Difference in Performance of EPI Pigs Fed Either Lipase-Predigested or Creon®-Supplemented Semielemental Diet.

Pancreatic enzyme replacement therapy (PERT) and fat predigestion are key in ensuring the optimal growth of patients with cystic fibrosis. Our study attempted to highlight differences between fat predigestion and conventional PERT on body composition of young pigs with exocrine pancreatic insufficiency (EPI). EPI and healthy pigs were fed with high-fat diet for six weeks. During the last two weeks of the study, all pigs received additional nocturnal alimentation with Peptamen AF (PAF) and were divided into three groups: H-healthy pigs receiving PAF; P-EPI pigs receiving PAF+PERT; and L-EPI pigs receiving PAF predigested with an immobilized microbial lipase. Additional nocturnal alimentation increased the body weight gain of EPI pigs with better efficacy in P pigs. Humerus length and area in pigs in groups L and P were lower than that observed in pigs in group H (p value 0.005-0.088). However, bone mineral density and strength were significantly higher in P and L as compared to that of H pigs (p value 0.0026-0.0739). The gut structure was improved in P pigs. The levels of neurospecific proteins measured in the brain were mainly affected in P and less in L pigs as compared to H pigs. The beneficial effects of the nocturnal feeding with the semielemental diet in the prevention of EPI pigs' growth/development retardation are differently modified by PERT or fat predigestion in terms of growth, bone properties, neurospecific protein distribution, and gut structure.

Maternal Immunoglobulins in Infants-Are They More Than Just a Form of Passive Immunity?

In the present review, we highlight the possible "extra-immunological" effects of maternal immunoglobulins (Ig) transferred to the blood circulation of offspring, either via the placenta before birth or via the colostrum/milk across the gut after birth in different mammalian species. Using the newborn pig as a model, since they are naturally born agammaglobulinemic, intravenously (i.v.) infused purified serum Ig rapidly improved the vitality, suckling behavior, and ensured the survival of both preterm and term piglets. In further studies, we found that proper brain development requires i.v. Ig supplementation. Studies have reported on the positive effects of i.v. Ig treatment in children with epilepsy. Moreover, feeding newborn pigs an elementary diet supplemented with Ig improved the gut structure, and recently a positive impact of enteral or parenteral Ig supplementation on the absorption of polyunsaturated fatty acids (PUFAs) was observed in the newborn pig. Summarized, our own results and those found in the literature, indicate the existence of important extra-immune effects of maternal Ig, in addition to the classical protective effects of transferred maternal passive immunity, including effects on the development of the brain, gut, and possibly other organ systems in the neonate. These additional properties of circulating Ig could have an impact on care guidelines for human neonates, especially those born prematurely with low plasma Ig levels.

Maternal HMB treatment affects bone and hyaline cartilage development in their weaned piglets via the leptin/osteoprotegerin system.

It has been demonstrated in animal studies that prenatal administration of ß-hydroxy-ß-methylbutyrate (HMB, metabolite of leucine) influences general growth and mechanical endurance of long bones in newborn offspring in sex-dependent manner. The present experiment was conducted to evaluate the effect of HMB treatment of pregnant sows on bone development in offspring at weaning. From 70th day until the 90th day of gestation, sows received either a basal diet (n = 12) or the same diet supplemented with HMB (n = 12) at the dose of 0.2 g/kg of body weight/day. Femora obtained from six males and females in each group weaned at the age of 35 days were examined. Maternal HMB treatment significantly enhanced body weight and changed bone morphology increasing femur mechanical strength in both sexes. Maternal HMB supplementation also elevated bone micro- and macroelement concentrations and enhanced content of proteoglycans in articular cartilage. Based on the obtained results, it can be concluded that maternal HMB supplementation in the mid-gestation period significantly accelerated bone development in both sexes by upregulation of a multifactorial system including leptin and osteoprotegerin. However, the sex (irrespective of the HMB treatment) was the factor which influenced the collagen structure in cartilages and trabecular bone, as demonstrated both by the Picrosirius red staining and performed analysis of thermal stability of collagenous tissues. The structural differences in collagen between males and females were presumably related to a different collagen maturity. No studies conducted so far provided a detailed morphological analysis of bone, articular cartilage, growth plate and the activities of the somatotropic and pituitary-gonadal axes, as well as leptin/osteoprotegerin system in weaned offspring prenatally treated with HMB. This study showed also the relationship between the maternal HMB treatment and bone osteometric and mechanical traits, hormones, and growth and bone turnover markers such as leptin, osteoprotegerin and insulin-like growth factor-1.

