Inhibition Effects of Acetylsalicylic Acid with Nitric Oxide (NO-ASA) on Neoplastic Changes in the Exocrine Pancreas Acinar Cells of the Azaserine Injected Rats.

BACKGROUND: Atypical acinar cell foci (AACF) seen in pancreatic cancer are fatal and have been studied with some causative agents. However, for the first time, the effect of acetylsalicylic acid with nitric oxide (NO-ASA) on AACF was examined in this study. Although NO-ASA has very successful inhibitory effects against some types of cancer, it has not been investigated whether they can exert their inhibition effects on AACFs. METHODS: For experimental purposes, 21 14-day-old male Wistar albino rats were used. Azaserine (30 mg/kg) was dissolved in 0.9% NaCl solution and injected intraperitoneally (i.p.) into 14 rats, except for the Control group (Cont) rats, for three weeks. Rats that were injected with azaserine once a week for three weeks and those that did not receive treatment were divided into experimental groups. 15 days after the end of the azaserine injection protocol, NO-ASA was applied to azaserine with NO-ASA (Az+NO-ASA) group rats three consecutive times with an interval of 15 days by gavage. At the end of the 5-month period, pancreatic tissue was dissected and weighed. Pancreas preparations prepared from histological sections were examined for AACF burden and analyzed via a video image analyzer. One-way analysis of variance (ANOVA) non-parametric statistical analyses were performed to test whether there was a difference between the averages of the experimental and Control groups. RESULTS: AACF burden in both groups injected with azaserine was found to be statistically significant in all categories compared to that of the Control group (p < 0.05). The average Calculated Estimated average AACF volume (mm3) values, the Calculated estimated average AACF diameter (µm), the Estimated average number of AACF per unit volume, AACF rate as a % of Calculated Organ Volume were higher in the AzCont group rats than in the Az+NO-ASA group, when compared, and there was an important level statistical difference between the groups (p < 0.05). It was determined that for all parameters AACFs load in Az+NO-ASA group rats were significantly reduced compared to that of AzCont group rats (p < 0.05). CONCLUSIONS: We observed that, as a result of the NO-ASA application, the experimental AACF focus ratio created by azaserine injection was significantly inhibited. The inhibitory effect of AACFs in Az+NO-ASA group rats may have resulted from the significant and independent chemopreventive and/or chemotherapeutic activity of NO-ASA against exocrine pancreatic AACF foci.

The effect of steroid therapy on pancreatic exocrine function in autoimmune pancreatitis.

BACKGROUND/OBJECTIVES: Autoimmune pancreatitis (AIP) is a steroid-responsive inflammatory disease of the pancreas. Few studies investigated pancreatic exocrine function (PEF) in patients suffering from AIP and no definitive data are available on the effect of steroids in PEF recovery. Aim of the study is the evaluation of severe pancreatic insufficiency (sPEI) prevalence in AIP at clinical onset and after steroid treatment. METHODS: 312 Patients with diagnosis of AIP between January 1st, 2010 and December 31st, 2020 were identified in our prospectively maintained register. Patients with a pre-steroid treatment dosage of fecal elastase-1 (FE-1) were included. Changes in PEF were evaluated in patients with available pre- and post-treatment FE (between 3 and 12 months after steroid). RESULTS: One-hundred-twenty-four patients were included, with a median FE-1 of 122 (Q1-Q3: 15-379) µg/g at baseline. Fifty-nine (47.6 %) had sPEI (FE-1<100 µg/g). Univariable analysis identified type 1 AIP, radiological involvement of the head of the pancreas (diffuse involvement of the pancreas or focal involvement of the head), weight loss, age and diabetes as associated with a greater risk of sPEI. However, at multivariable analysis, only the involvement of the head of the pancreas was identified as independent risk factor for sPEI. After steroids, mean FE-1 changed from 64 (15-340) to 202 (40-387) µg/g (P = 0.058) and head involvement was the only predictor of improvement of sPEI. CONCLUSION: The inflammatory involvement of the head of the pancreas is associated with PEF severity, as well as PEF improvement after treatment with steroids in patients with AIP.

MiR-204-5p Performs a Protective Effect on Cerulein-Induced Rat Pancreatic Acinar Cell AR42J Cell Damage by Targeting Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Gamma and Regulating PI3K/Hippo Pathways.

