Peroxisomal dysfunction unhinges cellular metabolism by causing the accumulation of toxic metabolic intermediates (e.g. reactive oxygen species, very -chain fatty acids, phytanic acid or eicosanoids) and the depletion of important lipid products (e.g. plasmalogens, polyunsaturated fatty acids), leading to various proinflammatory and devastating pathophysiological conditions like metabolic syndrome and age-related diseases including diabetes. Because the peroxisomal antioxidative marker enzyme catalase is low abundant in Langerhans islet cells, peroxisomes were considered scarcely present in the endocrine pancreas. Recently, studies demonstrated that the peroxisomal metabolism is relevant for pancreatic cell functionality. During the postnatal period, significant changes occur in the cell structure and the metabolism to trigger the final maturation of the pancreas, including cell proliferation, regulation of energy metabolism, and activation of signalling pathways. Our aim in this study was to (i) morphometrically analyse the density of peroxisomes in mouse endocrine versus exocrine pancreas and (ii) investigate how the distribution and the abundance of peroxisomal proteins involved in biogenesis, antioxidative defence and fatty acid metabolism change during pancreatic maturation in the postnatal period. Our results prove that endocrine and exocrine pancreatic cells contain high amounts of peroxisomes with heterogeneous protein content indicating that distinct endocrine and exocrine cell types require a specific set of peroxisomal proteins depending on their individual physiological functions. We further show that significant postnatal changes occur in the peroxisomal compartment of different pancreatic cells that are most probably relevant for the metabolic maturation and differentiation of the pancreas during the development from birth to adulthood.

While relatively ignored for years as vestigial, cilia have recently become the focus of intense interest as organelles that result in severe pathologies when disrupted. Here, we further establish a connection between cilia dysfunction and disease by showing that loss of polaris (Tg737), an intraflagellar transport (IFT) protein required for ciliogenesis, causes abnormalities in the exocrine and endocrine pancreas of the Tg737(orpk) mouse. Pathology is evident late in gestation as dilatations of the pancreatic ducts that continue to expand postnatally. Shortly after birth, the acini become disorganized, undergo apoptosis, and are largely ablated in late stage pathology. In addition, serum amylase levels are elevated and carboxypeptidase is abnormally activated within the pancreas. Ultrastructural analysis reveals that the acini undergo extensive vacuolization and have numerous 'halo-granules' similar to that seen in induced models of pancreatitis resulting from duct obstruction. Intriguingly, although the acini are severely affected in Tg737(orpk) mutants, cilia and Tg737 expression are restricted to the ducts and islets and are not detected on acinar cells. Analysis of the endocrine pancreas in Tg737(orpk) mutants revealed normal differentiation and distribution of cell types in the islets. However, after fasting, mutant blood glucose levels are significantly lower than controls and when challenged in glucose tolerance tests, Tg737(orpk) mutants exhibited defects in glucose uptake. These findings are interesting in light of the recently proposed role for polaris, the protein encoded by the Tg737 gene, in the hedgehog pathway and hedgehog signaling in insulin production and glucose homeostasis.

Arf4 is proposed to be a critical regulator of membrane protein trafficking in early secretory pathway. More recently, Arf4 was also implicated in regulating ciliary trafficking, however, this has not been comprehensively tested in vivo. To directly address Arf4's role in ciliary transport, we deleted Arf4 specifically in either rod photoreceptor cells, kidney, or globally during the early postnatal period. Arf4 deletion in photoreceptors did not cause protein mislocalization or retinal degeneration, as expected if Arf4 played a role in protein transport to the ciliary outer segment. Likewise, Arf4 deletion in kidney did not cause cystic disease, as expected if Arf4 were involved in general ciliary trafficking. In contrast, global Arf4 deletion in the early postnatal period resulted in growth restriction, severe pancreatic degeneration and early death. These findings are consistent with Arf4 playing a critical role in endomembrane trafficking, particularly in the pancreas, but not in ciliary function.

Type 1 diabetes (T1D) is widely considered to result from the autoimmune destruction of insulin-producing β cells. This concept has been a central tenet for decades of attempts seeking to decipher the disorder's pathogenesis and prevent/reverse the disease. Recently, this and many other disease-related notions have come under increasing question, particularly given knowledge gained from analyses of human T1D pancreas. Perhaps most crucial are findings suggesting that a collective of cellular constituents-immune, endocrine, and exocrine in origin-mechanistically coalesce to facilitate T1D. This review considers these emerging concepts, from basic science to clinical research, and identifies several key remaining knowledge voids.