Dietary 2-oxoglutarate prevents bone loss caused by neonatal treatment with maximal dexamethasone dose.

Synthetic glucocorticoids (GCs) are widely used in the variety of dosages for treatment of premature infants with chronic lung disease, respiratory distress syndrome, allergies, asthma, and other inflammatory and autoimmune conditions. Yet, adverse effects such as glucocorticoid-induced osteoporosis and growth retardation are recognized. Conversely, 2-oxoglutarate (2-Ox), a precursor of glutamine, glutamate, and collagen amino acids, exerts protective effects on bone development. Our aim was to elucidate the effect of dietary administered 2-Ox on bone loss caused by neonatal treatment with clinically relevant maximal therapeutic dexamethasone (Dex) dose. Long bones of neonatal female piglets receiving Dex, Dex+2-Ox, or untreated were examined through measurements of mechanical properties, density, mineralization, geometry, histomorphometry, and histology. Selected hormones, bone turnover, and growth markers were also analyzed. Neonatal administration of clinically relevant maximal dose of Dex alone led to over 30% decrease in bone mass and the ultimate strength ( P < 0.001 for all). The length (13 and 7% for femur and humerus, respectively) and other geometrical parameters (13-45%) decreased compared to the control ( P < 0.001 for all). Dex impaired bone growth and caused hormonal imbalance. Dietary 2-Ox prevented Dex influence and vast majority of assessed bone parameters were restored almost to the control level. Piglets receiving 2-Ox had heavier, denser, and stronger bones; higher levels of growth hormone and osteocalcin concentration; and preserved microarchitecture of trabecular bone compared to the Dex group. 2-Ox administered postnatally had a potential to maintain bone structure of animals simultaneously treated with maximal therapeutic doses of Dex, which, in our opinion, may open up a new opportunity in developing combined treatment for children treated with GCs. Impact statement The present study has showed, for the first time, that dietary 2-oxoglutarate (2-Ox) administered postnatally has a potential to improve/maintain bone structure of animals simultaneously treated with maximal therapeutic doses of dexamethasone (Dex). It may open the new direction in searching and developing combined treatment for children treated with glucocorticoids (GCs) since growing group of children is exposed to synthetic GCs and adverse effects such as glucocorticoid-induced osteoporosis and growth retardation are recognized. Currently proposed combined therapies have numerous side effects. Thus, this study proposed a new direction in combined therapies utilizing dietary supplementation with glutamine derivative. Impairment caused by Dex in presented long bones animal model was prevented by dietary supplementation with 2-Ox and vast majority of assessed bone parameters were restored almost to the control level. These results support previous thesis on the regulatory mechanism of nutrient utilization regulated by glutamine derivatives and enrich the nutritional science.

Dietary alpha-ketoglutarate increases cold tolerance in Drosophila melanogaster and enhances protein pool and antioxidant defense in sex-specific manner.

