Changes in digestive enzymes through developmental and molt stages in the spiny lobster, Panulirus argus

Changes in major digestive enzymes through developmental and molt stages were studied for the spiny lobster Panulirus argus. There were significant positive relationships between specific activity of trypsin and amylase enzymes and lobster size, whereas esterase and lipase specific activities decreased as lobsters aged. No relationship was found between amylase/trypsin ratio and lobster size. Positive trends were found, however, for trypsin/lipase and amylase/lipase ratios. Results suggest that changes in enzyme activity respond to the lobsters' physiological needs for particular dietary components although multivariate analysis suggested that enzyme activities could be not totally independent of diet. On the other hand, the pattern of changes of major enzyme activities through molt cycle was similar for most enzymes studied. Following molt, trypsin, chymotrypsin, amylase, and lipase activities gradually increased to maximal levels at late intermolt (C4) and premolt (D). There were no variations in the electrophoretic pattern of digestive enzymes through developmental and molt stages and thus, it is demonstrated that regulation is exerted quantitatively rather than qualitatively. Further studies on the effect of other intrinsic and extrinsic factors on digestive enzyme activities are needed to fully understand digestive abilities and regulation mechanisms in spiny lobsters.     

Control of the release of digestive enzymes in the larvae of the fall armyworm,Spodoptera frugiperda

There is a basal level of enzyme activity for trypsin, aminopeptidase, amylase, and lipase in the gut of unfed larval (L6) Spodoptera frugiperda. Trypsin activity does not decrease with non‐feeding, possibly because of the low protein levels in plants along with high amino acid requirements for growth and storage (for later reproduction in adults). Therefore, trypsin must always be present so that only a minimal protein loss via egestion occurs. Larvae, however, adjust amylase activity to carbohydrate ingestion, and indeed amylase activity is five‐fold higher in fed larvae compared to unfed larvae. Gut lipase activity is low, typical of insects with a high carbohydrate diet. A flat‐sheet preparation of the ventriculus was used to measure the release of enzymes in response to specific nutrients and known brain/gut hormones in S. frugiperda. Sugars greatly increase (>300%) amylase release, but starch has no effect. Proteins and amino acids have little or no effect on trypsin or aminopeptidase release. The control of enzyme release in response to food is likely mediated through neurohormones. Indeed, an allatostatin (Spofr‐AS A5) inhibits amylase and trypsin, and allatotropin (Manse‐ AT) stimulates amylase and trypsin release. Spofr‐AS A5 also inhibits ileum myoactivity and Manse‐AT stimulates myoactivity. The epithelial secretion rate of amylase and trypsin was about 20% of the amount of enzyme present in the ventricular lumen, which, considering the efficient counter‐current recycling of enzymes, suggests that the secretion rate is adequate to replace egested enzymes. © 2009 Wiley Periodicals, Inc.     

Control of the release of digestive enzymes in the caeca of the cricket Gryllus bimaculatus

Abstract In Gryllus bimaculatus, more digestive enzymes (amylase, trypsin, aminopeptidase) are secreted in the caecum of fed crickets than in unfed crickets, but the enzymes are released continuously at a basal rate in unfed animals. The rate of synthesis of the enzymes appears to parallel their rate of release. Digestive enzymes are released in response to a specific ratio of nutrients, although a high nutrient component in the food does not necessarily induce a high digestive enzyme release for that component. Rinsed flat‐sheet preparations of the caecum are incubated with specific nutrients (carbohydrates and proteins) and various concentrations of a neuropeptide (type‐A allatostatin), which affects generally the basal rates of secretion. Both maltose and glucose increase the release of amylase in vitro, but starch produces an inhibition of amylase release at lower concentrations. Bovine serum albumin (BSA), peptone and a mixture of amino acids have almost no effect on the release of aminopeptidase or carboxypeptidase, and only low concentrations of peptone increase trypsin release. High concentrations of both BSA and peptone strongly inhibit trypsin activity, perhaps by excess substrate binding to the trypsin active site. The allatostatin Grybi‐AST 5 elevates the release of amylase in vitro, but not of trypsin or aminopeptidase, in 2‐day‐old fed females. In the caeca from 1‐day‐old unfed crickets, both amylase and the trypsin release are stimulated in the presence of AST 5. The paracrine AST 5 is probably released from the gut endocrine cells and binds to the enzyme‐producing caecal cells.     

