cell protease II is the tripeptide Suc-Val-Pro-PheP(OPh)2 which corresponds to the sequence of an excellent p-nitroanilide substrate for several chymases. The valine derivative Z-ValP(OPh)2 is specific for elastases and reacts with human leukocyte elastase (HLE, 280 M-1 s-1) but not with chymotrypsin. The tripeptide Boc-Val-Pro-ValP(OPh)2, which has a sequence found in a good trifluoromethyl ketone inhibitor of HLE, is the best inhibitor for HLE (kobsd/[I] = 27,000 M-1 s-1) and porcine pancreatic elastase (PPE, kobsd/[I] = 11,000 M-1 s-1). The rates of inactivation of chymotrypsin by MeO-Suc-Ala-Ala-Pro-PheP(OPh)2 and PPE and HLE by MeO-Suc-Ala-Ala-Pro-ValP(OPh)2 were decreased 2-5-fold in the presence of the corresponding substrate, which demonstrates active site involvement. Only one of two diastereomers of Suc-Val-Pro-PheP(OPh)2 reacts with chymotrypsin (146,000 M-1 s-1), and the enzyme-inhibitor complex had one broad signal at 25.98 ppm in the 31P NMR spectrum corresponding to the Ser-195 phosphonate ester. Phosphonylated serine proteases are extremely stable since the half-time for reactivation was greater than 48 h for the inhibited elastases and 7.5-26 h for chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of rat mast cell protease I (chymase)

The amino acid sequence has been determined for rat mast cell protease I (RMCP I), a product of peritoneal mast cells. The active enzyme contains 227 residues, including three corresponding to the catalytic triad characteristic of serine protease (His-57, Asp-102, and Ser-195 in chymotrypsin). A computer search for homology indicates 73% and 33% sequence identity of RMCP I with rat mast cell protease II from mucosal mast cells and bovine chymotrypsin A, respectively. When the structure of RMCP I is compared to those of cathepsin G from human neutrophils and two proteases expressed in activated lymphocytes, 48-49% of the sequences are identical in each case. RMCP I has six half-cystine residues at the same positions as in RMCP II, cathepsin G, and the two lymphocyte proteases, suggesting disulfide pairs identical with those reported for RMCP II. A disulfide bond near the active site seryl residue and substrate binding site, present in pancreatic and plasma serine proteases, is not found in RMCP I or in the other cellular proteases. These results indicate that RMCP I and other chymotrypsin-like proteases of granulocyte and lymphocyte origin are more closely related to each other than to the pancreatic or plasma serine proteases.     

The phylogeny of trypsin-related serine proteases and their zymogens. New methods for the investigation of distant evolutionary relationships.

The sequence of all presently known trypsin-related serine proteases and their zymogens of animal and bacterial origin were optimally aligned on the basis of three different scoring schemes for amino acid comparisons. Sequence homology was found to extend into the activation peptides. The gaps resulting from the alignment of the sequences of the active enzymes formed the basis for a new procedure based on position and number of gaps, which allowed the correct topology of the evolutionary relationship of thrombin and the pancreatic enzymes trypsin, chymotrypsin and elastase to be determined. The procedure was applied in an analogous manner to changes in disulfide bridges as well as to a selected set of amino acid positions.Evolutionary distances between proteins were estimated by minimum, base differences as well as according to the stochastic model of evolution. These distances were used successfully to find the best topology of evolutionary relationships. The fact that the branch lengths in evolutionary trees were less affected by the number of sequences considered when evolutionary distances between contemporary sequences were measured in minimum base differences than when measured according to the stochastic model of evolution, suggested in our specific case, that minimum base differences yielded estimates of evolutionary distance closer to reality than the stochastic model of evolution.All these techniques combined yielded the following picture for the evolution of the four protease families. Prothrombin and the zymogens of the pancreatic serine proteases had a common ancestor with tryptic specificity. After the initial divergence, the gene for trypsinogen duplicated. Evidence was found that the duplicated gene underwent drastic changes for a short period of time to become eventually the common ancestor of chymotrypsin and elastase. The phylogenetic tree elaborated for these enzyme families and the methods introduced to determine its topology, should readily allow determination of the attachment site of branches leading to newly sequenced serine proteases, provided their amino acid sequence can be aligned fairly unambiguously. In addition, the consequences of the alignment of the different serine proteases for the relationship of zymogen to enzyme are discussed.     