Alpha-ketoglutarate (AKG) is an important intermediate in Krebs cycle which bridges the metabolism of amino acids and carbohydrates. Its effects as a dietary supplement on cold tolerance were studied in Drosophila melanogaster Canton S. Two-day-old adult flies fed at larval and adult stages with AKG at moderate concentrations (5-10mM) recovered faster from chill coma (0°C for 15min or 3h) than control ones. The beneficial effect of AKG on chill coma recovery was not found at its higher concentrations, which suggests hormetic like action of this keto acid. Time of 50% observed mortality after 2h recovery from continuous cold exposure (-1°C for 3-31h) (LTi50) was higher for flies reared on 10mM AKG compared with control ones, showing that the diet with AKG enhanced insect cold tolerance. In parallel with enhancement of cold tolerance, dietary AKG improved fly locomotor activity. Metabolic effects of AKG differed partly in males and females. In males fed on AKG, there were no differences in total protein and free amino acid levels, but the total antioxidant capacity, catalase activity and low molecular mass thiol content were higher than in control animals. In females, dietary AKG promoted higher total antioxidant capacity and higher levels of proteins, total amino acids, proline and low molecular mass thiols. The levels of lipid peroxides were lower in both fly sexes reared on AKG as compared with control ones. We conclude that both enhancement of antioxidant system capacity and synthesis of amino acids can be important for AKG-promoted cold tolerance in D. melanogaster. The involvement of AKG in metabolic pathways of Drosophila males and females is discussed.

Dietary 2-oxoglutarate mitigates gastrectomy-evoked structural changes in cartilage of female rats.

Gastrectomy (Gx) leads to osteopenia/osteoporosis in humans and animals. However, little is known about the influence of Gx on the cartilage in this regard. Recent studies have demonstrated a protective effect of 2-oxoglutaric acid (2-Ox) on bone and cartilage. Hence, the purpose of this study was to investigate whether 2-Ox can mitigate eventual Gx-induced cartilage impairment. Twenty female Sprague-Dawley rats were subjected to Gx and randomly divided into two groups: Gx + 2-Ox and Gx. Another 20 rats were sham-operated (ShO) and randomly divided into two groups: ShO + 2-Ox and ShO. The daily dose of 2-Ox administered to the rats in the drinking water was 0.43 g per 100 g rat. After eight weeks, rats were euthanized and femora and tibiae were collected. Histology and histomorphometry analyses of the articular cartilage and the growth plate were done. Gx resulted in a 32% (±44.5 femur, ±35.8 tibia) decrease in overall thickness of articular cartilage in both bones (femur: ShO 279.1 ± 48.5 vs. Gx 190.2 ± 38.4 µm, tibia: ShO 222.9 ± 50.3 µm vs. Gx 151.3 ± 52.6 µm) (in some zones up to 58 ± 28.0%), and in the growth plate up to 20% (±22.4) (femur: ShO 243.0 ± 34.0 vs. Gx 207.0 ± 33.7 µm, tibia: ShO 220.0 ± 24.6 µm vs. Gx 171.1 ± 16.1 µm). Gx altered the spatial distribution of thick and thin collagen fibers, and chondrocyte shape and size. 2-Ox administration prevented the reduction in both cartilages thickness (Gx + 2-Ox: articular cartilage 265.2 ± 53.8 µm, 235.6 ± 42.7 µm, growth plate 236.7 ± 39.2 µm, 191.3 ± 16.5 µm in femur and tibia, respectively), and abolished the spatial changes in collagen distribution and structure induced by Gx. Gx affects cartilage structure and thickness, however, 2-Ox administration mitigates these effects and showed protective and stimulatory properties. Our observations suggest that dietary 2-Ox can be used to offset some of the changes in hyaline cartilage, in particular articular cartilage, following bariatric surgeries.

Decreased insulin secretion and glucose clearance in exocrine pancreas-insufficient pigs.