OBJECTIVE: This research plans to address the function of miR-204-5p/tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) in cerulein-induced acute pancreatitis (AP). METHODS: Rat pancreatic acinar cell AR42J was stimulated by 100 nmol/L of cerulein to mimic the situation in AP. Gene Expression Omnibus database was used to select differentially expressed genes. StarBase database and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were used to select the target genes of miR-204-5p, which were further affirmed by dual luciferase assay. The biological behaviors of AR42J cells were measured by cell proliferation and flow cytometry assays. Quantitative real-time polymerase chain reaction and western blot assays were executed to assess YWHAG expression. The secretion of C-C Motif Chemokine Ligand 2/Timp metallopeptidase inhibitor 1 in AR42J cells was evaluated by enzyme-linked immunosorbent assay. The protein expression of YAP1/p-YAP1/PI3K/p-PI3K was measured by western blot. RESULTS: miR-204-5p expression was profoundly reduced in cerulein-induced AP model. YWHAG was upregulated in cerulein-induced AP model and related to C-C Motif Chemokine Ligand 2/Timp1. In addition to the negative association between miR-204-5p and YWHAG, the alleviation impact of miR-204-5p mimic on cerulein-induced AR42J cell damage was blocked by YWHAG overexpression and PI3K/Hippo signaling pathways activation. CONCLUSIONS: These observations indicated that the alleviation impact of miR-204-5p on cerulein-induced AR42J cell damage was mediated via YWHAG and PI3K/Hippo signaling pathways.

Proinflammatory cytokines inhibit thiamin uptake by human and mouse pancreatic acinar cells: involvement of transcriptional mechanism(s).

Thiamin (vitamin B1) plays critical roles in normal metabolism and function of all mammalian cells. Pancreatic acinar cells (PACs) import thiamin from circulation via specific carrier-mediated uptake that involves thiamin transporter-1 and -2 (THTR-1 and -2; products of SLC19A2 and SLC19A3, respectively). Our aim in this study was to investigate the effect(s) of proinflammatory cytokines on thiamin uptake by PACs. We used human primary (h)PACs, PAC 266-6 cells, and mice in vivo as models in the investigations. First, we examined the level of expression of THTR-1 and -2 mRNA in pancreatic tissues of patients with chronic pancreatitis and observed severe reduction in their expression compared with normal control subjects. Exposing hPACs and PAC 266-6 to proinflammatory cytokines (hyper IL-6, TNF-α, and IL-1ß) was found to lead to a significant inhibition in thiamin uptake. Focusing on hyper-IL-6 (which also inhibited thiamin uptake by primary mouse PACs), the inhibition in thiamin uptake was found to be associated with significant reduction in THTR-1 and -2 proteins and mRNA expression as well as in activity of the SLC19A2 and SLC19A3 promoters; it was also associated with reduction in level of expression of the transcription factor Sp1 (which is required for activity of these promoters). Finally, blocking the intracellular Stat3 signaling pathway was found to lead to a significant reversal in the inhibitory effect of hyper IL-6 on thiamin uptake by PAC 266-6. These results show that exposure of PACs to proinflammatory cytokines negatively impacts thiamin uptake via (at least in part) transcriptional mechanism(s).NEW & NOTEWORTHY Findings of the current study demonstrate, for the first time, that exposure of pancreatic acinar cells to proinflammatory cytokines (including hyper IL-6) cause significant inhibition in vitamin B1 (thiamin; a micronutrient that is essential for normal cellular energy metabolism) and that this effect is mediated at the level of transcription of the thiamin transporter genes SLC19A2 and SLC19A3.

Galactose protects pancreatic acinar cells from cerulein induced damage by regulating FGF21 and Klotho.

To investigate the effect and mechanism of galactose on cerulean-induced pancreatic acinar cell injury. Acute pancreatitis cell injury model was established by arbusin-induced pancreatic acinar cell AR42J injury; galactose (25, 50, 100 mmol / L) was used to treat the injured cells, and the optimal concentration was 50 mmol / L; cell counting kit (CCK-8), enzyme linked immunosorbent assay (ELISA) to detect cell survival rate and necrosis rate; flow cytometry and Western blotting (Western blot) to detect cell apoptosis and autologous phage-related gene (Beclin1) and microtubule-associated protein 1 light chain 3 (LC3), apoptosis-related protein B-cell lymphoma / leukemia-2 (Bcl-2), Bcl-2-related X gene (Bax), and fibroblasts Expression of growth factor 21 antibody (FGF21) and anti-aging gene Klotho. A pancreatic acinar cell injury model was successfully established with cerana (100 nmol / L); galactose (25, 50, 100 mmol/L) In a concentration-dependent manner, the inhibitory effect of ceriferin on AR42J injury was inhibited at an optimal concentration of 50 mmol / L. Compared with the ceriferin group, the apoptosis rate of AR42J cells in the galactose group was significantly reduced. table Significantly increased, Bcl-2, FGF21 and Klotho protein expression was significantly increased, Bax protein was significantly decreased; the FGF21 inhibitor can be significantly reduced on galactose these caerulein-induced AR42J cells. Galactose can inhibit the apoptosis and autophagy of pancreatic acinar cells induced by cerana, and its potential mechanism is to up-regulate FGF21 and Klotho, providing a new potential drug for the treatment of acute pancreatitis.

