Pancreatic Neurons Research Articles

8330 Pancreatic neurons in neuron-islet complexes and activation of these neurons promotes insulin secretion under high glucose level

Abstract Disclosure: Y. Liu: None. Z. Yang: None. Q. Feng: None. M. Hu: None. L. Sha: None. Islets are rich in pancreatic nerve innervation. Pancreatic ganglionic neurons, as an integrating center, receive central and peripheral nerve innervation regulating endocrine and exocrine of pancreas. Neurons in pancreatic ganglia have been studied intensely in our lab and a few other labs. Neurons located in islets forming neuron-islet complexes have been demonstrated. However, the properties and function of these neurons has not been studied. In this study, pancreatic neurons in the neuron-islet complex and the effects of activation of these neurons on insulin release were investigated. Methods: Eight week old rats were used. Pancreas was digested and a method was developed to identify neuron-islet complexes from islets. Results: CHAT and TUBB3 were co-localized in nearly all neurons in the neuron-islet complex indicating that these neurons were mainly cholinergic neurons. A small portion (<10%) of TUBB3 positive cells were also positive for nNOS. Nicotinic tartrate was used to activate neurons in the complexes. After one hour of administration of nicotinic tartrate, with 5.6 mM glucose, ACh level was increased significantly from 15.26±5.46 μM to 23.24±4.18 μM and insulin level were slightly increased from 37.21±2.04 μM to 39.27±1.23μM (P > 0.05). With 16.7 mM glucose, ACh level were increased significantly from 23.83±2.28 μM to 30.92±1.03 μM and insulin increased significantly from 41.93±1.30 μM to 49.04±1.68 μM. Conclusion: The neurons in neuron-islet complexes are mainly cholinergic neurons and activation of these neuron under high glucose level stimulate insulin secretion. Presentation: 6/2/2024

Innervation of the pancreas in development and disease.

The autonomic nervous system innervates the pancreas by sympathetic, parasympathetic and sensory branches during early organogenesis, starting with neural crest cell invasion and formation of an intrinsic neuronal network. Several studies have demonstrated that signals from pancreatic neural crest cells direct pancreatic endocrinogenesis. Likewise, autonomic neurons have been shown to regulate pancreatic islet formation, and have also been implicated in type I diabetes. Here, we provide an overview of recent progress in mapping pancreatic innervation and understanding the interactions between pancreatic neurons, epithelial morphogenesis and cell differentiation. Finally, we discuss pancreas innervation as a factor in the development of diabetes.

169-OR: Magnetogenetic Stimulation of Pancreatic Nerves Regulates Blood Glucose

There is substantial evidence that neural regulation plays a critical role in glucose metabolism and hormone release. Targeting neural populations innervating the pancreas could provide an alternative therapy for diabetes. However, our understanding of the precise physiological roles of pancreatic nerves is incomplete because we lack tools to study these populations in a non-invasive, targeted, temporally controlled manner. Here, we developed and validated a novel magnetogenetic neuromodulatory construct in vitro and in vivo then targeted defined pancreatic nerves to assess their roles in glucose metabolism and hormone secretion. The construct, coding for TRPV1 ion channel fused to a nanobody binding endogenous ferritin (NbTRPV1), was transfected into neural cell lines and primary neurons. Magnetic stimulation significantly increased intracellular calcium in cells expressing NbTRPV1. For in vivo delivery, the construct was packaged into AAV and infused into the pancreas through the pancreatic duct, using serotypes and promoters to target β cells or pancreatic nerves. Ex vivo calcium imaging confirmed magnet activation of pancreas-projecting neurons expressing NbTRPV1. In vivo, targeting NbTRPV1 to β cells improved glucose tolerance in male and female mice, validating the efficacy of this construct to regulate cell activity in the pancreas of freely moving mice. Magnet treatment of ChAT-CRE mice expressing NbTRPV1 in parasympathetic pancreatic neurons significantly improved glucose tolerance and increased insulin secretion. Conversely, targeting vagal sensory nerves from the pancreas in Advillin-iCRE mice impaired glucose tolerance with no significant effect on hormone secretion. These data demonstrate for the first time a method for remote, temporally controlled, targeted modulation of pancreatic islets and nerves to regulate blood glucose. This approach can be used to study the contribution of pancreatic nerves in a wide range of diseases, from diabetes to pancreatic cancer. Disclosure R. Li: None. M. Jimenez gonzalez: None. L. E. Pomeranz: None. G. J. Schwartz: None. S. Stanley: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-17-ACE-31 to S.S.); National Institutes of Health (R01NS097184, OT2OD024912)