SECRETION OF LIPASES IN THE DIGESTIVE TRACT OF THE CRICKET Gryllus bimaculatus

Little is known concerning the sites and the ratios of the lipase secretions in insects, therefore we undertook an examination of the lipase secretion of fed and unfed adult female Gryllus bimaculatus. The ratio of triacylglyceride lipase, diacylglyceride lipase, and phosphatidylcholine lipase secreted by fed females in the caecum and ventriculus is 1:1.4:0.4. These activities decrease in the caecum by 30–40% in unfed females. The total lipase activity (TLA) in the caecum is about 10 times that in the ventriculus. Minimal lipase secretion occurs before and during the final moult, and remains at this level in unfed crickets, indicating a basal secretion rate. In 2‐day‐old fed females, about 10% of the TLA in the entire gut is found in the crop, about 70% in the caecum, 20% in the ventriculus, and 3% in the ileum. Lipases in the ventriculus are recycled back to the caecum and little is lost in the feces. Oleic acid stimulated in vitro lipase secretion, but lipids did not. Feeding stimulated lipase secretion, starvation reduced lipase secretion, but this does not prove a direct prandal regulation of secretion, because feeding also induced a size and volume increase of the caecum.     

Activity, release and flow of digestive enzymes in the cricket, Gryllus bimaculatus

Abstract.  In the cricket Gryllus bimaculatus , proventricular pressure forces a nutrient fluid from the ground food-brei through a sieve at the base of the caecae, and the combination of secreted enzymes and water causes a rapid inflation of the caecae on the first day after imaginal ecdysis. The ceacal region of the midgut is the primary site for the secretion of digestive enzymes. Proteases and amylase flow from the caecae into the mostly empty crop on day 1, and carbohydrate and protein digestion starts as soon as food is present (6 h). Thereafter, much of the amylase activity (but not protease) in the crop is synthesized and released by the crop tissues themselves. Regurgitating proteases and amylases from the caecae into the crop after day 1 is most likely accomplished by temporarily halting proventricular peristalsis and allowing the caecal muscles to contract, forcing caecal contents, including enzymes, forward. The total activity of digestive enzymes in the caecae is virtually identical in 2-day-old fed and unfed females, indicating little or no secretagogue (prandial) regulation of enzyme secretion. Most of the digestive enzymes in the ventricular endoperitrophic space may originate from the mucus dragged from the caecae. Lipase activity is low in all gut regions in both starved and fed females. Head ligation or injection of trypsin modulating oostatic factor, allatostatin A or B fails to indicate any involvement of nerves or hormones in the release of digestive enzymes in the caecae. Gryllus bimaculatus appears to secrete digestive enzymes continuously, and a considerable loss of enzymes may occur at certain times through egestion.     

