Amylase mRNA Research Articles

Skeletal Muscle Myokine Expression in Critical Illness, Association With Outcome and Impact of Therapeutic Interventions.

Muscle expresses and secretes several myokines that bring about benefits in distant organs. We investigated the impact of critical illness on muscular expression of irisin, kynurenine aminotransferases, and amylase; association with clinical outcome; and impact of interventions that attenuate muscle wasting/weakness. We studied critically ill patients who participated in 2 randomized controlled trials (EPaNIC/NESCI) and documented time profiles in critically ill mice. Included in the study were 174 intensive care unit (ICU) patients (day 8 ± 1) vs 19 matched controls, and 60 mice subjected to surgery/sepsis vs 60 pair-fed healthy mice. Interventions studied included 7-day neuromuscular electrical stimulation (NMES), and withholding parenteral nutrition (PN) in the first ICU week (late PN) vs early PN. The main outcome measures were FNDC5 (irisin- precursor), KYAT1, KYAT3, and amylase mRNA expression in skeletal muscle. Critically ill patients showed 34% to 80% lower mRNA expression of FNDC5, KYAT1, and amylases than controls (P < .0001). Critically ill mice showed time-dependent reductions in all mRNAs compared with healthy mice (P ≤ .04). The lower FNDC5 expression in patients was independently associated with a higher ICU mortality (P = .015) and ICU-acquired weakness (P = .012), whereas the lower amylase expression in ICU survivors was independently associated with a longer ICU stay (P = .0060). Lower amylase expression was independently associated with a lower risk of death (P = .048), and lower KYAT1 expression with a lower risk of weakness (P = .022). NMES increased FNDC5 expression compared with unstimulated muscle (P = .016), and late PN patients had a higher KYAT1 expression than early PN patients (P = .022). Expression of the studied myokines was affected by critical illness and associated with clinical outcomes, with limited effects of interventions that attenuate muscle wasting or weakness.

Loss of LAT1 sex-dependently delays recovery after caerulein-induced acute pancreatitis.

BACKGROUNDThe expression of amino acid transporters is known to vary during acute pancreatitis (AP) except for LAT1 (slc7a5), the expression of which remains stable. LAT1 supports cell growth by importing leucine and thereby stimulates mammalian target of rapamycin (mTOR) activity, a phenomenon often observed in cancer cells. The mechanisms by which LAT1 influences physiological and pathophysiological processes and affects disease progression in the pancreas are not yet known.AIMTo evaluate the role of LAT1 in the development of and recovery from AP.METHODSAP was induced with caerulein (cae) injections in female and male mice expressing LAT1 or after its knockout (LAT1 Cre/LoxP). The development of the initial AP injury and its recovery were followed for seven days after cae injections by daily measuring body weight, assessing microscopical tissue architecture, mRNA and protein expression, protein synthesis, and enzyme activity levels, as well as by testing the recruitment of immune cells by FACS and ELISA.RESULTSThe initial injury, evaluated by measurements of plasma amylase, lipase, and trypsin activity, as well as the gene expression of dedifferentiation markers, did not differ between the groups. However, early metabolic adaptations that support regeneration at later stages were blunted in LAT1 knockout mice. Especially in females, we observed less mTOR reactivation and dysfunctional autophagy. The later regeneration phase was clearly delayed in female LAT1 knockout mice, which did not regain normal expression of the pancreas-specific differentiation markers recombining binding protein suppressor of hairless-like protein (rbpjl) and basic helix-loop-helix family member A15 (mist1). Amylase mRNA and protein levels remained lower, and, strikingly, female LAT1 knockout mice presented signs of fibrosis lasting until day seven. In contrast, pancreas morphology had returned to normal in wild-type littermates.CONCLUSIONLAT1 supports the regeneration of acinar cells after AP. Female mice lacking LAT1 exhibited more pronounced alterations than male mice, indicating a sexual dimorphism of amino acid metabolism.