1764-P: Specific Viral Targeting of Peripheral Pancreatic Innervation

The brain and peripheral organs communicate through the peripheral nervous system (PNS) comprised of autonomic (sympathetic and parasympathetic) and sensory nerves. Pancreas is densely innervated by the PNS and their cell bodies reside in several ganglia, including nodose (NG), coeliac (CG) dorsal root ganglia (DRG) and intrapancreatic ganglia. Pancreatic innervation is altered in diabetes. Until now, we have lacked the ability to identify and specifically target pancreatic nerves and modulate their function to determine the physiological roles of pancreatic nerves. Here, we delivered retrograde tracers into the pancreas and quantified pancreas-projecting neurons in NG/DRG/CG with 3D visualization and tissue clearing. Next, we assessed the efficiency of different adeno-associated virus (AAV) serotypes, delivery routes and promoters to target construct expression in defined pancreatic nerves. Specific AAV serotypes selectively target pancreas-projecting efferent CG neurons or afferent NG neurons. We utilized Cre-dependent AAV to deliver activating chemogenetic constructs into pancreatic parasympathetic nerves. Targeted activation of the pancreatic parasympathetic neurons improved glucose tolerance and increased insulin secretion. Our studies demonstrate that specific AAV serotypes can target defined pancreatic innervation. Targeted modulation of pancreatic parasympathetic nerves can clarify crucial roles of peripheral nerves in pancreatic function and pathophysiology. Disclosure M. Jimenez Gonzalez: None. R. Li: None. L.E. Pomeranz: None. G.J. Schwartz: None. S. Stanley: None. Funding American Diabetes Association (1-17-ACE-31 to S.S.); National Institutes of Health (1R01NS097184, OT2OD024912); Alexander and Alexandrine Sinsheimer Scholar Award

2197-P: SEL1L-HRD1 Endoplasmic Reticulum-Associated Degradation Is Required for Maintaining Glucagon Production in Pancreatic Alpha Cells

Endoplasmic reticulum-associated degradation (ERAD) is a key component of cellular protein quality control, an essential process for pancreatic islet endocrine cells, which synthesize peptide prohormones in high quantities. Two highly conserved ER membrane proteins comprise the core ERAD machinery: HRD1, a ubiquitin ligase that retrotranslocates and ubiquitinates misfolded proteins for proteasomal degradation, and SEL1L, an adaptor protein that identifies ERAD target proteins and is required for HRD1 stability and function. Pancreatic alpha cells, intestinal L-cells, and certain neurons synthesize and differentially cleave proglucagon into tissue-specific hormones, including glucagon and glucagon-like peptide 1 (GLP-1). To investigate the role of ERAD in these cells, we targeted SEL1L deletion with the Cre-lox system, using mice expressing Cre recombinase under the proglucagon promoter. We found that Sel1LGcg mice showed normal growth and glucose homeostasis in vivo. Oral glucose tolerance was equally impaired by administration of the GLP-1 receptor antagonist exendin 9-39 in both Sel1LGcg and control mice, suggesting intact GLP-1 signaling in Sel1LGcg mice. Glucagon secretion in vivo was not significantly different following a 24 hour fast or insulin-induced hypoglycemia. Interestingly, pancreatic glucagon content was reduced by half in Sel1LGcg mice, despite unchanged pancreatic weight. By immunofluorescence, pancreatic islet morphology in Sel1LGcg mice was normal, though α-cells demonstrated increased expression of the ER chaperone BiP and ERAD-associated proteins OS9 and XTP3-B. By transmission electron microscopy, a large subset of α-cells showed significant ER dilation and fewer, smaller glucagon granules. Our data demonstrate that SEL1L-HRD1 ERAD is required for maintaining normal glucagon expression and ER morphology in pancreatic α-cells but does not entirely disrupt proglucagon signaling in vivo. Disclosure R.B. Reinert: None. N. Shrestha: None. Y. Ji: None. L. Qi: None. Funding National Institutes of Health (5T32DK007245)