Clinical Studies of Human Pancreatic Enzyme Synthesis

Very little is known of the effects of diet and disease on panceratic enzyme syntheis in humans as conventional tests measure the secretory response to secreagogues, such as CCK, and secretion may be unrelated to synthesis because of the masking effect of a large intracellular pool of stored enzymes (zymogens). In order to obtain information on enzyme synthesis, as well as secretion, we have measured the incorporation characteristics of isotopically labelled amino acids (e.g., ¹⁴C or ¹³C leucine tracer) into amylase and trypsin protein, extracted by affinity chromatography from duodenal secretions during pancreatic stimulation with CCK-8The results of our studies in healthy volunteers and patients have suggested that (a) it takes between 75 and 101 min for the participation of newly synthesized pancreatic enzymes in the digestive process, and that zymogen stores are replaced at a rate of between 12 percent and 47 percent per hour in normal healthy subjects, (b) the synthesis and production rates of trypsin and amylase are parallel in healthy subjects, but can diverge under stressful conditions such as hypersecretory states, post-acute pancreatitis and protein malnutrition, (c) hyperphagia stimulates the synthesis of enzymes whilst malnutrition diminishes the synthesis of trypsin to a greater extent than amylase, (d) intravenous glucose and amino acids exert negative feedback control on the synthesis and release of amylase and trypsin, and (e) the decreased secretion of pancreatic enzymes in Type 1 insulin-dependent diabetics is more a consequence of defective enzyme release from zymogen stores than defective synthesis.In conclusion, our results indicate that changes in pancreatic enzyme secretion noted in patients do not always reflect changes in enzyme synthesis, and that the production of individual enzymes may diverge under certain circumstances. Based on the methodology described, it should be possible to develop more sensitive clinical tests of pancreatic function that provide information not only on the abiltiy of the pancreas to secrete enzymes under certain disease states, but also information on the gland''s synthetic activity     

饲料中添加磷虾水解物对大菱鲆幼鱼生长性能、体组成及相关酶活性的影响

选取初始体重为(9.46±0.01) g的大菱鲆(Scophthalmus maximus L.)为研究对象, 以30%鱼粉组为对照, 分别添加5%磷虾水解物(LKH)和10%磷虾水解物(HKH)以替代鱼粉蛋白, 配制3组等氮等脂的饲料, 在室内流水养殖系统进行为期10周的养殖实验, 旨在探究饲料中添加磷虾水解物对大菱鲆幼鱼生长性能、体组成及相关酶活性的影响。结果显示, HKH组大菱鲆幼鱼的特定生长率(SGR)、饲料效率(FE)、蛋白质效率比(PER)、蛋白质沉积率(PPV)均显著高于对照组(P<0.05), 但摄食率(FI)显著低于对照组(P<0.05); LKH、HKH组大菱鲆幼鱼肌肉总氨基酸含量和必需氨基酸含量显著高于对照组(P<0.05); LKH、HKH组肝脏谷草转氨酶(GOT)活性显著高于对照组(P<0.05), 而血清GOT活性与对照组无显著性差异(P>0.05); HKH组肝脏谷丙转氨酶(GPT)活性显著高于对照组和LKH组(P<0.05), 但血清GPT活性显著低于LKH组和对照组(P<0.05); HKH组肠道淀粉酶活性显著高于对照组(P<0.05), 同时随着磷虾水解物添加量的升高, 肠道及幽门盲囊胰蛋白酶活性先降低后升高, 且在HKH组显著高于对照组(P<0.05)。综上结果表明, 在饲料中添加10%磷虾水解物能够提高大菱鲆幼鱼的生长性能, 促进其氨基酸代谢和消化吸收。     

The digestive system of the “stick bug” Cladomorphus phyllinus (Phasmida,Phasmatidae): A morphological,physiological and biochemical analysis

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data.     

Physiological effects of enteral and parenteral feeding on pancreaticobiliary secretion in humans

In the nutritional management of digestive disorders, it is important to know the relative secretory and metabolic responses to enteral and parenteral feeding. Twenty-seven healthy volunteers were studied while receiving either oral drinks or duodenal infusions of a complex formula diet, duodenal or intravenous infusions of elemental (protein as free amino acids, low fat) formulae, or saline. Pancreaticobiliary secretory responses were measured by nasoduodenal polyethylene glycol perfusion and aspiration, while monitoring blood hormone and nutrient levels. Diets were matched for protein (1.5 g x kg(-1) x d(-1)) and energy (40 kcal x kg(-1) x d(-1)). Compared with placebo, all oroenteral diets stimulated amylase, lipase, trypsin, and bile acid secretion and increased plasma concentrations of gastrin and cholecystokinin, whereas intravenous feeding did not. The complex formula produced a similar response whether given as drinks or duodenal infusions. Changing the duodenal formula to elemental reduced enzyme secretion by 50%, independently of CCK. Higher increases in plasma insulin, glucose, and amino acids were noted with intravenous feeding. Delivering food directly to the intestine by a feeding tube does not reduce pancreaticobiliary secretion. Enteral "elemental" formulae diminish, but only intravenous feeding avoids pancreatic stimulation. Intravenous administration impairs metabolic clearance.     