Docosahexaenoic acid inhibits ethanol/palmitoleic acid-induced necroptosis in AR42J cells.

Fatty acid ethyl esters (FAEEs), non-oxidative metabolites of ethanol, are the main causative agents of severe acute pancreatitis resulting from alcohol abuse. Pancreatic acinar cells exposed to ethanol in combination with the fatty acid palmitoleic acid (EtOH/POA) display increased levels of palmitoleic acid ethyl ester and cell death. Oxidative stress and acinar cell necroptosis are implicated in the pathology of severe acute pancreatitis. Docosahexaenoic acid (DHA) serves as a powerful anti-oxidant that reduces pancreatic inflammation and improves the outcomes of patients with acute pancreatitis. We investigated whether treatment of EtOH/POA, as an in vitro model of alcoholic pancreatitis, increases reactive oxygen species (ROS), necroptosis-regulating proteins, and cell death by increasing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and intracellular calcium. Also, we investigated whether DHA inhibits EtOH/POA-induced alterations in pancreatic acinar AR42J cells. As a result, EtOH/POA increased intracellular and mitochondrial ROS levels, NADPH oxidase activity, necroptosis-regulating proteins, and cell death, which was inhibited by NADPH oxidase inhibitor apocynin, the Ca2+ chelator BAPTA, and DHA. However, DHA did not reduce EtOH/POA-induced increases in Ca2+ oscillation or levels in AR42J cells. Furthermore, EtOH/POA induced mitochondrial dysfunction by reducing mitochondrial membrane polarization and hence, adenosine triphosphate (ATP) production. DHA treatment attenuated EtOH/POA-induced mitochondrial dysfunction. In conclusion, DHA inhibits EtOH/POA-induced necroptosis by suppressing NADPH oxidase activity, reducing ROS levels, preventing mitochondrial dysfunction, and inhibiting activation of necroptosis-regulating proteins in AR42J cells.

Cholecystokinin-1 receptor agonist induced pathological findings in the exocrine pancreas of non-human primates.

BACKGROUND AND AIMS: Cholecystokinin (CCK) may potentially be used to treat obesity. However, it is well-known to induce acute pancreatitis and pancreas neoplasia in rodents, but not in primates. Here we report the nonclinical safety profile of a long-acting CCK-1 receptor (CCK-1R) agonist, NN9056, in rats and monkeys to support a First-in-Man clinical trial with NN9056. METHODS: Thirteen-week toxicological studies were conducted in rats and non-human primates followed by histopathological evaluation of affected tissues. NN9056 was characterised in vitro, and CCK-1R expression was assessed by in situ hybridization in cynomolgus monkey and human pancreas tissues. RESULTS: Affinity and potency of NN9056 was comparable to native sulphated CCK-8 (CCK-8) across species on the CCK-1R while it had no effect on the CCK-2 receptor (CCK-2R). In situ hybridization demonstrated abundant expression of CCK-1Rs in the exocrine pancreas of the rat. In contrast, it was only discreetly expressed on pancreatic acinar cells in the periphery of scattered lobules in monkeys. A similar expression pattern was observed in human pancreas. 13-weeks daily dosing with NN9056 produced the expected pancreatic pathological findings in rats. In monkeys, NN9056 increased pancreas weight and induced histopathological changes despite the low expression level of CCK-1Rs. CONCLUSION: Surprisingly, chronic CCK-1R activation constitutes a risk for pancreatitis and trophic actions on the exocrine pancreas in monkeys. Since similar CCK-1R expression patterns were found in pancreas of monkeys and humans this risk is likely translatable to humans and clinical development of NN9056 was therefore halted.