Sensory Innervation of the Pancreatic Islet

Sensory innervation of viscera is an important component for proper maintenance of body homeostasis. Visceral stimuli are detected by free nerve endings of the vagus nerve and are transmitted to the hindbrain via sensory neurons. However, the signals that activate vagal sensory neurons in the periphery are not known. The objective of this study is to identify and characterize the population of vagal sensory neurons that innervates the pancreas. Using immunohistochemistry for sensory neuronal marker substance P, we show that sensory innervation targets islet periphery. Upon islet damage (in NOD model or following streptozotocin (STZ) treatment), density of islet sensory innervation increases. Expression of the immediate early gene c-Fos, shows that hindbrain neurons in the nucleus of the solitary tract (NTS) respond to severe islet inflammation induced by STZ. We assessed response profile of pancreatic sensory neurons using in-vivo calcium imaging of mouse vagal sensory ganglion (nodose). This method allows us to monitor in real time the response profile of sensory neurons and identify pancreas-specific subpopulations by chemical stimulation of the exteriorized pancreas. We have identified sensory neurons that respond to pancreatic application of serotonin (5HT) and cholecystokinin (CCK). The observed response profile suggests that these neuronal populations are tuned to sense differentially endocrine and exocrine pancreas. While endocrine sensory neurons are sensing local 5HT that is co-released with insulin, exocrine neurons sense CCK a hormone that stimulates release of digestive enzymes. Altogether our data suggest that pancreatic sensory neurons could be important regulators of pancreatic physiology. By taking a snapshot of endocrine and exocrine pancreatic microenvironments and sending this information to the brain, sensory neurons might trigger feedback loops and help the organ to be tuned to physiological needs. Disclosure M. Makhmutova: None. J. Weitz: None. R. Rodriguez Diaz: None. J. Almaca: None. E. Bernal-Mizrachi: None. A. Caicedo: None.

TRPV1 gene polymorphisms in patients with diabetes compared with healthy individuals

Type 1 diabetes is an autoimmune disease that is caused by pancreatic islet inflammation. Various studies on non-obese diabetic mice have shown associations between transient receptor vanilloid type-1 (TRPV1) gene polymorphism in pancreatic sensory neurons and the incidence of insulitis and insulin resistance. Inhibition of these neurons improves insulitis and prevents the onset of autoimmune diabetes. The aim of this study was to investigate the genetic variation of TRPV1 gene in type 1 diabetic patients and healthy controls. The genotypes of three single nucleotide polymorphisms (SNPs) of the TRPV1 gene (rs222749, rs222447, and rs224534) were investigated in 50 patients with type 1 diabetes and 50 healthy controls using PCR-restriction fragment length polymorphism analysis and DNA sequencing. Then, the frequencies of TRPV1 gene variants were compared between patient and control groups. The results showed that there was no significant difference in allele frequency of the three TRPV1 gene SNPs between patients with type 1 diabetes and healthy controls. Likewise, distribution of genotypes did not significantly differ between patients and controls. In this study, no significant association was found between TRPV1 gene polymorphisms and the presence of type 1 diabetes.

Abstract B69: Tuft cell signaling can induce pancreatic inflammation and promote pancreatic ductal adenocarcinoma