Morphological and biochemical changes in the pancreas associated with acute systemic hypoxia

This study aimed to investigate the changes associated with acute systemic hypoxia in the endocrine system, particularly in pancreatic tissues. The investigation was based on macroscopic, pathohistological, biochemical, and molecular biological findings in cell lines and human cadavers. The results showed that cases of death due to asphyxia more frequently showed severe subcapsular/interstitial hemorrhage versus the other causes of death. Histological examination showed that asphyxia cases were associated with severe morphological changes. Although measured insulin levels in the asphyxia were higher compared to other causes of death, no differences were noted for the glucagon and amylase levels with regard to the cause of death. Increased blood insulin levels were not associated with macro- and micromorphological changes, and did not show any association with glucose or cortisol levels. The experiment conducted under hypoxic conditions in cultured cells demonstrated that insulin mRNA expression and insulin protein levels peaked at 10 min after hypoxia exposure. However, there were no changes in either the amylase mRNA or protein levels. Corticosterone level peaked at 120 min after exposure to hypoxic conditions. Overall, acute systemic hypoxic conditions can directly affect the mechanisms involved in pancreatic insulin secretion.

Siam weed (Chromolaena odorata) meal as digestive enhancer and replacer of DORB in the diet ofL. rohita: Effect on growth, digestive enzymes and amylase gene expression

A 60-day feeding trial was conducted to evaluate growth response, digestive enzyme activities, digestibility and amylase gene expression of Labeo rohita fingerling fed with Siam weed (Chromolaena odorata) meal (SWM) by gradually replacing de-oiled rice bran (DORB). One hundred and eighty fingerlings were randomly distributed into four distinct experimental groups in triplicates. Four semi-purified iso-nitrogenous diets (330 g kg−1 CP) with different combinations of SWM and DORB such as T1 (diet with 300 g kg−1 DORB + 0.0 g kg−1 SWM), T2 (diet with 200.0 g kg−1 DORB + 100.0 g kg−1 SWM), T3 (diet with 100.0 g kg−1 DORB + 200.0 g kg−1 SWM) and T4 (diet with 0.0 g kg−1 DORB +300.0 g kg−1 SWM) were prepared and fed to satiation level. Among the various groups, T4 showed a significant reduction (p < 0.05) in feed intake than the other experimental groups. T3 group exhibited highest (p < 0.05) WG, SGR, PER compared to T1, T2 and T4 groups. Significantly higher (p < 0.05) amylase, protease, trypsin activities and amylase mRNA expression while lower chymotrypsin activity were recorded in the T3 group compared to the control group. In vitro and in vivo digestibility and metabolic enzyme activities were significantly higher (p < 0.05) for T3 diet fed fish compared to other groups. Overall results revealed that SWM can effectively include up to 300.0 g kg−1 by successfully replacing equal amount of DORB without compromising the growth. Hence, it could be used as an alternate ingredient in the diet of Labeo rohita fingerlings.

Abstract C56: Bmi1 is widely expressed in acini and regulates Kras-driven transcription factor networks in early pancreatic neoplasia