Responses of the exocrine pancreatic secretion to spontaneous feeding in rats with bile-pancreatic juice diversion

The regulatory response of the exocrine pancreas was examined in rats under unanesthetized and unrestrained conditions. The previous study demonstrated that the pancreatic protease secretion increased 2-fold after spontaneous feeding of a low protein diet in chronically bile-pancreatic cannulated rats (normal rats) whose bile-pancreatic juice (BPJ) was returned to the duodenum. In the present study, we observed the response of the exocrine pancreatic secretion to spontaneous feeding of a low protein diet in rats with chronic diversion of BPJ from the proximal small intestine for 6 days (bypass rat) whose diverted BPJ was returned to the upper ileum. During BPJ diversion, the dry weight and the protein content of the pancreas were increased 2-fold, compared with normal rats. Also, the levels of trypsinogen and chymotrypsinogen in the pancreas were increased several times, but amylase was decreased. The basal secretion of enzymes after a 24-hr fast was enhanced in bypass rats in proportion to the pancreatic enzyme contents. After spontaneous feeding of 8% casein fat-free diet, the increases in the pancreatic secretion of bypass rats were much smaller than those of normal rats. In contrast, the increase of BPJ flow of bypass rats after feeding was greater than that of normal rats. These findings represent that the chronic diversion of BPJ exerts hypergrowth of pancreas and hypersecretion of proteases in the fasting state, and less sensitivity of pancreatic enzyme secretion to dietary feeding.     

Effect of level of dietary protein on arginine-stimulated citrulline synthesis. Correlation with mitochondrial N-acetylglutamate concentrations.

Increases in dietary protein have been reported to increase the rate of citrulline synthesis and the level of N-acetylglutamate in liver. We have confirmed this effect of diet on citrulline synthesis in rat liver mitochondria and show parallel increases in N-acetylglutamate concentration. The magnitude of the effect of arginine in the suspending medium on citrulline synthesis was also dependent on dietary protein content. Mitochondria from rats fed on a protein-free diet initially contained low levels of N-acetylglutamate, and addition of arginine increased the rate of its synthesis. Citrulline synthesis and acetylglutamate content in these mitochondria increased more than 5-fold when 1 mM-arginine was added. A diet high in protein results in mitochondria with increased N-acetylglutamate and a high rate of citrulline synthesis; 1 mM-arginine increased citrulline synthesis in such mitochondria by only 36%. The concentration of arginine in portal blood was 47 microM in rats fed on a diet lacking protein, and 182 microM in rats fed on a diet containing 60% protein, suggesting that arginine may be a regulatory signal to the liver concerning the dietary protein intake. The rates of citrulline synthesis were proportional to the mitochondrial content of acetylglutamate in mitochondria obtained from rats fed on diets containing 0, 24, or 60% protein, whether incubated in the absence or presence of arginine. Although the effector concentrations are higher than the Ka for the enzymes, these results support the view that concentrations of both arginine and acetylglutamate are important in the regulation of synthesis of citrulline and urea. Additionally, the effects of dietary protein level (and of arginine) are exerted in large part by way of modulation of the concentration of acetylglutamate.     

Accumulation of a metallo-carboxypeptidase inhibitor in leaves of wounded potato plants

Mechanical wounding of young potato plants induces over a two fold increase in inhibitory activity against the bovine pancreatic metalloexopeptidase carboxypeptidase A. This increase in inhibitory activity in both wounded and unwounded leaves parallels the increases of two inhibitors of bovine serine endopeptidases, trypsin and chymotrypsin. This suggests that the Proteinase Inhibitor Inducing Factor is regulating the synthesis and accumulation of inhibitors of two different mechanistic classes of proteases found in animals and microorganisms. These increases in antiproteolytic activities due to wounding support the hypothesis that this response is part of a defense mechanism directed against plant pests.     

Expression of c-myc oncogene in rat liver by a dietary manipulation

After rats deprived of protein for several days are fed a meal containing protein, hepatic DNA replication is induced. When nuclear DNA synthesis is stimulated in the normally quiescent rat liver by a dietary manipulation, we examined the changes of the steady-state levels of messenger RNA for c-myc. Levels of c-myc mRNA are gradually elevated approximately 4 to 5-fold above normal in the livers of rats that are fed for several days a diet that lacks protein. After a nutritional shift from a protein-free diet to a diet containing 50% casein, the levels of c-myc mRNA decrease rapidly by 2 h and returned to approximately basal levels after 8 h. Our results suggest that c-myc expression during the prereplicative stage of liver is likely to reflect events associated with entry and progression of hepatocytes into the cell cycle.