Abstract The tuft cell is a chemosensory cell type that can interpret signals from luminal surfaces throughout the body in order to activate downstream effectors. One clear effector pathway of the tuft cell promotes a type-two immune response in the intestine after parasitic infection, suggesting that this cell population has the ability to communicate with the immune system. Furthermore, tuft cells have also been shown to activate sensory nerve fibers to lead to the production of a neuroinflammatory signal in the nasal epithelium. Therefore, tuft cells can promote effector inflammatory responses through direct immune cell signaling and through sensory nerve fibers. Pancreatic ductal adenocarcinoma (PDA) is characterized by a robust stromal reaction, a large component of which is composed of an immune response. It has been previously studied that this inflammatory response can function to promote both the initiation and progression of PDA. One mechanism by which inflammatory cells can be recruited to the pancreas is through neuroinflammatory pathways. Work with collaborators has shown that pancreatic sensory neurons are required for the progression of PDA, as ablation of this cell type significantly prolongs survival. Interestingly, tuft cells in the metaplastic pancreas have been shown to appear during transdifferentiation and progression to PDA. These tuft cells also possess key signaling elements that have the potential to mediate the response to extracellular signals, such as α-gustducin and TRPM5. Metaplastic tuft cells also produce the proteins necessary to make or release prostaglandins, acetylcholine and ATP, all of which can stimulate either a direct immunomodulatory signal or activate sensory nerve fibers. This leads us to hypothesize that tuft cells are a key mediator of signals to the stromal immune compartment and to sensory neurons in order to promote a robust immune response. In order to answer this question we are using chemosensory knockout animals to ablate critical signaling components of the chemosensory cascade to study the progression of PDA. Citation Format: Megan T. Hoffman, Shan Gao, Howard Crawford.{Authors}. Tuft cell signaling can induce pancreatic inflammation and promote pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B69.

Stem Cell Therapy: Challenges Ahead

Stem cells have generated great interest for their potential therapeutic use because of their capacity to self-renew indefinitely and to generate all cell lineages (pluripotency). Many diseases such as neurodegenerative disorders or diabetes are caused by loss of functionality or deficiency of a particular cell type. Stem cells differentiated into a specific cell type such as pancreatic β-cells or neurons, for example, thus hold great promise for regenerative medicine. However, many challenges have to be overcome before stem cell therapy can become a viable clinical approach.

Neuroplastic Changes Occur Early in the Development of Pancreatic Ductal Adenocarcinoma

Perineural tumor invasion of intrapancreatic nerves, neurogenic inflammation, and tumor metastases along extrapancreatic nerves are key features of pancreatic malignancies. Animal studies show that chronic pancreatic inflammation produces hypertrophy and hypersensitivity of pancreatic afferents and that sensory fibers may themselves drive inflammation via neurogenic mechanisms. Although genetic mutations are required for cancer development, inflammation has been shown to be a precipitating event that can accelerate the transition of precancerous lesions to cancer. These observations led us to hypothesize that inflammation that accompanies early phases of pancreatic ductal adenocarcinoma (PDAC) would produce pathologic changes in pancreatic neurons and innervation. Using a lineage-labeled genetically engineered mouse model of PDAC, we found that pancreatic neurotrophic factor mRNA expression and sensory innervation increased dramatically when only pancreatic intraepithelial neoplasia were apparent. These changes correlated with pain-related decreases in exploratory behavior and increased expression of nociceptive genes in sensory ganglia. At later stages, cells of pancreatic origin could be found in the celiac and sensory ganglia along with metastases to the spinal cord. These results demonstrate that the nervous system participates in all stages of PDAC, including those that precede the appearance of cancer.

Distribution pattern of CART-containing neurons and cells in the human pancreas

Cocaine- and amphetamine-regulated transcript (CART) has been shown to play a critical role in appetite suppression, cell survival, thermoregulation, glucose sensing, stimulation of hormone secretion, as well as for the regulatory function of the islets of Langerhans. Although the principal site of CART synthesis has already been reported, our knowledge of the subject is mainly based on and limited to research conducted on animals owing to difficulties in obtaining human samples. Therefore, the primary goal of the reported study was an attempt to identify and localize CART in healthy human pancreas. Nineteen deceased subjects (donors of organs) with normal pancreas and alimentary tract were used in the study. After determination of brain death and confirmation of death by the relevant doctors committee, pancreas samples, about 1cm long, were collected from each corpse (the same part of the pancreas) after the organs were harvested for transplantation. Paraffin sections were made and stained with hematoxylin and eosin and then subjected to CART immunohistochemistry. In the normal pancreas of human adults, CART is mainly present in both nerve fibers and in nerve cell bodies in pancreatic ganglia. In addition to pancreatic neurons, immunoreactivity to CART was also seen in islet endocrine cells. This is the first report on the presence of CART-IR structures in the normal human pancreas. CART should be now added to the numerous regulatory peptides that are involved in the complex regulation of pancreatic endocrine and exocrine processes.