Abstract Rare acinar cells expressing Bmi1, a component of the Polycomb Repressor Complex 1 (PRC1), represent a reservoir of cells contributing to pancreatic repair during injury and stress. This is marked by acinar-ductal metaplasia (ADM), a transition of acinar cells to a more duct-like phenotype, which is seen in pancreatitis and early stages of Kras-driven pancreatic neoplasia. The mechanism behind Kras-driven ADM at the genomic level is not completely understood. Developmental transcription factor (TF) networks regulate and maintain the fate of cells and rely on appropriate chromatin context for their activity. Deletion of Bmi1 represses Kras-driven ADM and neoplasia in genetically engineered mouse models (GEMMs) of PDA. Thus, we hypothesized that Kras reprograms the acinar fate TF network to promote PDA in a Bmi1-dependent epigenetic context. We used GEMMs to study the expression of Bmi1 in the acinar compartment and its role in early pancreatic neoplasia. We combined an acinar cell-specific tamoxifen-inducible Cre-recombinase under the elastase promoter (Ela-CreER) with the Kras G12D oncogenic allele and the conditional Bmi1 knockout allele and the Rosa26 tdTomato reporter to generate Ela-CreER, KrasLSL-G12D/+, Bmi1, R26 tdTomato mice and their littermate controls. We also generated Ela-CreER, Bmi1GFP, R26tdTomato and Bmi1-CreER, R26 tdTomato mice to quantify the fraction of acinar cells expressing Bmi1. We activated the Cre-recombinase in 6- to 8-week-old Ela-CreER mice with 5 tamoxifen gavages (4mg/day) prior to induction of acute pancreatitis by intraperitoneal caerulein administration (8 hourly injections x 2 days) one week after the first tamoxifen dose. We FACS sorted tdTomato+ cells from untreated mice (0 hr) and one week (168 hr) after pancreatitis induction and analyzed RNA levels of 8 TFs, reported to play a role in pancreatic development, homeostasis, and neoplasia. Expression levels of two lineage markers (amylase and elastase) were also measured by RT-qPCR. Lineage tracing in Bmi1-CreER and Ela-CreER, Bmi1GFP mice revealed expression of Bmi1 in vast majority of acinar cells. RT-qPCR of tdTomato+ cells in the Ela-CreER models indicated a loss of acinar-specific TFs in mice expressing mutant Kras one week after induction of pancreatitis consistent with cells undergoing ADM. Genetic ablation of Bmi1 in the presence of mutant Kras restored elastase and amylase mRNA levels. At the morphologic level, loss of Bmi1 also led to recovery of acinar cells from ADM and correlated with an increase in RNA expression of three key acinar-specific fate TFs—Mist1, Hnf1a, and Nr5a2. The mRNA levels of other key acinar TFs—Pdx1 and Ptf1a/p48—did not recover with the loss of Bmi1 in the presence of KrasG12D. Together, our data suggest that Bmi1 is expressed in the majority of acinar cells and regulates oncogenic Kras-driven ADM by altering master TF gene regulatory networks. Its deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis. Citation Format: Joyce K. Thompson, Emily Wu, Osama Alkhalili, Howard C. Crawford, Marina Pasca di Magliano, Filip Bednar. Bmi1 is widely expressed in acini and regulates Kras-driven transcription factor networks in early pancreatic neoplasia [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C56.

High Pancreatic Amylase Expression Promotes Adiposity in Obesity-Prone Carbohydrate-Sensitive Rats

We have reported large differences in adiposity (fat mass/body weight) gain between rats fed a low-fat, high-starch diet, leading to their classification into carbohydrate "sensitive" and "resistant" rats. In sensitive animals, fat accumulates in visceral adipose tissues, leading to the suggestion that this form of obesity could be responsible for rapid development of metabolic syndrome. We investigated whether increased amylase secretion by the pancreas and accelerated starch degradation in the intestine could be responsible for this phenotype. Thirty-two male Wistar rats (7-wk-old) were fed a purified low-fat (10%), high-carbohydrate diet for 6 wk, in which most of the carbohydrate (64% by energy) was provided as corn starch. Meal tolerance tests of the Starch diet were performed to measure glucose and insulin responses to meal ingestion. Indirect calorimetry combined with use of 13C-labelled dietary starch was used to assess meal-induced changes in whole body and starch-derived glucose oxidation. Real-time polymerase chain reaction was used to assess mRNA expression in pancreas, liver, white and brown adipose tissues, and intestine. Amylase activity was measured in the duodenum, jejunum, and ileum contents. ANOVA and regression analyses were used for statistical comparisons. "Resistant" and "sensitive" rats were separated according to adiposity gain during the study (1.73%±0.20% compared with 4.35%±0.36%). Breath recovery of 13CO2 from 13C-labelled dietary starch was higher in "sensitive" rats, indicating a larger increase in whole body glucose oxidation and, conversely, a larger decrease in lipid oxidation. Amylase mRNA expression in pancreas, and amylase activity in jejunum, were also higher in sensitive rats. Differences in digestion of starch can promote visceral fat accumulation in rats when fed a low-fat, high-starch diet. This mechanism may have important implications in human obesity.