The effect of exocrine pancreatic function on the glucose-mediated insulin response and glucose utilization were studied in an exocrine pancreas-insufficient (EPI) pig model. Five 10-week-old EPI pigs after pancreatic duct ligation and 6 age-matched, non-operated control pigs were used in the study. Blood glucose, plasma insulin and C-peptide concentrations were monitored during meal (MGTT), oral (OGTT) and intravenous (IVGTT) glucose tolerance tests. Upon post-mortem examination, the pancreatic remnants of the EPI pigs showed acinar fibrotic atrophy but normal islets and ß-cell morphology. The EPI pigs displayed increased fasting glucose concentrations compared with control animals (6.4 ± 0.4 versus 4.8 ± 0.1 mmol l(-1) , P < 0.0001) but unchanged insulin concentrations (2.4 ± 0.6 versus 2.1 ± 0.2 pmol l(-1) ). During the OGTT and IVGTT, the EPI pigs showed slower, impaired glucose utilization, with the disruption of a well-timed insulin response. Plasma C-peptide concentrations confirmed the delayed insulin response during the IVGTT in EPI pigs. Oral pancreatic enzyme supplementation (PES) of EPI pigs improved glucose clearance during IVGTT [AUC(glucose) 1295 ± 70 mmol l(-1) × (120 min) in EPI versus 1044 ± 32 mmol l(-1) × (120 min) in EPI + PES, P < 0.0001] without reinforcing the release of insulin [AUC(C-peptide) 14.4 ± 3.8 nmol l(-1) × (120 min) in EPI versus 6.4 ± 1.3 nmol l(-1) × (120 min) in EPI + PES, P < 0.002]. The results suggest the existence of an acino-insular axis regulatory communication. The presence of pancreatic enzymes in the gut facilitates glucose utilization in an insulin-independent manner, indicating the existence of a gut-derived pancreatic enzyme-dependent mechanism involved in peripheral glucose utilization.

A piglet with surgically induced exocrine pancreatic insufficiency as an animal model of newborns to study fat digestion.

The maldigestion and malabsorption of fat in infants fed milk formula results due to the minimal production of pancreatic lipase. Thus, to investigate lipid digestion and absorption and mimic the situation in newborns, a young porcine exocrine pancreatic insufficient (EPI) model was adapted and validated in the present study. A total of thirteen EPI pigs, aged 8 weeks old, were randomised into three groups and fed either a milk-based formula or a milk-based formula supplemented with either bacterial or fungal lipase. Digestion and absorption of fat was directly correlated with the addition of lipases as demonstrated by a 30% increase in the coefficient of fat absorption. In comparison to the control group, a 40 and 25% reduction in total fat content and 26 and 45% reduction in n-3 and n-6 fatty acid (FA) content in the stool was observed for lipases 1 and 2, respectively. Improved fat absorption was reflected in the blood levels of lipid parameters. During the experiment, only a very slight gain in body weight was observed in EPI piglets, which can be explained by the absence of pancreatic protease and amylase in the gastrointestinal tract. This is similar to newborn babies that have reduced physiological function of exocrine pancreas. In conclusion, we postulate that the EPI pig model fed with infant formula mimics the growth and lipid digestion and absorption in human neonates and can be used to elucidate further importance of fat and FA in the development and growth of newborns, as well as for testing novel formula compositions.

Melanoidins isolated from heated potato fiber (Potex) affect human colon cancer cells growth via modulation of cell cycle and proliferation regulatory proteins.

Melanoidins are brown, nitrogen containing, high molecular weight end products of Maillard reaction with poorly established activity towards tumor cells. The goal of present study was to verify whether both heated potato fiber Potex extract (180°C for 2h) and melanoidins isolated from the extract exerts growth-inhibiting activity in human colon cancer cells in vitro. The cells of LS180 colon cancer cell line were tested upon treatment with roasted potato fiber extract (AM4) as well as with high (HMW) and low (LMW) molecular weight fractions isolated from the extract, since both may be regarded as/or contain melanoidins. The tested compounds at concentration of 1000 µg/ml reduced cell growth down to 45%, 69% and 54%, respectively. Furthermore, deregulated ERK1/2 signaling was revealed upon treatment. Moreover, multiple alternations in cell cycle regulators activity were found (i.e. cyclinD1, cyclin-dependent kinase 4 and 6, p21, p27, p53, pRb) leading to cell cycle cessation in G0 phase. Importantly, LMW compounds revealed markedly stronger potential to alter specific molecular targets comparing to HMW compounds. Summarizing, the results emphasize that both high and low molecular weight melanoidins contribute to antiproliferative activity of heated potato fiber in LS180 colon cancer cells in vitro.