Aqueous extraction from dachengqi formula granules reduces the severity of mouse acute pancreatitis via inhibition of pancreatic pro-inflammatory signalling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Dachengqi decoction (DCQD) belongs to a family of purgative herbal formulas widely used in China for the treatment of acute pancreatitis (AP). AP is a prevalent digestive disease currently without an effective pharmacological intervention. Formula granules have become the preferred method for delivery of herbal formulation in China given its benefit of potency retention, dosing precision and ease of use. The efficacy of DCQD formula granules (DFGs) in experimental AP models has not been investigated. AIM OF THE STUDY: To analyse and compare the differences in chemical composition of DFGs, with their aqueous extraction (AE) and chloroform extraction (CE) derivatives. To assess their efficacy on severity and targeted pancreatic pro-inflammatory signalling pathways in freshly isolated acinar cells and two models of experimental AP. MATERIAL AND METHODS: UPLC-Q-TOF-MS was used to analyse chemical components of DFGs and their extractions. Freshly isolated mouse pancreatic acinar cells were treated with taurolithocholic acid 3-sulphate disodium salt (TLCS, 500 µM) with or without DFGs, AE and CE. Apoptotic and necrotic cell death pathway activation was measured by caspase 3/7 (10 µl/mL) and propidium iodide (PI, 1 µM), respectively, using a fluorescent plate reader. Necrotic acinar cells were also counted by epifluorescence microscopy. Mice received either 7 intraperitoneal injections of caerulein (50 µg/kg) at hourly intervals or retrograde infusion of TLCS (3 mM, 50 µl) to induce AP (CER-AP and TLCS-AP, respectively). In CER-AP, mice received oral gavage of DFGs (2.1, 4.2 and 5.2 g/kg), AE (0.6, 1.2, and 2.4 g/kg) and CE (4, 9 and 17 mg/kg), or matched DFGs (1.8 g/kg) and AE (1 g/kg) for 3 times at 2-hourly intervals, or a single intraperitoneal injection of DCQD-related monomers rhein (20 mg/kg), narigeinine (25 mg/kg), and honokiol (5 mg/kg) begun at the 3rd injection of caerulein. In TLCS-AP, DFGs (4.2 g/kg) were given orally at 1, 3 and 5 h post-surgery. Disease severity and pancreatic pro-inflammatory markers were determined. RESULTS: The main effective anthraquinones and their glycosides, flavonoids and their glycosides, polyphenols and lignans were found in the DFGs. A higher proportion of polar components including glycosides attached to anthraquinones, phenols and flavonoids was found in AE. Conversely, lower polar components containing methoxy substituted flavonoids and anthraquinones were more abundant in CE. DFGs were given at 4.2 g/kg, a consistent reduction in the pancreatic histopathology score and severity indices was observed in both CER-AP and TLCS-AP. In vitro, AE significantly reduced both apoptotic and necrotic cell death pathway activation, while CE increased TLCS-induced acinar cell necrosis. In vivo, AE at dose of 1.2 g/kg consistently reduced pancreatic histopathological scores and myeloperoxidase in the CER-AP that were associated with suppressed expression of pro-inflammatory meditator mRNAs and proteins. CE increased lung myeloperoxidase and failed to protect against CER-AP in all dosages. AE was demonstrated to be more effective than DFGs in reducing pancreatic histopathological scores and myeloperoxidase. CONCLUSIONS: AE from DFGs alleviated the severity of mouse AP models via an inhibition of pancreatic pro-inflammatory signalling pathways. Efficacy of AE on experimental AP was more potent than its original DFGs and DCQD monomers.

Leucine improves α-amylase secretion through the general secretory signaling pathway in pancreatic acinar cells of dairy calves.

The present study aimed to elucidate the mechanisms by which leucine impacts the secretion of pancreatic enzymes, especially amylase, by studying the proteomics profiles of pancreatic acinar (PA) cells from dairy cows. PA cells, the experimental model, were treated with four concentrations of leucine (0, 0.23, 0.45, and 0.90 mM). The abundance of different proteins in the four leucine treatment groups was detected. Label-free proteomic analysis enabled the identification of 1,906 proteins in all four treatment groups, and 1,350 of these proteins showed common expression across the groups. The primary effects of leucine supplementation were increased (P < 0.05) citrate synthase and ATPase activity, which enlarged the cytosolic ATP pool, and the upregulation of secretory protein 61 (Sec61) expression, which promoted protein secretion. In summary, these results suggest that leucine increases citrate synthase in the TCA cycle and ATPase activity and promotes the Sec signaling pathway to increase the exocrine function of PA cells.

Role of nutrient-driven O-GlcNAc-post-translational modification in pancreatic exocrine and endocrine islet development.