Regulation of pancreas development and enzymatic gene expression by duodenal infusion of leucine and phenylalanine in dairy goats

This study aimed to investigate the effect of a duodenal infusion of leucine and phenylalanine on pancreatic development and enzyme gene expression in dairy goats. Sixteen healthy yearling Guanzhong dairy goats of similar weights (29.7 ± 1.4 kg) had surgical duodenal intubation, and were used in a completely random design in this experimental. The animals were randomly divided into the 4 treatment groups, with 4 goats in each group: control, 3 g/d leucine (3 Leu), 9 g/d leucine (9 Leu) and 2 g/d phenylalanine (2 Phe), dissolved in and infused into the duodenum for 21 days. The goats were fed twice daily (0800 and 1800) with 350 g (dry matter) of total mixed rations, and were all slaughtered on the last day of the experimental period for pancreatic tissue sampling. The results showed that leucine and phenylalanine infusion did not affect pancreas weight or protein concentration (P > 0.05). However, 9 Leu treatment reduced pancreatic DNA concentration, and both 9 Leu and 2 Phe treatments increased the pancreatic protein:DNA ratio (P < 0.05). Leucine and phenylalanine infusions increased pancreatic amylase activity (P < 0.05), whereas had no effect on the total trypsin and lipase activities (P > 0.05). Treatment with 9 Leu and 2 Phe increased amylase mRNA levels (P < 0.05), and 2 Phe increased lipase mRNA levels (P < 0.05). Trypsin mRNA levels were unaffected by leucine and phenylalanine infusion (P > 0.05). The phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 was increased by leucine infusion (P < 0.05), but not by phenylalanine infusion (P > 0.05). Leucine infusion also increased the free leucine concentration in the pancreas (P < 0.05), and had no effect on glucose concentrations (P > 0.05). In conclusion, duodenal infusion of an appropriate amount of leucine and/or phenylalanine can effectively improve the expression and secretion of pancreatic amylase in goats, which is important for improving intestinal starch digestion in ruminant animals. The regulation of the pancreatic amylase expression by leucine and phenylalanine mainly occurs at the transcriptional and/or translational level in goats.

Functional effects of proinflammatory factors present in Sj\xf6gren\u2019s syndrome salivary microenvironment in an in vitro model of human salivary gland

Primary Sjögren’s syndrome (pSS) is an autoimmune exocrinopathy in which the role that the immune response plays in reducing exocrine gland function, including the glandular microenvironment of cytokines, has not been fully understood. Epithelial cells from biopsies of human parotid gland (HPG) were used to establish a model of human salivary gland in vitro. In this model, the functional consequences of several proinflammatory soluble factors present in the pSS glandular microenvironment were assessed. Stimulation with isoproterenol and calcium produced a significant increase in the basal activity of amylase in the HPG cell supernatants. Under these conditions, the presence of TNF-α and CXCL12 increased amylase mRNA cellular abundance, but reduced the amylase activity in the cell-free supernatant in a dose-dependent manner. IL-1β and IFN-γ, but not TGF-β, also diminished amylase secretion by HPG cells. These results suggest that the glandular microenvironment of cytokine, by acting post-transcriptionally, may be responsible, at least in part, for the reduced exocrine function observed in pSS patients. These data may help to a better understanding of the pathogenesis of SS, which in turn would facilitate the identification of new therapeutic targets for this disorder.

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased pancreatic trypsin mRNA levels by 40%, 44% and 28%, respectively. Supplementation with NSP enzyme and 160 mg/kg protease decreased pancreatic trypsin mRNA levels by 13%. Pancreatic lipase and amylase mRNA expression were significantly elevated in treated animals compared to the control group (p<0.05). These results suggest that the amount of NSP enzyme and acid protease in the diet significantly affects digestive function, endogenous digestive-enzyme activity and mRNA expression in broilers.