Synergistic Role of TRPV1 and TRPA1 in Pancreatic Pain and Inflammation

The transient receptor potential (TRP) channels TRPV1 and TRPA1 have each been associated with regulation of efferent properties of primary afferent neurons that initiate neurogenic inflammation and are required for the development of inflammatory hyperalgesia. To evaluate the role of these channels in producing pain during pancreatic inflammation, we studied pancreatic nodose ganglion (NG) and dorsal root ganglion (DRG) sensory neurons (identified by content of retrograde tracer) and behavioral outcomes in a mouse model of acute pancreatitis. Pancreatic inflammation was induced by 8 hourly injections of cerulein (50 μg/kg). The extent of inflammation, pancreatic neuron TRP channel expression and function and excitability, and pain-related behaviors were evaluated over the course of the following week. Histology and myeloperoxidase activity confirmed pancreatic inflammation that was associated with increased excitability and messenger RNA expression of the TRP channels in NG and DRG pancreatic neurons. Calcium imaging of pancreatic NG and DRG neurons from mice given cerulein revealed increased responses to TRP agonists. TRPV1 and TRPA1 antagonists attenuated cerulein-induced pain behaviors and pancreatic inflammation; they had a synergistic effect. Pancreatic inflammation significantly increased the expression and functional properties of TRPV1 and TRPA1, as well as the excitability of pancreatic sensory neurons in vagal and spinal pathways. TRP channel antagonists acted synergistically to reverse pancreatic inflammation and associated pain behaviors; reagents that target interactions between these channels might be developed to reduce pain in patients with acute pancreatitis.

911 Characterization of TRPV1 and TRPA1 in Pancreatic Nodose and Dorsal Root Ganglion Neurons

911 Characterization of TRPV1 and TRPA1 in Pancreatic Nodose and Dorsal Root Ganglion Neurons

Culture of Porcine Fetal Pancreatic Neurons

Pancreatic neurons have not been cultured commonly. Cultured neurons can be continuously observed, and their external environment is easy to be controlled. We report here a simple method for separating and cultivating neuronal cells from pancreas. Pancreata of fetal swine were digested with collagenase. Clusters were collected with a sieve and digested with trypsin. Digested clusters were collected and cultured in Dulbecco modified Eagle medium containing serum and basic fibroblast growth factor. Cultured cells were investigated morphologically. Cultured cells formed spiderweblike colonies. These cells were distinguished into Schwann cells and 2 types of neurons. The neurons were positive on immunocytochemical staining with antigrowth-associated protein-43 and cytochemical staining for cholinesterase. One type of neuron was located in the central cluster and had very long processes extending radially. The other type of neuron was sparsely scattered, had long processes, and was connected to other neurons. The neurotransmitter of these neurons was concluded to be acetylcholine. Using our method, neuronal cells were readily cultured from pancreatic tissue. These cells will be useful in elucidating the physiology and pharmacology of pancreatic neurons.

Intron 1 sequences are required for pancreatic expression of the human proglucagon gene

The mammalian proglucagon gene is expressed in pancreatic islet A-cells, intestinal L-cells, and select neurons of the brain, where posttranslational processing results in the liberation of a unique profile of peptides. Despite the importance of proglucagon-derived peptides in human biology, little is known about the regulation of the human gene, as the rat gene has been the preferred model for understanding the regulation of proglucagon gene expression. Previously, we have shown that although the immediate promoter region of the rat proglucagon gene is sufficient for expression in pancreatic islet cells, the homologous human proglucagon promoter sequences are not sufficient. We have now used a comparative genomic approach to identify noncoding sequences near the human proglucagon gene that are conserved among mammals, and thus potentially are regulatory sequences. Our alignments identified three evolutionarily conserved noncoding regions (ECR), one is the immediate promoter region (ECR1), the second is about 5 kb 5' to the mRNA start site (ECR2), and the third is near the 3' end of the first intron (ECR3). Our in vitro transient transfection assays with reporter gene constructs that include the human ECR3 support expression in rodent islet cell lines. Complementary studies with transgenic mice possessing a reporter gene regulated by a human proglucagon gene promoter-intron 1 (including ECR3) sequences express the reporter gene in the pancreas, as well as the intestine and selected neurons. These studies suggest that conserved sequences within intron 1 of the human proglucagon gene are important for expression in the pancreas.