Nutrient control of release of pancreatic enzymes in yellowtail (Seriola quinqueradiata): involvement of CCK and PY in the regulatory loop

Cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptides are key regulators of pancreatic enzyme secretion in vertebrates. CCK stimulates enzyme secretion whereas peptide Y (PY), a NPY-related peptide, plays an antagonistic role to that of CCK. In fish, very little is known about how different nutrients affect the synthesis of CCK and PY in the digestive tract, and the mechanism by which CCK and PY actually regulate digestive enzyme secretion is not well understood. In order to determine how different nutrients stimulate the synthesis of CCK and PY in yellowtail (Seriola quinqueradiata), CCK and PY mRNA levels in the digestive tract were measured after oral administration of a single bolus of either phosphate-buffered saline (PBS: control), starch (carbohydrate), casein (protein), oleic acid (fatty acid) or tri-olein (triglyceride). In addition, in order to confirm the synthesis and secretion of digestive enzymes, the mRNA levels and enzymatic activities of three digestive enzymes (lipase, trypsin and amylase) were also analyzed. Casein, oleic acid and tri-olein increased the synthesis of lipase, trypsin and amylase, while starch and PBS did not affect the activity of any of these enzymes. CCK mRNA levels rose, while PY mRNA levels were reduced in fish administered casein, oleic acid and tri-olein. These results suggest that in yellowtail, CCK and PY maintain antagonistic control of pancreatic enzyme secretion after intake of protein and/or fat.     

Two hypotheses on the feedback regulation of pancreatic enzyme secretion

We review the mechanisms underlying the feedback regulation of pancreatic enzyme secretion in response to a meal. Pancreatic enzyme secretion in the rat and pig is known to be regulated by a negative feedback mechanism mediated by intestinal trypsin and chymotrypsin. Such a mechanism has recently been noted in humans. The presence of these enzymes in the small intestine suppresses pancreatic enzyme secretion, whereas their removal increases it. Two novel peptides have been proposed to account for the stimulation of pancreatic enzyme secretion in response to feeding trypsin inhibitor. One was assumed to be present in rat pancreatic juice and the other to be spontaneously secreted from the rat small intestine. In either case, trypsin and trypsin inhibitors do not directly interact with the luminal surface of the small intestine, but their actions are mediated by a trypsin-sensitive, cholecystokinin-releasing peptide. This is a novel explanation of the well-recognized stimulation of pancreatic enzyme secretion in response to dietary protein intake.     

REGULATION OF AMYLASE,CELLULASE AND CHITINASE SECRETION IN THE DIGESTIVE TRACT OF THE TWO‐SPOTTED FIELD CRICKET,Gryllus bimaculatus

The secretion of amylase and cellulase in Gryllus bimaculatus is determined by increased food intake, whereby shortly after molting food consumption increases. About half of the standing amylase concentration (activity) in the endothelial cells can be secreted within 30 min. The peak of amylase and cellulase secretion that occurs in the photophase is related to the feeding peak in the previous scotophase. The secretion of chitinase on the other hand is primarily controlled by the molting cycle. Only amylase secretion was affected by calcium in the incubation medium, suggesting an apocrine release mechanism. Refeeding experiments (after 5 days without food) suggest that the release of amylase in response to a nutrient in the lumen (glucose) is not due to simple stimulation of exocytosis, but rather a stimulation of synthesis.     