The effect of dietary administration of 2-oxoglutaric acid on the cartilage and bone of growing rats.

2-Oxoglutaric acid (2-Ox), a precursor to hydroxyproline - the most abundant amino acid in bone collagen, exerts protective effects on bone development during different stages of organism development; however, little is known about the action of 2-Ox on cartilage. The aim of the present study was to elucidate the influence of dietary 2-Ox supplementation on the growth plate, articular cartilage and bone of growing rats. A total of twelve male Sprague-Dawley rats were used in the study. Half of the rats received 2-oxoglutarate at a dose of 0·75 g/kg body weight per d in their drinking-water. Body and organ weights were measured. Histomorphometric analyses of the cartilage and bone tissue of the femora and tibiae were conducted, as well as bone densitometry and peripheral quantitative computed tomography (pQCT). Rats receiving 2-Ox had an increased body mass (P<0·001) and absolute liver weight (P=0·031). Femoral length (P=0·045) and bone mineral density (P=0·014), overall thickness of growth plate (femur P=0·036 and tibia P=0·026) and the thickness of femoral articular cartilage (P<0·001) were also increased. 2-Ox administration had no effect on the mechanical properties or on any of the measured pQCT parameters for both bones analysed. There were also no significant differences in histomorphometric parameters of tibial articular cartilage and autofluorescence of femoral and tibial growth plate cartilage. Dietary supplementation with 2-Ox to growing rats exerts its effects mainly on cartilage tissue, having only a slight influence on bone. The effect of 2-Ox administration was selective, depending on the particular bone and type of cartilage analysed.

Can 2-oxoglutarate prevent changes in bone evoked by omeprazole?

OBJECTIVE: Proton-pump inhibitors, such as omeprazole, are widely used in the prevention and treatment of gastroesophageal diseases. However, an association between proton-pump inhibitors and the increased risk of bone fractures has been observed, especially in patients treated for extended periods. Conversely, 2-oxoglutarate, a precursor of hydroxyproline, the most abundant amino acid in bone collagen, counteracts the bone loss. The aim of the present study was to elucidate the influence of omeprazole on bone and investigate whether dietary 2-oxoglutarate supplementation could prevent the effects of omeprazole. METHODS: Eighteen male Sprague-Dawley rats were used. Rats received omeprazole in the diet and 2-oxoglutarate in the drinking water. Body and organ weights and serum concentrations of cholecystokinin and gastrin were measured. The femurs, tibias, and calvarias were collected. Histomorphometric analysis of bone and cartilage tissues was conducted. Bone densitometric and peripheral quantitative computed tomographic analyses of the femur and tibia were performed. RESULTS: Omeprazole decreased the femur and tibia weights, the mechanical properties of the femur, the volumetric bone density and content, the trabecular and cortical bone mineral content, the total, trabecular, and cortical bone areas, the mean cortical thickness, and the periosteal circumference of the femur. Omeprazole had a minor effect on the examined bone morphology and exerted negligible effects on the cartilage. 2-Oxoglutarate lowered the gastrin concentration. CONCLUSIONS: Omeprazole treatment exerts its effects mostly on bone mineralization and cancellous bone, adversely affecting bone properties. This adverse effect of omeprazole was not markedly abolished by 2-oxoglutaric acid, which acted as an anti-hypergastrinemic agent.