Alpha-adrenergic modulation of synaptic transmission in rabbit pancreatic ganglia

Pancreatic ganglia contain noradrenergic nerve terminals whose role in ganglionic transmission is unknown. Intracellular recordings from rabbit pancreatic neurons were used to study the effects of α-adrenergic agonists and antagonists on ganglionic transmission and to determine if endogenously released norepinephrine contributed to synaptic depression. Significant regional differences in α adrenergic effects were observed. In neurons from ganglia of the head/neck region norepinephrine or selective α 2 agonists presynaptically inhibited ganglionic transmission and this effect was antagonized by the α 2 antagonist yohimbine. In the majority of cells membrane hyperpolarization accompanied presynaptic inhibition during superfusion of α 2 agonists. Repetitive nerve stimulation evoked a presynaptic post-train depression (PTD) of ganglionic transmission in all neurons tested. A combination of nisoxetine (selective inhibitor of the norepinephrine transporter) and tyramine (releaser of endogenous catecholamines) increased PTD. Pretreatment with clonidine inhibited synaptic transmission and abolished PTD while yohimbine did not affect it. Pretreatment with guanethidine (≥ 3.5 h) also failed reduce PTD while neurons unresponsive to α 2 adrenoceptor agonists routinely exhibited PTD, implying the presence of other inhibitory neurotransmitters sharing a common presynaptic mechanism with α 2 agonists. In the majority of neurons from ganglia of the body region superfusion of norepinephrine or the selective α 1 agonist phenylephrine evoked membrane depolarization and facilitated ganglionic transmission. These effects were antagonized by the α 1 antagonist prazosin. The remaining neurons exhibited either α 2-mediated synaptic inhibition or no-response. In conclusion, inhibitory α 2 and excitatory α 1 adrenoceptors exist in pancreatic ganglia and predominate in the head/neck and body, respectively. Norepinephrine, released during repetitive nerve stimulation, may contribute to synaptic depression in the head/neck region and appeared to share a common mechanism with other, unidentified neurotransmitters mediating synaptic depression in both regions. These differences indicate a functional heterogeneity of pancreatic sympathetic innervation that may reflect the reported regional differences in exocrine and endocrine cells.

Electrophysiological effects of GABA on cat pancreatic neurons.

In mammalian peripheral sympathetic ganglia GABA acts presynaptically to facilitate cholinergic transmission and postsynaptically to depolarize membrane potential. The GABA effect on parasympathetic pancreatic ganglia is unknown. We aimed to determine the effect of locally applied GABA on cat pancreatic ganglion neurons. Ganglia with attached nerve trunks were isolated from cat pancreata. Conventional intracellular recording techniques were used to record electrical responses from ganglion neurons. GABA pressure microejection depolarized membrane potential with an amplitude of 17.4 +/- 0.7 mV. Electrically evoked fast excitatory postsynaptic potentials were significantly inhibited (5.4 +/- 0.3 to 2.9 +/- 0.2 mV) after GABA application. GABA-evoked depolarizations were mimicked by the GABA(A) receptor agonist muscimol and abolished by the GABA(A) receptor antagonist bicuculline and the Cl(-) channel blocker picrotoxin. GABA was taken up and stored in ganglia during preincubation with 1 mM GABA; beta-aminobutyric acid application after GABA loading significantly (P < 0.05) increased depolarizing response to GABA (15.6 +/- 1.0 vs. 7.8 +/- 0.8 mV without GABA preincubation). Immunolabeling with antibodies to GABA, glial cell fibrillary acidic protein, protein gene product 9.5, and glutamic acid decarboxylase (GAD) immunoreactivity showed that GABA was present in glial cells, but not in neurons, and that glial cells did not contain GAD, whereas islet cells did. The data suggest that endogenous GABA released from ganglionic glial cells acts on pancreatic ganglion neurons through GABA(A) receptors.

Pituitary adenylate cyclase activating peptide (PACAP) in the enteropancreatic innervation.