西江鲤仔稚鱼生长及消化酶活性变化

为了探究了鲤(Cyprinus carpio)仔稚鱼的生长过程中主要消化酶活性变化规律, 实验测定了鲤从孵化出膜到40 日龄(日龄, Day after hatching)仔稚鱼期间的生长、可溶性蛋白含量和几种消化酶活性变化。结果显示: 仔稚鱼全长以及体重在15 日龄后增速加快, 特定生长率为14.81%。淀粉酶、脂肪酶、胰蛋白酶, 糜乳蛋白酶、碱性磷酸酶以及氨基肽酶在1 日龄仔鱼体内均能检测到活性。在仔稚鱼发育过程中, 其可溶性蛋白含量先下降后上升。仔鱼摄食前消化酶的活性出现一定的上升, 随着仔稚鱼消化系统发育以及营养方式的转变, 其消化酶活性从3到25 日龄处于不断的变化状态, 而25 日龄到实验结束鲤仔稚鱼的消化酶处于一种相对稳定的状态, 标志着其消化功能趋于完善。根据不同发育时期鲤消化酶活性的变化, 设计有机可腐化的材料制备人工鱼巢以提高人工鱼巢的生态修复功能, 降低饥饿对仔稚鱼成活率的影响。     

Digestive enzymes and metabolic profile of Pseudoplatystoma corruscans (Teleostei: Siluriformes) in response to diet composition

Digestive enzyme responsiveness to feeding and associated adjustments of metabolism can be used to derive nutritionally effective diet formulations. Juvenile pintado (Pseudoplatystoma corruscans) were fed different diets. After feeding, fish were killed and blood, liver and white muscle were collected to evaluate metabolites. Stomach along with anterior, middle and posterior intestine were sampled for enzyme analysis. Non-specific protease, trypsin, chymotrypsin, amylase and lipase were assayed. Crude protein (CP) did not induce proteolytic activity; highest protease activities were observed in the stomach. Amylase was higher in the stomach in fish feeding on diets containing 13-25% starch. Lipase activity was observed along the gastrointestinal tract, with the highest activities observed in the middle section. The metabolic profile of white muscle was not affected by CP. In contrast, some plasma and liver metabolites were altered concomitant with changes in the digestive enzymes. Amino acid catabolism was increased. Digestion in pintado was responsive to cornstarch, reflected in intermediary metabolism; proteolytic activities of the digestive tract seem to be sufficient to deal with large amounts of dietary protein. As a result, we are able to recommend a balance between protein and energetic compounds, such as lipids and carbohydrates, in the diet to optimize fish growth.     

Dietary protein requirement for captive juvenile green turtles (Chelonia mydas)

Head-starting programs are extremely important for restoring the population of sea turtles in wild whereas husbandry conditions and feeding regimens of captive turtles are still limited. In the current study, the optimal dietary protein requirement for green turtle (Chelonia mydas) was investigated to support rearing in head-starting programs. Twenty-five-day-old turtles (44.5–46.2 g body weight, n = 45) were randomly distributed into 15 experimental plastic tanks, comprising three treatment replications of 3 turtles each. They were fed fishmeal-based feeds containing different levels of protein (30%, 35%, 40%, 45%, and 50%) for 8 weeks. At the end of feeding trial, growth performance (specific growth rate = 1.86% body weight/day) and feed utilization (protein efficiency ratio = 3.30 g gain/g protein) were highest in turtles fed with 40% protein in feed (p < .05). These nutritional responses were significantly supported by specific activities of fecal digestive enzymes, especially trypsin, chymotrypsin, amylase, and the amylase/trypsin ratio. Also, this dietary level improved the deposition of calcium and phosphorus in carapace, supporting a hard carapace and strong healthy bones. There were no negative effects in general health status of reared turtles, as indicated by hematological parameters. Based on a broken-line analysis between dietary protein levels and specific growth rate, the optimal protein level for green turtles was estimated as 40.6%. Findings from the current study support the use of artificial diets of specific protein levels to rear captive green turtle before release to natural habitats.     