Dietary lipid requirements of larval genetically improved farmed tilapia,Oreochromis niloticus(L.), and effects on growth performance, expression of digestive enzyme genes, and immune response

The present study was performed to investigate the effects of dietary lipid level on growth performance, antioxidant defense, and mRNA levels of digestive enzymes and immune genes in larval genetically improved farmed tilapia (Oreochromis niloticus). The results show that larvae fed lipid levels of 4–11.5% had greater weight gain (WG), feed efficiency, protein efficiency ratio and survival than larvae fed the other dietary lipid levels (P < 0.05). No difference whole-body crude protein, ash or moisture contents were found between treatments (P > 0.05) but lipid contents increased as dietary lipid level was increased. The 8% and 11.5% dietary lipid levels had stimulatory effects on trypsin mRNA level compared with those of larvae fed the 15% and 18.5% lipid diets. Intestinal and liver amylase mRNA levels decreased in larvae fed the 18.5% lipid diet, following the decrease in dietary starch. Analysis of the secondary curve equation for WG indicated that the optimal dietary lipid level in larval tilapia was 8.56%. In addition, the 4–11.5% lipid levels improved superoxide dismutase and glutathione peroxidase activities and increased mRNA levels of heat shock protein70, hepcidin antimicrobial peptide-1, and C-type lysozyme in the liver of larvae (P < 0.05). A 192-h Streptococcus iniae challenge study was conducted after the 35-day feeding trial, and the results showed that post-challenge mortality of larvae fed the 11.5% lipid level was the lowest and enhanced the immune activities.

Effects of lysine and methionine supplementation on growth, body composition and digestive function of grass carp (Ctenopharyngodon idella) fed plant protein diets using high-level canola meal

A 76-day feeding trial was carried out to evaluate the effects of Lysine and Methionine supplementation on growth and digestive capacity of grass carp (Ctenopharyngodon idella) fed plant protein diets using high-level canola meal (CM). Fish with initial average weight 103.9 ± 0.6 g were fed three extruded diets. Fish meal (FM) diet was formulated as the normal control with 40 g kg−1 FM and 300 g kg−1 CM; CM diet was prepared by replacing all FM with CM (total 340 g kg−1) without Lys or Met supplementation; CM supplement (CMS) diet was similar to CM diet but was supplemented with essential amino acids (EAA) to ensure the levels of Lys and Met similar to those in the FM diet. Feed intake, feed efficiency and specific growth rate of the grass carp fed CMS and FM diets were similar (P > 0.05), but higher than those of the grass carp fed CM diet (P < 0.05). The hepatosomatic index, relative gut length, intestosomatic index and intestinal folds height were significantly improved in fish fed FM and CMS diets as compared to CM diet (P < 0.05). Lower activities of trypsin, lipase and amylase in hepatopancreas were observed in fish fed CM diet (P < 0.05). Three hundred and forty gram per kilogram CM without Lys or Met supplementation significantly decreased trypsin, lipase and amylase mRNA levels in hepatopancreas (P < 0.05). These results indicated that the high supply of CM (340 g kg−1) in plant protein (200 g kg−1 soybean meal and 100 g kg−1 cottonseed meal) diets decreased digestive ability through decreasing digestive enzyme activities and enzyme gene's expressions of grass carp, and these side effects can be reversed by supplementing Lys and Met. Therefore, CM could be high level used in a plant protein blend-based extruded diet for grass carp as long as EAA were supplemented.

Biomarkers of radiation exposure in children with neuroblastoma treated with 131I-mIBG.