Alpha-ketoglutarate (AKG) inhibits proliferation of colon adenocarcinoma cells in normoxic conditions.

BACKGROUND AND OBJECTIVE: Alpha-ketoglutarate (AKG), a key intermediate in Krebs cycle, is an important biological compound involved in the formation of amino acids, nitrogen transport, and oxidation reactions. AKG is already commercially available as a dietary supplement and its supplementation with glutamine, arginine, or ornithine alpha-ketoglutarate has been recently considered to improve anticancer immune functions. It is well documented that AKG treatment of Hep3B hepatoma cells in hypoxia induced HIF-alpha (hypoxia-inducible factor) degradation and reduced vascular endothelial growth factor (VEGF) synthesis. Moreover, AKG showed potent antitumor effects in murine tumor xenograft model, inhibiting tumor growth, angiogenesis, and VEGF gene expression. However, the mechanisms of its anticancer activity in normoxia have not been examined so far. RESULTS: Here, we report that in normoxia, AKG inhibited proliferation of colon adenocarcinoma cell lines: Caco-2, HT-29, and LS-180, representing different stages of colon carcinogenesis. Furthermore, AKG influenced the cell cycle, enhancing the expression of the inhibitors of cyclin-dependent kinases p21 Waf1/Cip1 and p27 Kip1. Moreover, expression of cyclin D1, required in G1/S transmission, was decreased, which accompanied with the significant increase in cell number in G1 phase. AKG affected also one the key cell cycle regulator, Rb, and reduced its activation status. CONCLUSION: In this study for the first time, the antiproliferative activity of AKG on colon adenocarcinoma Caco-2, HT-29, and LS-180 cells in normoxic conditions was revealed. Taking into consideration an anticancer activity both in hypoxic and normoxic conditions, AKG may be considered as a new potent chemopreventive agent.

Potato fiber protects the small intestinal wall against the toxic influence of acrylamide.

OBJECTIVE: Acrylamide is a neurotoxic, genotoxic substance present in many commonly consumed food products and has been shown to have carcinogenic effects in rodents. The protective effects (if any) of potato fiber preparations, composed of cell wall material from potatoes, against the toxic influence of dietary acrylamide on the small intestinal wall were investigated. METHODS: Male mice of the BALB/c strain were used in the study. Acrylamide was administered to the mice in their drinking water (0.5 mg/kg of body weight per day) and one of two types of potato fiber preparations (heated or raw potato fiber preparation) was added to their feed (2% addition to their feed). Histomorphometry of the small intestinal wall, hemoglobin adducts of acrylamide, animal weight, and feed and water consumption analyses were performed. RESULTS: Acrylamide altered the morphology and histology of the small intestinal wall, decreasing proliferation, myenteron and submucosal thicknesses, villus length, fractal dimension, crypt depth, crypt number, and the small intestinal absorptive surface. Conversely, apoptosis, hemoglobin adduct levels, intensity of epithelium staining, enterocyte number, villus epithelial thickness, and crypt width and parameters associated with nerve ganglia were increased. The two potato fiber preparations that were used abolished the negative influences of acrylamide on the small intestinal wall and had no influence on the hemoglobin adduct levels of acrylamide. CONCLUSION: The negative impact of acrylamide on the histologic structure, regeneration, and innervation of the small intestinal wall and the absorptive function of the small intestinal mucosa can be abolished by dietary potato fiber preparations.

Antiproliferative activity of melanoidins isolated from heated potato fiber (potex) in glioma cell culture model.