Although the developing pancreas is exquisitely sensitive to nutrient supply in utero, it is not entirely clear how nutrient-driven post-translational modification of proteins impacts the pancreas during development. We hypothesized that the nutrient-sensing enzyme O-GlcNAc transferase (Ogt), which catalyzes an O-GlcNAc-modification onto key target proteins, integrates nutrient-signaling networks to regulate cell survival and development. In this study, we investigated the heretofore unknown role of Ogt in exocrine and endocrine islet development. By genetic manipulation in vivo and by using morphometric and molecular analyses, such as immunofluorescence imaging and single cell RNA sequencing, we show the first evidence that Ogt regulates pancreas development. Genetic deletion of Ogt in the pancreatic epithelium (OgtKOPanc) causes pancreatic hypoplasia, in part by increased apoptosis and reduced levels of of Pdx1 protein. Transcriptomic analysis of single cell and bulk RNA sequencing uncovered cell-type heterogeneity and predicted upstream regulator proteins that mediate cell survival, including Pdx1, Ptf1a and p53, which are putative Ogt targets. In conclusion, these findings underscore the requirement of O-GlcNAcylation during pancreas development and show that Ogt is essential for pancreatic progenitor survival, providing a novel mechanistic link between nutrients and pancreas development.

Effect of thyroid hormones on rat exocrine pancreas morphology and function.

AIM: The influence of thyroid hormones on exocrine pancreas function is poorly understood, and limited to the postnatal development period. Here, we evaluated the effects of hypo- and hyperthyroidism on the morphology and enzyme content of this tissue. MAIN METHODS: To induce hypothyroidism male Wistar rats were subjected to a thyroidectomy (Tx) or sham operated (SO). After 40 days, some of the Tx and SO rats were treated with T3 for 7 days. Following euthanization, the pancreas was removed and evaluated for morphology, as well as amylase, lipase and trypsin content, using histological and immunoreactive techniques analyses, respectively. Serum amylase levels were also evaluated. KEY FINDINGS: The pancreatic acinar cells of Tx rats were smaller, exhibited reduced Haematoxyllin stained areas, and contained lower amylase and lipase levels, indicative of low cell activity. Tx rats also presented higher collagen levels, and high trypsin content in pancreatic extracts. Interestingly, T3 administration reversed the observed acinar cell alterations and restored pancreatic enzyme content, by augmenting amylase and lipase and attenuating trypsin levels, but failed to change collagen content. Increased levels of lipase and decreased trypsin were also observed in T3-treated SO rats. SIGNIFICANCE: Thyroid hormones play an important role in acinar cell morphology and function. In the hypothyroid state there is a decrease in pancreatic enzyme levels that is restored with T3 treatment. In addition to participating in insulin sensitivity and glycemic control, THs also modulate enzyme expression and activity in the exocrine pancreas, consequently, delivering metabolic substrates to specific organs and tissues.

Group II p21-activated kinase, PAK4, is needed for activation of focal adhesion kinases, MAPK, GSK3, and ß-catenin in rat pancreatic acinar cells.

PAK4 is the only member of the Group II p21-activated kinases (PAKs) present in rat pancreatic acinar cells and is activated by gastrointestinal hormones/neurotransmitters stimulating PLC/cAMP and by various pancreatic growth factors. However, little is known of the role of PAK4 activation in cellular signaling cascades in pancreatic acinar cells. In the present study, we examined the role of PAK4's participation in five different cholecystokinin-8 (CCK-8)-stimulated signaling pathways (PI3K/Akt, MAPK, focal adhesion kinase, GSK3, and ß-catenin), which mediate many of its physiological acinar-cell effects, as well as effects in pathophysiological conditions. To define PAK4's role, the effect of two different PAK4 inhibitors, PF-3758309 and LCH-7749944, was examined under experimental conditions that only inhibited PAK4 activation and not activation of the other pancreatic PAK, Group I PAK2. The inhibitors' effects on activation of these five signaling cascades by both physiological and pathophysiological concentrations of CCK, as well as by 12-O-tetradecanoylphobol-13-acetate (TPA), a PKC-activator, were examined. CCK/TPA activation of focal adhesion kinases(PYK2/p125FAK) and the accompanying adapter proteins (paxillin/p130CAS), Mek1/2, and p44/42, but not c-Raf or other MAPKs (JNK/p38), were mediated by PAK4. Activation of PI3K/Akt/p70s6K was independent of PAK4, whereas GSK3 and ß-catenin stimulation was PAK4-dependent. These results, coupled with recent studies showing PAK4 is important in pancreatic fluid/electrolyte/enzyme secretion and acinar cell growth, show that PAK4 plays an important role in different cellular signaling cascades, which have been shown to mediate numerous physiological and pathophysiological processes in pancreatic acinar cells.NEW & NOTEWORTHY In pancreatic acinar cells, cholecystokinin (CCK) or 12-O-tetradecanoylphobol-13-acetate (TPA) activation of focal adhesion kinases (p125FAK,PYK2) and its accompanying adapter proteins, p130CAS/paxillin; Mek1/2, p44/42, GSK3, and ß-catenin are mediated by PAK4. PI3K/Akt/p70s6K, c-Raf, JNK, or p38 pathways are independent of PAK4 activation.