Reports of exocrine-to-endocrine reprogramming through expression or stabilization of the transcription factor neurogenin 3 (NGN3) have generated renewed interest in harnessing pancreatic plasticity for therapeutic applications. NGN3 is expressed by a population of endocrine progenitor cells that give rise exclusively to hormone-secreting cells within pancreatic islets and is necessary and sufficient for endocrine differentiation during development. In the adult human pancreas, NGN3 is expressed by dedifferentiating exocrine cells with a phenotype resembling endocrine progenitor cells and the capacity for endocrine differentiation in vitro. Neurotrophic tyrosine kinase receptor type 2 (TRKB), which regulates neuronal cell survival, differentiation and plasticity, was identified as highly overexpressed in the NGN3 positive cell transcriptome compared to NGN3 negative exocrine cells. This study was designed to determine if NGN3 is regulated by TRKB signaling in the adult human exocrine pancreas.

To elucidate the etiology of type 1 diabetes, the affected pancreas needs to be thoroughly characterized. Pancreatic innervation has been suggested to be involved in the pathology of the disease and a reduction of sympathetic innervation of the islets was recently reported. In the present study, we hypothesized that parasympathetic innervation would be altered in the type 1 diabetes pancreas. Human pancreatic specimens were obtained from a unique cohort of individuals with recent onset or long standing type 1 diabetes. Density of parasympathetic axons was assessed by immunofluorescence and morphometry. Our main finding was a reduced density of parasympathetic axons in the exocrine, but not endocrine compartment of the pancreas in individuals with recent onset type 1 diabetes. The reduced density of parasympathetic axons in the exocrine compartment could have functional implications, e.g. be related to the exocrine insufficiency reported in type 1 diabetes patients. Further studies are needed to understand whether reduced parasympathetic innervation is a cause or consequence of type 1 diabetes.

In order to purify and characterize nestin-positive cells in the developing pancreas a transgenic mouse was generated, in which the enhanced green fluorescent protein (EGFP) was driven by the nestin second intronic enhancer and upstream promoter. In keeping with previous studies on the distribution of nestin, EGFP was expressed in the developing embryo in neurones in the brain, eye, spinal cord, tail bud and glial cells in the small intestine. In the pancreas there was no detectable EGFP at embryonic day 11.5 (E11.5). EGFP expression appeared at E12.5 and increased in intensity through E14.5, E18.5 and post-natal day 1. Flow cytometry was used to quantify and purify the EGFP positive population in the E15.5 pancreas. The purified (96%) EGFP-expressing cells, which represent 20% of the total cell population, were shown by RT/PCR to express exocrine cell markers (amylase and P48) and endocrine cell markers (insulin 1, insulin 2, and Ngn3). They also expressed, at a lower level, PDX-1, Isl-1, and the islet hormones pancreatic polypeptide, glucagon and somatostatin as well as GLUT2, the stem cell marker ABCG2 and PECAM, a marker of endothelial cells. It was further shown by immunocytochemistry of the E15.5 pancreas that EGFP colocalised in separate subpopulations of cells that expressed nestin, insulin and amylase. These results support the conclusion that nestin expressing cells can give rise to both endocrine and exocrine cells. The ability to purify these putative progenitor cells may provide further insights into their properties and function.

Alcoholism causes an imbalance of endoplasmic reticulum (ER) homeostasis in pancreatic acini. In those cells, the ER is involved in the synthesis and folding of pancreatic enzymes. Ubiquitin-fold modifier 1 (Ufm1) is part of a novel ubiquitin-like modification system involved in maintaining ER homeostasis. Among the components of the Ufm1 system, Regulator of C53 and DDRGK1 (RCAD) has recently been identified as a Ufm1-specific E3 ligase that promotes ufmylation of DDRGK1, an RCAD-interacting protein. We determined the importance of RCAD in the proper synthesis and secretion of pancreatic enzymes using mice with genetically deleted RCAD. The pancreas of RCAD-deficient mice was of normal size and histology. Using quantitative PCR and Western blotting, we found that amylase was upregulated in pancreas organs from RCAD-knockout (KO) mice. Constitutive amylase secretion was much higher in isolated pancreatic acini from RCAD KO mice, whereas CCK-stimulated amylase secretion was disturbed. RCAD deficiency caused a downregulation in expression of ER chaperone BiP, which affected ER homeostasis and activated both apoptosis and trypsin. We also found that both RCAD and DDRGK1 transcript levels were upregulated in pancreatic acini from alcohol-preferring rats. Elevated expression of RCAD and DDRGK1 was associated with increased ER stress and UPR activation. Because of the lack of BiP expression, caspase 3 and trypsin activation we enhanced in RCAD-deficient pancreatic acini upon treatment with ethanol and CCK. In conclusion, the RCAD/BiP pathway is required for proper synthesis and secretion of pancreatic enzymes. In alcoholism, increased levels of components of the Ufm1 system could prevent the deleterious effects of alcohol in the pancreas by regulating BiP levels.NEW & NOTEWORTHY RCAD/BiP pathway is required for the proper synthesis and secretion of amylase from pancreatic acini, as well as for the maintenance of the ER homeostasis. In alcoholism, the exocrine pancreas could increase the levels of components of the Ufm1 system to protect itself from alcohol's deleterious effects by regulating the expression of ER chaperone BiP.