Potex constitutes a potato fiber preparation widely used as an ingredient to meat and bakery products which thermal treatment results in creation of new compounds. Melanoidins are high molecular weight brown end products of Maillard reaction, and few data presenting tumor cell growth inhibiting activity of melanoidins have been reported. Thus, in present study we utilized water extract of Potex roasted (180 °C for 2 h), whose chemical characterization revealed the presence of melanoidin complexes. Heated Potex extract inhibited C6 glioma cell proliferation in a dose-dependent manner measured by MTT method. High molecular weight components present in initial extract were responsible for stronger antiproliferative effect compared with low molecular weight fraction. Impaired MAPK (mitogen-activated protein kinase) and Akt signaling was found in cells treated with the extract. Moreover, flow cytometry analyses revealed the extract to induce G1/S arrest in glioma cells. Simultaneously, Western blot analysis showed elevated levels of p21 protein with concomitant decrease of cyclin D1. In conclusion, observed antiproliferative activity of melanoidins present in heated Potex was linked to disregulated MAPK and Akt signaling pathways, as well as to cell cycle cessation. These results suggest potential application of Potex preparation as a functional food ingredient and chemopreventive agent.

Dietary supplementation with phytohemagglutinin in combination with alpha-ketoglutarate limits the excretion of nitrogen via urinary tract.

The aim of the study was to evaluate the effect of both phytohaemagglutinin (PHA) alone, and in combination with alpha-ketoglutaric acid (AKG), on nitrogen elimination via the urinary tract as opposed to the gastrointestinal tract of rats. In experiment 1, rats were assigned to one of two experimental groups, (1) Control and (2) PHA, whilst in experiment 2, rats were assigned to one of three experimental groups, (1) Control, (2) AKG, and (3) AKG+PHA. AKG was administered via drinking water, while PHA was administered via a stomach tube. The stock solution of crude PHA in 0.9 % NaCl, was (20 % w/v) in water: 50 mg PHA/ml, 20 ml/kg body wt. Rats were 7 weeks old at the start of the experiments. Significantly lower daily weight gains in the AKG+PHA and PHA groups (p<0.05) were observed compared to the Control and AKG groups. Increased duodenal crypt depth (138 % ; p<0.05) was noticeable in the AKG+PHA group cf Controls; however, there was no significant difference in the thickness of the tunica mucosa. In the AKG+PHA group, the expression of neuropeptide Y (NPY) in the granula of neuronal cells of the submucosal parasympathetic ganglia was noticeable, although no expression was found in goblet cells. Finally, significant reduction in N excretion in urine was observed in the AKG+PHA, compared with the Control groups (p< 0.05). It is concluded that a combined PHA and AKG treatment stimulated the small bowel growth via enhanced epithelial turnover, reduced the N excreted in urine and increased the N in faeces.

Food and secretagogue stimulation decrease the digestive enzyme content remaining in the rat pancreas.

The aim of the study reported here was to investigate changes in the digestive enzyme content in the pancreas after food and secretagogue stimulation. Rats from which food had been withheld overnight were either fed (between 6 and 8 a.m.) or not before euthanasia and pancreatic excision (at 8 a.m.: 21 not fed and 21 fed) and at 4 (12 p.m.: six not fed and six fed) and 8 h later (4 p.m.: six not fed and six fed). Another 16 rats were anesthetized, fitted with jugular vein and pancreatic duct catheters, and infused with the secretagogues, CCK-33 and secretin, during 1.5 h of pancreatic juice collection before euthanasia and pancreatic excision. The pancreata were homogenized, and total soluble protein and individual enzyme (trypsin and amylase) tissue contents were analyzed. Results indicated lower amounts of protein and enzymes remaining in the pancreata of the fed, compared with non-fed rats. Enzyme values indicated recovery within four hours in fed rats, but non-fed rats also had increased values during daytime. High enzyme secretion during the high dose of hormonal stimulation was reflected in lower enzyme values remaining in the pancreas, compared with that in response to low-dose stimulation. Results indicated that stimulation of the pancreas, either by food ingestion or exogenous secretagogues, lowers the amounts of digestive enzymes remaining in the pancreas, and imply that stimulation and circadian rhythms influence the pancreatic enzyme content at euthanasia. This finding should be borne in mind in interpretation of data from pancreatic studies.