10060 Background: Neuroblastoma (NB) is the most common solid extra-cranial tumor of childhood. 131I-mIBG is a targeted radiopharmaceutical for patients with advanced NB and is one of the most active agents in this treatment resistant population. To date, no clinically relevant biomarkers of response or toxicity have been reported in patients treated with 131I-mIBG. Methods: Patients with relapsed or refractory NB who received 131I-mIBG at UCSF were eligible to participate in this prospective correlative study. Blood samples were obtained at baseline and 72 hours after 131I-mIBG infusion. We quantified a panel of biomarkers shown to be effected in patients receiving other forms of radiation, including: serum amylase; plasma Flt-3 ligand; plasma monocyte colony stimulating factor (MCSF); and CDKN1A mRNA in mononuclear cells. Extent of modulation of each marker was evaluated using paired t-tests at hour 0 and 72. We assessed potential differential modulation between groups based on response (PR/CR vs SD/PD), toxicity (grade 3 non-hematologic toxicity; grade 4 neutropenia or thrombocytopenia) or administration of combination therapy (131I-mIBG alone vs in combination with vincristine/irinotecan or vorinostat) using unpaired t-tests. Results: 26 patients (21 male; median age 7 years) participated and received a median 131I-mIBG dose of 18 mCi/kg. 16 patients received 131I-mIBG in combination. We observed robust modulation of amylase (median fold increase from baseline 3.1; p &lt;0.0001), Flt-3 ligand (median fold increase from baseline 3.6; p=0.0005), and CDKN1A mRNA (median fold decrease from baseline 4.3, p=0.0002). No differences were noted in MCSF (p=0.80). Patients receiving combination 131I-mIBG had more robust modulation of Flt-3 ligand (mean difference 296 pg/mL; p=0.02) compared to single agent 131I-mIBG. No correlations with clinical response or toxicity were found. Conclusions: Amylase, Flt-3 ligand, and CDKN1A mRNA all show robust modulation after 131I-mIBG treatment. Patients receiving 131I-mIBG in combination demonstrated the largest changes in plasma Flt-3 ligand. In this initial analysis, no associations were found between response or toxicity and the proposed biomarkers.

Genetic ontogeny of pancreatic enzymes in Labrus bergylta larvae and the effect of feed type on enzyme activities and gene expression

A newly cultivated wrasse species, Labrus bergylta, have shown great potential for use in Atlantic salmon (Salmo salar) farms in the battle against sea lice (Lepeoptheirus salmonis) infections. Hatchery reared L. bergylta were studied from 2 to 55 DPH to examine the molecular basis of digestive ontogeny related to the pancreas. An isolated feeding trial was performed on 27-34 DPH larvae to compare the effect of diet on enzyme activity and the possible exogenous contribution by live feed. The following genes coding for key pancreatic enzymes were analyzed by qPCR: trypsin, Cyp7 A1, BAL, sPLA(2) 1B, amylase and pancreatic chitinase. Enzyme activity was measured on trypsin, neutral lipase, sPLA(2), amylase and chitinase in fed and unfed larvae. We did not observe any effects of the formulated diet v.s. rotifers on enzyme activities of neutral lipase, chitinase and sPLA(2). However, a probable feed-dependency was observed at a transcriptional level, where rotifers seem to stimulate upregulation. The regulation of BAL was the only exception, where an upregulation was observed after weaning both in the ontogeny series and the experimental part. Our data on pancreatic chitinase and amylase mRNA levels suggest the importance of carbohydrates in the diet of early larval and juvenile L. bergylta.

Sorafenib-induced loss of polarity of zymogen granules in pancreatic acinar cells.

236 Background: Sorafenib is an oral multi-kinase inhibitor which is regarded as a key drug for HCC and RCC. It has been unexpectedly found that the compound causes an increase of serum pancreatic enzyme levels without clinically recognized pancreatitis. The reason for this event is not well understood yet. The aim of this study was to clarify the mechanisms involved in this phenomenon. Methods: Eight-week old BALB/cA male mice were used in in vivo studies. Sorafenib tosylate was administered per os once daily at a dose of 150 mg/kg body weight. Control mice were given vehicle alone. Mice were sacrificed 24 hr after 1-, 2-, 3- and 7-day administration of the compound, and blood samples and pancreatic tissue samples were obtained (n=5 for each group). The tissue samples were used for hematoxylin and eosin (HE) staining, immunohistochemistry, electron microscopy (EM), western blot and RT-quantitative PCR studies. Results: Serum amylase levels were elevated after sorafenib administration. The amylase level hit the peak after 2-day administration, and then gradually decreased. By HE staining, the control group without sorafenib showed a basophilic stained area in the baso-lateral site of the acinar cells. In contrast, the acinar cytoplasm after 2-day administration of sorafenib was totally eosinophilic. The typical findings of acute pancreatitis were not seen in the both group. By EM examination, zymogen granules (ZGs) of the sorafenib group spread into basal site of the acinar cells. ZGs mounted up on both of apical and baso-lateral plasma membrane and showed exocytosis. The levels of amylase mRNA were not elevated by sorafenib. In addition the expression of N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) proteins was not changed. Conclusions: The results suggest that the amount of acinar amylase production was not changed but the distribution of ZGs was altered by sorafenib. Sorafenib seemed to cause temporary loss of polarity of ZGs secretion in acinar cells by blocking apical exocytosis. Acute pancreatitis was not evident; thus the current model was not similar to the pancreatitis model caused by the supra-maximal secretagogue stimulation which blocks the apical exocytosis.