Akt1 signalling supports acinar proliferation and limits acinar-to-ductal metaplasia formation upon induction of acute pancreatitis.

Molecular signalling mediated by the phosphatidylinositol-3-kinase (PI3K)-Akt axis is a key regulator of cellular functions. Importantly, alteration of the PI3K-Akt signalling underlies the development of different human diseases, thus prompting the investigation of the pathway as a molecular target for pharmacologic intervention. In this regard, recent studies showed that small molecule inhibitors of PI3K, the upstream regulator of the pathway, reduced the development of inflammation during acute pancreatitis, a highly debilitating and potentially lethal disease. Here we investigated whether a specific reduction of Akt activity, by using either pharmacologic Akt inhibition, or genetic inactivation of the Akt1 isoform selectively in pancreatic acinar cells, is effective in ameliorating the onset and progression of the disease. We discovered that systemic reduction of Akt activity did not protect the pancreas from initial damage and only transiently delayed leukocyte recruitment. However, reduction of Akt activity decreased acinar proliferation and exacerbated acinar-to-ductal metaplasia (ADM) formation, two critical events in the progression of pancreatitis. These phenotypes were recapitulated upon conditional inactivation of Akt1 in acinar cells, which resulted in reduced expression of 4E-BP1, a multifunctional protein of key importance in cell proliferation and metaplasia formation. Collectively, our results highlight the critical role played by Akt1 during the development of acute pancreatitis in the control of acinar cell proliferation and ADM formation. In addition, these results harbour important translational implications as they raise the concern that inhibitors of PI3K-Akt signalling pathways may negatively affect the regeneration of the pancreas. Finally, this work provides the basis for further investigating the potential of Akt1 activators to boost pancreatic regeneration following inflammatory insults. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pyridoxine and pancreatic acinar cells: transport physiology and effect on gene expression profile.

Pyridoxine (vitamin B6), an essential micronutrient for normal cell physiology, plays an important role in the function of the exocrine pancreas. Pancreatic acinar cells (PACs) obtain vitamin B6 from circulation, but little is known about the mechanism involved in the uptake process; limited information also exists on the effect of pyridoxine availability on the gene expression profile in these cells. We addressed both these issues in the current investigation using mouse-derived pancreatic acinar 266-6 cells (PAC 266-6) and human primary PACs (hPACs; obtained from organ donors), together with appropriate physiological and molecular (RNA-Seq) approaches. The results showed [3H]pyridoxine uptake to be 1) pH and temperature (but not Na+) dependent, 2) saturable as a function of concentration, 3) cis-inhibited by unlabeled pyridoxine and its close structural analogs, 4) trans-stimulated by unlabeled pyridoxine, 5) regulated by an intracellular Ca2+/calmodulin-mediated pathway, 6) adaptively-regulated by extracellular substrate (pyridoxine) availability, and 7) negatively impacted by exposure to cigarette smoke extract. Vitamin B6 availability was found (by means of RNA-Seq) to significantly (FDR < 0.05) modulate the expression profile of many genes in PAC 266-6 cells (including those that are relevant to pancreatic health and development). These studies demonstrate, for the first time, the involvement of a regulatable and specific carrier-mediated mechanism for pyridoxine uptake by PACs; the results also show that pyridoxine availability exerts profound effects on the gene expression profile in mammalian PACs.

Effect of Docosahexaenoic Acid on Ca2+ Signaling Pathways in Cerulein-Treated Pancreatic Acinar Cells, Determined by RNA-Sequencing Analysis.