Insectivore to frugivore: ontogenetic changes in gut morphology and digestive enzyme activity in the characid fish Brycon guatemalensis from Costa Rican rain forest streams

Brycon guatemalensis , a Neotropical characid fish, consumes an entirely terrestrial diet, shifting from eating insects as juveniles to fruits and leaves as adults. Juvenile and larger‐sized fish collected in the Rio Puerto Viejo at the La Selva Biological Station in Costa Rica were studied to test the hypotheses that, with ontogeny, (1) relative gut length increases, (2) pyloric caeca arrangement and number remain unchanged and (3) pepsin, trypsin and lipase activities decrease, while α‐amylase activity increases. These hypotheses were mainly supported in that larger fish had longer guts, unchanged pyloric caeca arrangement but fewer caeca, and, at both environmental and standard temperatures for the enzyme assays, lower pepsin and trypsin activities but higher α‐amylase activities than the juveniles. Only lipase, among the digestive enzymes, exhibited the unexpected outcome of either not differing significantly in activity (per g of tissue) between juveniles and larger fish or being significantly higher (per mg of protein) in larger fish. The overall results support the view that B. guatemalensis is specialized morphologically and biochemically to function first as a carnivore and then as a herbivore during its life history.     

IN VITRO ANTIVIRAL ACTIVITY OF AN ALKALINE TRYPSIN FROM THE DIGESTIVE JUICE OF Bombyx mori LARVAE AGAINST NUCLEOPOLYHEDROVIRUS

Alkaline trypsin protein of molecular mass 25,436 Da purified from the digestive juice of Bombyx mori larvae indicated strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) under in vitro conditions. Partial N-terminal amino acid sequence of the protein was determined and the cDNA was cloned based on the amino acid sequence. A homology search of the deduced amino acid sequence of the cDNA showed 55% identity with Helicoverpa armigera trypsin and the active site of this protein was completely conserved. Hence, the protein was designated B. mori trypsin (Bmtryp). The results suggest that Bmtryp, an insect digestive enzyme, can be a potential antiviral factor against BmNPV at the initial site of viral infection.     

Digestive enzymes of two freshwater fishes (Limia vittata and Gambusia punctata) with different dietary preferences at three developmental stages

The variation of activity of some digestive enzymes was studied in three age groups of two freshwater endemic fishes from Cuba: Limia vittata and Gambusia punctata. Trypsin, chymotrypsin and amylase activities showed a different pattern between both species. Trypsin and chymotrypsin activity increased with the age of fishes, while amylase activity decreased. The highest activity of trypsin and chymotrypsin was registered in G. punctata while the highest amylase activity was detected in L. vittata. Zymograms revealed proteases with molecular masses similar to trypsin and chymotrypsin reported for other fish species. Amylase electrophoresis showed the presence of this enzyme; in L. vittata amylase zymograms showed two bands with molecular masses of 175 and 100 kDa and in G. punctata four bands of 175, 100, 46 and 30 kDa respectively were found. The activity of the digestive enzymes can be used as an effective indicator of the feeding habits and the development of the digestive tracts in L. vittata and G. punctata.     

Localization of pancreatic enzyme secretion-stimulating activity and trypsin inhibitory activity in zymogen granule of the rat pancreas

The intracellular localization of pancreatic enzyme secretion-stimulating activity in rat pancreas was investigated. We found and purified a pancreatic enzyme secretion-stimulating peptide from rat bile/pancreatic juice. The peptide is trypsin-sensitive (showing temporary trypsin inhibitory activity), and it is hypothesized that it acts as a trypsin-sensitive mediator in the feedback regulation of diet-induced pancreatic enzyme secretion. The zymogen granule fraction was purified 5-fold by ultracentrifugation by the Percoll density gradient method. The purity of the zymogen granule fraction was determined from the specific amylase activity and electron microscopic morphology. The specific enzyme activities of amylase and trypsin and the trypsin inhibitory activity increased in parallel during the purification, and the pancreatic enzyme secretion-stimulating activity was also localized in the zymogen granule fraction. These results suggest that the pancreatic enzyme secretion-stimulating peptide originates from the acinar cells, and that it is secreted through exocytosis of zymogen granules into the small intestine, its ratio to trypsin thus remaining constant. This idea supports our hypothesis that the stimulating peptide acts as a mediator for the feedback regulation of pancreatic enzyme secretion by trypsin.