Two chronic dietary studies, conducted years apart, with ammonium perfluorooctanoate (APFO) in Sprague Dawley rats have been previously reported. Although both included male 300 ppm dietary dose groups, only the later study, conducted in 1990-1992 by Biegel et al., reported an increase in proliferative lesions (hyperplasia and adenoma) of the acinar pancreas. An assessment of the significance of the differences between both studies requires careful consideration of: the diagnostic criteria for proliferative acinar cell lesions of the rat pancreas (for example, the diagnosis of pancreatic acinar cell hyperplasia versus adenoma is based on the two-dimensional size of the lesion rather than distinct morphological differences); the basis for those criteria in light of their relevance to biological behavior; and the potential diagnostic variability between individual pathologists for difficult-to-classify lesions. A pathology peer review of male exocrine pancreatic tissues from the earlier study, conducted in 1981-1983 by Butenhoff et al., was undertaken. This review identified an increase in acinar cell hyperplasia but not adenoma or carcinoma in the earlier study. Both studies observed a proliferative response in the acinar pancreas which was more pronounced in the study by Biegel et al. Definitive reasons for the greater incidence of proliferative lesions in the later study were not identified, but some possible explanations are presented herein. The relevance of this finding to human risk assessment, in the face of differences in the biological behavior of human and rat pancreatic proliferative lesions and the proposed mechanism of formation of these lesions, are questionable.

Dietary fat overload (typical to obesity) increases the risk of pancreatic pathologies through mechanisms yet to be defined. We previously showed that saturated dietary fat induces pancreatic acinar lipotoxicity and cellular stress. The endoplasmic reticulum (ER) of exocrine pancreas cells is highly developed and thus predisposed to stress. We studied the combination of saturated and unsaturated FAs in metabolic and pancreatitis like cerulein (CER)-induced stress states on cellular ER stress.Exocrine pancreas AR42J and rat primary exocrine acinar cells underwent acute (24 h) challenge with different FAs (saturated, monounsaturated) at different concentrations (250 and 500 µM) and in combination with acute CER-induced stress, and were analyzed for fat accumulation, ER stress unfolded protein response (UPR) and immune and enzyme markers. Acute exposure of AR42J and pancreatic acinar cells to different FAs and their combinations increased triglyceride accumulation. Palmitic acid significantly dose-dependently enhanced the UPR, immune factors and pancreatic lipase (PL) levels, as demonstrated by XBP1 splicing and elevation in UPR transcripts and protein levels (Xbp1,Atf6, Atf4, Chop, Tnfα, Tgfβ and Il-6). Exposure to high palmitic levels in a CER-induced stress state synergistically increased ER stress and inflammation marker levels. Exposure to oleic acid did not induce ER stress and PL levels and significantly decreased immune factors in an acute CER-induced stress state. Combination of oleic and palmitic acids significantly reduced the palmitic-induced ER stress, but did not affect the immune factor response. We show that combination of monounsaturated and saturated FAs protects from exocrine pancreatic cellular ER stress in both metabolic and CER-induced stress.