Intracellular Ca2+ homeostasis is commonly disrupted in acute pancreatitis. Sustained Ca2+ release from internal stores in pancreatic acinar cells (PACs), mediated by inositol triphosphate receptor (IP3R) and the ryanodine receptor (RyR), plays a key role in the initiation and propagation of acute pancreatitis. Pancreatitis induced by cerulein, an analogue of cholecystokinin, causes premature activation of digestive enzymes and enhanced accumulation of cytokines and Ca2+ in the pancreas and, as such, it is a good model of acute pancreatitis. High concentrations of the omega-3 fatty acid docosahexaenoic acid (DHA) inhibit inflammatory signaling pathways and cytokine expression in PACs treated with cerulein. In the present study, we determined the effect of DHA on key regulators of Ca2+ signaling in cerulein-treated pancreatic acinar AR42 J cells. The results of RNA-Sequencing (RNA-Seq) analysis showed that cerulein up-regulates the expression of IP3R1 and RyR2 genes, and that pretreatment with DHA blocks these effects. The results of real-time PCR confirmed that DHA inhibits cerulein-induced IP3R1 and RyR2 gene expression, and demonstrated that DHA pre-treatment decreases the expression of the Relb gene, which encodes a component of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcriptional activator complex, and the c-fos gene, which encodes a component of activator protein-1 (AP-1) transcriptional activator complex. Taken together, DHA inhibits mRNA expression of IP3R1, RyR2, Relb, and c-fos, which is related to Ca2+ network in cerulein-stimulated PACs.

In utero and postnatal VX-770 administration rescues multiorgan disease in a ferret model of cystic fibrosis.

Cystic fibrosis (CF) is a multiorgan disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). In patients with CF, abnormalities initiate in several organs before birth. However, the long-term impact of these in utero pathologies on disease pathophysiology is unclear. To address this issue, we generated ferrets harboring a VX-770 (ivacaftor)-responsive CFTR G551D mutation. In utero VX-770 administration provided partial protection from developmental pathologies in the pancreas, intestine, and male reproductive tract. Homozygous CFTR G551D/G551D animals showed the greatest VX-770-mediated protection from these pathologies. Sustained postnatal VX-770 administration led to improved pancreatic exocrine function, glucose tolerance, growth and survival, and to reduced mucus accumulation and bacterial infections in the lung. VX-770 withdrawal at any age reestablished disease, with the most rapid onset of morbidity occurring when withdrawal was initiated during the first 2 weeks after birth. The results suggest that CFTR is important for establishing organ function early in life. Moreover, this ferret model provides proof of concept for in utero pharmacologic correction of genetic disease and offers opportunities for understanding CF pathogenesis and improving treatment.

Diabetes mellitus induced by PD-1 and PD-L1 inhibitors: description of pancreatic endocrine and exocrine phenotype.

AIMS: Programmed cell death-1 and programmed death ligand 1 (PD-1/PD-L1) inhibitors restore antitumor immunity, but many autoimmune side-effects have been described. Diabetes mellitus is a rare complication, and little data concerning its pathophysiology and phenotype have been published. This study aimed to describe both pancreatic endocrine and exocrine functions, immunological features and change in pancreas volume in subjects with diabetes mellitus induced by PD-1 and PD-L1 inhibitors. METHODS: We analyzed the data of six subjects treated with immunotherapy who presented acute diabetes. RESULTS: There were five men and one woman. Median age was 67 years (range 55-83). Three subjects were treated with nivolumab, two with pembrolizumab and one with durvalumab. Median time to diabetes onset after immunotherapy initiation was 4 months (range 2-13). Four patients presented fulminant diabetes (FD); none of these had type 1 diabetes (T1D)-related autoantibodies, none of them had T1D or FD-very high-risk HLA class II profiles. The bi-hormonal endocrine and exocrine pancreatic failure previously reported for one FD patient was not found in other FD subjects, but glucagon response was blunted in another FD patient. Pancreas volume was decreased at diabetes onset in 2 FD patients, and all patients presented a subsequent decrease of pancreas volume during follow-up. CONCLUSIONS: In the patients presented herein, immunotherapy-induced diabetes was not associated with T1D-related autoantibodies. The hormonal and morphological analysis of the pancreatic glands of these six cases contributes to the understanding of the underlying and probably heterogeneous mechanisms. There is a need to find biomarkers to identify patients at risk to develop these new forms of diabetes at early stages of the process to prevent ketoacidosis and to evaluate preventive strategies.

Diabetes of the exocrine pancreas.