Effect of enzyme preparation on egg production, nutrient retention, digestive enzyme activities and pancreatic enzyme messenger RNA expression of late-phase laying hens

This experiment was conducted to study the effect of dietary inclusion of an enzyme preparation including phytase, xylanase, cellulase, α-amylase and acidic protease on egg production, nutrient retention, digestive enzyme activities and pancreatic enzyme mRNA expression. Three hundred and sixty ISA Brown laying hens were randomly allocated to two groups with 6 replicates of 30 birds each and fed a maize–soybean meal diet with or without enzyme supplementation (ES) from 60 to 68wk of age. There were no differences in hen performance between the two groups. The coefficient of total tract apparent retention (CTTAR) of ether extract (EE) was increased (P<0.05) by ES. Birds fed the diet supplemented with enzymes tended to have higher lipase and protease activity in duodenal and jejunal digesta (P<0.10) than control birds. There were no significant effects of ES on pancreatic enzyme activities. The mRNA expression of amylase in pancreas tended to be downregulated by ES as compared with the control group (P=0.097), whereas that of pancreatic trypsinogen was not affected. It is concluded that ES did not affect productive performance of laying hens, but improved nutrient retention and tended to increase enzyme activities in the intestinal contents. The tendency for lower pancreatic amylase mRNA expression with ES, not coupled with a reduction in pancreatic enzyme activities suggests that amylase expression may be regulated not only at transcriptional level but also at post-transcriptional level.

Pancreatic exocrine enzyme-producing cell differentiation via embryoid bodies from human embryonic stem cells

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all- trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.

Molecular ontogenesis of digestive capability and associated endocrine control in Atlantic cod (Gadus morhua) larvae

We have profiled the expression of twelve genes, in order to provide an overview on the molecular ontogeny of digestive capability with the associated endocrine control during Atlantic cod (Gadus morhua) larval development. Enzyme activity levels for the key digestive enzyme, trypsin, was also measured. Specifically, transcripts for trypsin, amylase, lipolytic enzymes: bile salt activated lipase (BAL), phospholipase A2 (PLA2) and Acyl CoA dehydrogenase (ACADM), regulatory peptides: neuropeptide Y (NPY), orexin (OX) cholecystokinin (CCK) and cocaine and amphetamine-related transcript (CART), the somatotropic factors: growth hormone (GH), preprosomatostatin 1 (PPSS1) and thyroid hormone receptors (TRα and TRβ) were analyzed using quatitative (real-time) polymerase chain reaction (qPCR). Trypsin and BAL mRNA levels peaked at approximately day 17 and 25 post-hatch, respectively, and thereafter displayed a decreasing pattern until metamorphosis. GH mRNA levels decreased moderately from 3 to 33dph, and thereafter, an increase was observed until 46dph. TRα mRNA levels showed a fluctuating pattern peaking at day 39 post-hatch. TRβ mRNA levels were too low to obtain quantitative measurements. Amylase mRNA slightly increased from day 3 to 17 post-hatch, and thereafter showed a steady decrease until day 60. Interestingly, PLA2 mRNA expression showed a consistent increase throughout the study period, indicating an increasingly important role during larval development. Overall, data from this study indicate that cod larvae show differential developmental mode of expression patterns for key genes and endocrine factors that regulate digestive capability, growth and development. These data are discussed in relation to larval trypsin enzyme activity and previous reports for other teleost species.