Despite progress in the field of decellularization and recellularization, the outcome for pancreas has not been adequate. This might be due to the challenging dual nature of pancreas with both endocrine and exocrine tissues. We aimed to develop a novel and efficient cold-perfusion method for decellularization of porcine pancreas and recellularize acellular scaffolds with human fetal pancreatic stem cells. Decellularization of whole porcine pancreas at 4°C with sodium deoxycholate, Triton X-100 and DNase efficiently removed cellular material, while preserving the extracellular matrix structure. Furthermore, recellularization of acellular pieces with human fetal pancreatic stem cells for 14 days showed attached and proliferating cells. Both endocrine (C-peptide and PDX1) and exocrine (glucagon and α-amylase) markers were expressed in recellularized tissues. Thus, cold-perfusion can successfully decellularize porcine pancreas, which when recellularized with human fetal pancreatic stem cells shows relevant endocrine and exocrine phenotypes. Decellularized pancreas is a promising biomaterial and might translate to clinical relevance for treatment of diabetes.

CD1d is a nonpolymorphic MHC class I-like molecule which presents nonpeptide ligands, e.g. glycolipids, to NKT cells. These cells are known to have multiple effects on innate and adaptive immune responses and on the development of pathological conditions. In order to analyze CD1d expression and function in the rat, the first rat CD1d-specific monoclonal antibodies (mAbs) were generated.

Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.

Histopathological heterogeneity in human pancreas has been well documented; however, functional evidence at the tissue level is scarce. Herein we investigated in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in no diabetes (ND, n=15), single islet autoantibody-positive (1AAb+, n=7), and type 1 diabetes donors (T1D, <14 months duration, n=5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features were comparable across the regions in ND. In T1D, insulin secretion and beta-cell volume were significantly reduced within all regions, while glucagon and enzymes were unaltered. Beta-cell volume was lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ were consistent across PH, PB and PT. This study supports low inter-regional variation in pancreas slice function and potentially, increased metabolic demand in 1AAb+.

Abnormal glucagon secretion and functional alterations of the exocrine pancreas have been described in patients with type 1 diabetes (T1D), but their respective anatomical substrata have seldom been investigated. Our aim was to develop an automated morphometric analysis process to characterize the anatomy of α-cell and exocrine pancreas in patients with T1D, using the publicly available slides of the Network for Pancreatic Organ Donors (nPOD).

Sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor-based incretin therapy intended for the treatment of type 2 diabetes mellitus (T2DM), has not been linked to adverse effects on the pancreas in prospective clinical trials or in nonclinical toxicology studies. To further assess potential pancreatic effects, sitagliptin was studied in the male Zucker diabetic fatty (ZDF) rat model of T2DM. Following 3 months of oral dosing with vehicle, or sitagliptin at doses 3- to 19-fold above the clinically therapeutic plasma concentration, which increased active plasma glucagon-like peptide-1 levels up to approximately 3-fold, or following 3 months of oral dosing with metformin, a non-incretin-based reference T2DM treatment, the pancreas of male ZDF rats was evaluated using qualitative and quantitative histopathology techniques. In the quantitative evaluation, proliferative index was calculated in exocrine pancreatic ducts and ductules using computer-based image analysis on sections stained by immunohistochemistry for cytokeratin (a cytoplasmic epithelial cell marker) and Ki-67 (a nuclear marker of recent cell division). Relative to controls, sitagliptin treatment did not alter disease progression based on detailed clinical signs and clinical pathology assessments. Sitagliptin treatment did not result in pancreatitis or any adverse effect on the pancreas based on a qualitative histopathology evaluation. Proliferative index did not increase with sitagliptin treatment based on quantitative assessment of more than 5000 sections of pancreas, where control group means ranged from 0.698-0.845% and sitagliptin-treated group means ranged from 0.679-0.701% (P = .874). Metformin treatment was similarly evaluated and found not to have adverse effects on pancreas.

Exocrine pancreatic cancer is an aggressive disease with an exceptionally high mortality rate. Genetic analysis suggests a causative role for environmental factors, but consistent epidemiological support is scarce and no biomarkers for monitoring the effects of chemical pancreatic carcinogens are available. With the objective to identify common traits for chemicals inducing pancreatic tumors we studied the National Toxicology Program (NTP) bioassay database. We found that male rats were affected more often than female rats and identified eight chemicals that induced exocrine pancreatic tumors in males only. For a hypothesis generating process we used a text mining tool to analyse published literature for suggested mode of actions (MOA). The resulting MOA analysis suggested inflammatory responses as common feature. In cell studies we found that all the chemicals increased protein levels of the inflammatory protein autotaxin (ATX) in Panc-1, MIA PaCa-2 or Capan-2 cells. Induction of MMP-9 and increased invasive migration were also frequent effects, consistent with ATX activation. Testosterone has previously been implicated in pancreatic carcinogenesis and we found that it increased ATX levels. Our data show that ATX is a target for chemicals inducing pancreatic tumors in rats. Several lines of evidence implicate ATX and its product lysophosphatidic acid in human pancreatic cancer. Mechanisms of action may include stimulated invasive growth and metastasis. ATX may interact with hormones or onco- or suppressor-genes often deregulated in exocrine pancreatic cancer. Our data suggest that ATX is a target for chemicals promoting pancreatic tumor development.