Diabetes of the exocrine pancreas (DEP) is a form of diabetes that occurs due to pancreatic disease. It is far more common than has been previously considered, with a recent study showing 1.8% of adults with new-onset diabetes should have been classified as DEP. The majority is misdiagnosed as type 2 diabetes mellitus (T2DM). Patients with DEP exhibit varying degrees of exocrine and endocrine dysfunction. Damage to the islet of Langerhans effects the secretion of hormones from the ß, α, and pancreatic polypeptide cells; the combination of low insulin, glucagon, and pancreatic polypeptide contributes to rapid fluctuations in glucose levels. This form of "brittle diabetes" may result in the poorer glycemic control observed in patients with DEP, when compared with those with T2DM. Diabetes of the exocrine pancreas has a different natural history to other forms of diabetes; patients are more likely to require early insulin initiation compared with those with T2DM. Therefore, individuals with DEP should be advised about the symptoms of decompensated hyperglycemia, although they are less likely to develop ketoacidosis. Clinicians should screen for DEP in patients with acute or chronic pancreatitis, following pancreatic resection, or with co-existing cystic fibrosis or hemochromatosis. Incident diabetes may herald the onset of pancreatic ductal carcinoma in a small subset of patients. Once identified, patients with DEP can benefit from specific lifestyle advice, pancreatic enzyme replacement therapy, metformin treatment, appropriate insulin dosing, and monitoring. Further research is needed to establish the ideal treatment regimens to provide optimal clinical outcomes for this unique form of diabetes.

Randomized controlled study of the effect of octreotide on pancreatic exocrine secretion and pancreatic fistula after pancreatoduodenectomy.

BACKGROUND: Octreotide is known to decrease the rate of postoperative complication after pancreatic resection by diminishing exocrine function of the pancreas. The aim of this study was to evaluate the effect of octreotide in decreasing exocrine excretion of pancreas and preventing pancreatic fistula. MATERIALS AND METHODS: Prospective randomized trial was conducted involving 59 patients undergoing pancreaticoduodenectomy for either malignant or benign tumor, 29 patients were randomized to receive octreotide; 30 patients allotted to placebo. All pancreaticojejunal anastomosis was performed with external stent of negative-pressured drainage and the amount of pancreatic juice through the external stent was measured until postoperative 7th day. Pancreatic fistula was recorded. RESULTS: There were no differences in demographics, pancreatic texture and pancreatic duct diameter between the octreotide and placebo group. The median output of pancreatic juice was not significantly different between both groups during 7 days after surgery. When the patients were stratified according to the diameter of pancreatic duct (duct ≤5 mm, > 5 mm), there were no significant differences in daily amount of pancreatic juice, however, when stratified according to pancreatic texture, median output of pancreatic juice was significantly lower in patients with hard pancreas compared with those with soft pancreas from 5 day to 7 day after surgery (p < 0.05). No significant differences in pancreatic fistula and postoperative complications were found between the octreotide and placebo groups. CONCLUSIONS: Prophylactic octreotide is not effective to inhibit the exocrine secretion of the remnant pancreas and does not decrease the incidence of pancreatic fistula after pancreaticoduodenectomy.

Association of fatty pancreas with pancreatic endocrine and exocrine function.

AIM: The purpose of this study was to clarify whether fatty pancreas might lead to impaired pancreatic endocrine or exocrine function. MATERIAL AND METHODS: The study involved 109 participants who had undergone the glucagon stimulation test and N-benzoyl-L-tyros-p-amino benzoic acid (BT-PABA) test to assess pancreatic function as well as unenhanced abdominal computed tomography (CT). Pancreatic endocrine impairment was defined as ΔC peptide immunoreactivity less than 2 [mmol/L] in the glucagon stimulation test, and pancreatic exocrine impairment was defined as a urinary PABA excretion rate less than 70% on the BT-PABA test. We defined as the mean CT value of pancreas / CT value of spleen (P/S ratio) as a marker to assess fatty pancreas. We analyzed the association between fatty pancreas and pancreatic impairment using the logistic regression model. The odds ratio (OR) is shown per 0.1 unit. RESULTS: Pancreatic endocrine function was impaired in 33.0% of the participants, and 56.9% of those were regarded as having pancreatic exocrine impairment. The P/S ratio was significantly correlated with pancreatic endocrine impairment in univariate analysis (OR = 0.61, 95% confidence interval (CI) = 0.43-0.83, P = 0.0013) and multivariate analysis (OR = 0.38, 95% CI = 0.22-0.61, P < .0001) for all participants. Similar significant relationships were observed in both univariate (OR = 0.70, 95% CI = 0.49-0.99, P = 0.04) and multivariate (OR = 0.39, 95% CI = 0.21-0.66, P = 0.0002) analyses for the participants without diabetes (n = 93). The amount of pancreatic fat was not associated with exocrine impairment in univariate analysis (OR = 0.80, 95% CI = 0.59-1.06, P = 0.12). CONCLUSION: Fatty pancreas was associated with pancreatic endocrine impairment but did not have a clear relationship with pancreatic exocrine impairment.