The Hexosamine Biosynthesis Pathway Is Essential for Pancreatic Beta Cell Development

Pancreatic exocrine and endocrine cells develop during embryonic life from endodermal progenitors. This process depends on activation of a hierarchy of transcription factors. Although information is available regarding the mesodermal signals controlling pancreas development, little is known about the role of environmental factors such as nutrients, including glucose, that also may impact development. Previously, we showed that glucose plays an important and specific role in beta cell development by activating the transition of Neurogenin3-positive endocrine progenitors into beta cells. Here, we examined the implication of glucose metabolism and more precisely the role of the hexosamine biosynthesis pathway (HBP) to understand the mechanisms by which glucose regulates beta cell development. We have established an in vitro model of endocrine and exocrine cells development from embryonic day 13.5 rat pancreases in a manner that replicates in vivo pancreas development perfectly. Using this model, we tested the effect of selective inhibitors and activators of the HBP and found that the HBP has a modest effect on cell proliferation and exocrine cell differentiation. On the other hand, beta cell development is tightly controlled by the HBP. Specifically, HBP activators increase beta cell development, whereas inhibitors repress such development. Importantly, both the HBP and glucose control the same steps in beta cell development.

Pancreatic Regenerating Gene I and Acinar Cell Differentiation

Pancreatic regenerating gene I (reg I) has been implicated in cellular differentiation. Acinar cells can transdifferentiate into other pancreatic-derived cells, and we postulated that changes in intracellular levels of reg I would affect the state of differentiation. We transfected AR42J cells with a plasmid containing the entire coding sequence of reg I and isolated clones with complementary DNA in sense (SS) or antisense (AS) orientation. Levels of messenger RNA (mRNA) and protein expression were examined by Western blotting and real-time polymerase chain reaction. Expression of reg I was confirmed in SS or AS clones. AR42J transfected with SS demonstrated more acinarlike phenotype, whereas those transfected with AS showed a less differentiated state. Specifically, amylase mRNA and protein levels increased in SS cells, whereas AS cells showed increased pancreatic and duodenal homeobox 1 (Pdx1) and insulin mRNAs and cytokeratin protein. Conversely, cytokeratin and Pdx1 were depressed in SS cells. These data demonstrate that in acinar cells, reg I overexpression is linked to acinar cell differentiation, whereas inhibition of reg I leads to beta cell and possibly ductal phenotype. Reg I expression in acinar cells is important in maintaining pancreatic cell lineage, and when decreased, cells can dedifferentiate and move toward becoming other pancreatic cells.

Transplanted Human Cord Blood Cells Generate Amylase-Producing Pancreatic Acinar Cells in Engrafted Mice

Pancreatic acinar cells and hepatocytes arise from the same cell population located within the embryonic endoderm. It has been reported that a multipotent population of liver cells is capable of differentiating into pancreatic cells. Recent studies revealed that murine and human hematopoietic cells could generate hepatocytes in vivo. Based on this developmental proximity between liver and pancreatic acinar cells, we examined whether human cord blood (CB) cells can generate pancreatic cells in vivo using a murine xenograft model. We transplanted 1 x 10 CD34 human CB cells into "conditioned" newborn nonobese diabetic-severe combined immunodeficiency/beta-2 microglobulin-null mice via facial vein injection and, 3 to 4 months later, examined the pancreata from recipient mice showing high-level human multilineage hematopoietic engraftment in the bone marrow. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses revealed human amylase mRNA and protein expression, respectively, in the pancreata from recipient mice. Using fluorescence in situ hybridization, we identified human alpha-satellite, DNA-positive cells with a morphology characteristic of pancreatic acinar cells. We also identified cells in paraffin sections of the pancreata that expressed amylase mRNA, had morphological characteristics of acinar cells, and contained human but not mouse centromeric DNA. These findings establish that human umbilical CB cells are capable of generating pancreatic acinar cells via a nonfusion mechanism.