Pancreatic ganglia are innervated by neurons in the gut and are formed by precursor cells that migrate into the pancreas from the bowel. The innervation of the pancreas, therefore, may be considered an extension of the enteric nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in a subset of enteric neurons. We investigated the presence of PACAP in the enteropancreatic innervation in guinea pigs, and the response of pancreatic neurons to PACAP-related peptides. PACAP immunoreactivity was found in nerve fibers in both enteric and pancreatic ganglia and in nerve bundles that travelled between the duodenum and pancreas. PACAP-immunoreactive nerve fibers were densely distributed in the pancreatic ganglia, where they surrounded a subset of cholinergic cell bodies. Pancreatic ganglia did not contain PACAP-immunoreactive cell bodies; however, neuronal perikarya with PACAP immunoreactivity were found in the myenteric plexus of the duodenum. These cells co-stored vasoactive intestinal peptide (VIP). PACAP depolarized pancreatic neurons. Pancreatic neurons were also depolarized by VIP; however, PACAP was more efficacious at depolarizing pancreatic cells than VIP. These findings are consistent with the view that the PACAP effects were mediated through PACAP-selective (PAC1) receptors. PACAP-responsive neurons displayed PAC1 receptor immunoreactivity, which was also found in islet cells and enteric neurons. These results provide support for the hypothesis that PACAP modulates reflex activity between the gut and pancreas. The excitatory effect of PACAP would be expected to potentiate pancreatic secretion.

Vagal-enteric interface: vagal activation-induced expression of c-Fos and p-CREB in neurons of the upper gastrointestinal tract and pancreas.

Many gastrointestinal and pancreatic functions are under strong modulatory control by the brain via the vagus nerve. To start identifying location and neurochemical phenotype of the enteric neurons receiving functional vagal efferent input, we activated vagal preganglionic neurons either by electrical or chemical stimulation and examined the expression of phosphorylated CREB (c-AMP response element binding protein) and the immediate early gene c-Fos. There was no spontaneous expression of both markers in the pancreas and considerable spontaneous expression of p-CREB but not Fos in the upper GI-tract. Unilateral electrical vagal stimulation-induced p-CREB was found in 40% of neurons in the head of the pancreas. Fos expression was found in 70-90% of neurons in the esophagus and stomach, in 20-30% of myenteric plexus neurons and 5-15% in submucosal neurons of the proximal duodenum. Double-labeling experiments showed that a majority of pancreatic neurons and about 25-35% of neurons in the stomach and duodenum contain NADPH-diaphorase and that many of these receive functional vagal input. Other neurons that can be vagally activated contain gastrin-releasing peptide or calretinin. Chemical stimulation of the dorsal surface of the caudal brainstem with the stable TRH analog RX77368 resulted in selective activation of vagal efferents with expression of Fos in a small number of gastric myenteric plexus neurons. The results demonstrate the suitability of this method to investigate magnitude and local distribution of vagal input to the enteric nervous system as well as specificity of its neurochemically coded pathways. They represent the first step in the identification of function-specific units of parasympathetic vagal outflow.

Pituitary Adenylate Cyclase-Activating Polypeptide Prevents Cytokine-Induced Cytotoxicity via Inhibition of Inducible Nitric Oxide Synthase Expression in βTC Cells

Type 1 diabetes mellitus is an autoimmune disease resulting from apoptotic destruction of pancreatic β-cells. The activation of inducible nitric oxide synthase (iNOS) by inflammatory cytokines is considered a mediator of destruction in β-cells. Recent findings showed that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), whose distribution was identified in pancreatic neurons, inhibited nitric oxide (NO) production in cytokine-activated macrophages. In the present study, we investigated the cytoprotective effect of PACAP in the cytokine-exposed mice β-cell line, βTC cells. 1 × 10−8 M PACAP inhibited the reduction of cell viability, NO production, expression of iNOS mRNA, and iNOS promoter activity caused by the combination of three proinflammatory cytokines. Selective iNOS inhibitor also showed the cytoprotective effect in βTC cells. These data suggested that PACAP has a cytoprotective effect in cytokine-treated β-cells through inhibition of iNOS transcription.