Using a dominant-negative mutant receptor (DNR) approach in transgenic mice, we have functionally inactivated transforming growth factor-beta (TGF-beta) signaling in select epithelial cells. The dominant-negative mutant type II TGF-beta receptor blocked signaling by all three TGF-beta isoforms in primary hepatocyte and pancreatic acinar cell cultures generated from transgenic mice, as demonstrated by the loss of growth inhibitory and gene induction responses. However, it had no effect on signaling by activin, the closest TGF-beta family member. DNR transgenic mice showed increased proliferation of pancreatic acinar cells and severely perturbed acinar differentiation. These results indicate that TGF-beta negatively controls growth of acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. In contrast, such abnormalities were not observed in the liver. Additional abnormalities in the pancreas included fibrosis, neoangiogenesis and mild macrophage infiltration, and these were associated with a marked up-regulation of TGF-beta expression in transgenic acinar cells. This transgenic model of targeted functional inactivation of TGF-beta signaling provides insights into mechanisms whereby loss of TGF-beta responsiveness might promote the carcinogenic process, both through direct effects on cell proliferation, and indirectly through up-regulation of TGF-betas with associated paracrine effects on stromal compartments.

Exocrine pancreatic insufficiency (EPI) is a syndrome characterized by insufficient synthesis of pancreatic enzymes leading to clinical symptoms of malabsorption and maldigestion. There are no studies about ultrasonographic appearance of the pancreas with EPI in dogs. The purpose of this retrospective study was to describe ultrasound features of the pancreas during EPI in this species. Dogs with history and clinical signs of maldigestion, serum canine trypsin-like immunoreactivity (cTLI) values <5 µg/L, and abdominal ultrasound exam were included in the study. Size, shape, margin, echogenicity, echostructure, and pancreatic duct appearance of the right pancreatic lobe were valued. Additional sonographic intestinal findings were recorded. Thirty-four dogs were included. The mean pancreatic thickness in our population was significantly lower than the mean reference values of healthy dogs. In 68% of dogs, the pancreas had a normal ultrasound appearance. Ultrasonographic intestinal abnormal findings were identified in 85% of dogs and were suggestive of inflammatory bowel disease. Despite the fact that EPI is a functional diagnosis, ultrasound evaluation should be considered among the useful tests. The finding of a normal but thinned pancreas associated with sonographic intestinal signs of inflammatory bowel disease in dogs with typical history and supportive clinical signs could suggest a diagnosis of EPI.

Epiplakin1 (Eppk1) is a plakin family gene with its function remains largely unknown, although the plakin genes are known to function in interconnecting cytoskeletal filaments and anchoring them at plasma membrane-associated adhesive junction. Here we analyzed the expression patterns of Eppk1 in the developing and adult pancreas in the mice. In the embryonic pancreas, Eppk1+/Pdx1+ and Eppk1+/Sox9+ pancreatic progenitor cells were observed in early pancreatic epithelium. Since Pdx1 expression overlapped with that of Sox9 at this stage, these multipotent progenitor cells are Eppk1+/Pdx1+/Sox9+ cells. Then Eppk1 expression becomes confined to Ngn3+ or Sox9+ endocrine progenitor cells, and p48+ exocrine progenitor cells, and then restricted to the duct cells and a cells at birth. In the adult pancreas, Eppk1 is expressed in centroacinar cells (CACs) and in duct cells. Eppk1 is observed in pancreatic intraepithelial neoplasia (PanIN), previously identified as pancreatic ductal adenocarcinoma (PDAC) precursor lesions. In addition, the expansion of Eppk1-positive cells occurs in a caerulein-induced acute pancreatitis, an acinar cell regeneration model. Furthermore, in the partial pancreatectomy (Px) regeneration model using mice, Eppk1 is expressed in "ducts in foci", a tubular structure transiently induced. These results suggest that Eppk1 serves as a useful marker for detecting pancreatic progenitor cells in developing and regenerating pancreas.