Phylogenetic study of serotonin-immunoreactive structures in the pancreas of various vertebrates

Summary The distribution pattern of serotonin (5HT) in the pancreas was studied immunohistochemically by using a 5HT monoclonal antibody in various vertebrates including the eel, bullfrog, South African clawed toad, turtle, chicken, mouse, rat, guinea-pig, cat, dog and human. In all species examined, except the bullfrog, 5HT-like immunoreactivity was observed in nerve fibers, in endocrine cells, or in both. Positive nerve fibers were found in the eel, turtle, mouse, rat and guinea-pig. These fibers ran mainly along the blood vessels and partly through the gap between the exocrine glands. In the eel and guinea-pig, positive fibers invaded the pancreatic islet. Occasionally, these positive fibers were found adjacent to the surface of both exocrine and endocrine cells, suggesting a regulatory role of 5HT in pancreatic function. 5HT-positive endocrine cells were observed in the pancreas of all species except for the bullfrog and rat. In the eel and in mammals such as the mouse, guinea-pig, cat, dog and human, 5HT-positive cells were mainly observed within the pancreatic islet. In the South African clawed toad, turtle and chicken, the positive cells were mainly in the exocrine region. The present study indicates that the distribution patterns of 5HT in the pancreas varies considerably among different species.     

Phylogenic study of calcitonin gene-related peptide-immunoreactive structures in the pancreas

 The immunohistochemical localization of calcitonin gene-related peptide was examined, at both light and electron microscopic levels, in the pancreas of various vertebrates, including the eel, bullfrog, turtle, chicken, mouse, rat, guinea pig, dog, monkey, and human. Immunoreactive staining was observed in nerve fibers in every animal species examined, but positive endocrine cells were limited to the rat, monkey, and human. The density of the positive endocrine cells varied considerably among the three species (monkey > rat > human). Positive nerve fibers were distributed throughout the parenchyma, being particularly rich around pancreatic ducts, and near large or small blood vessels. In four species (eel, mouse, rat, and dog), positive nerve fibers formed a dense network in the islet region. There were positive varicose nerve fibers around exocrine cells. These fibers, varying in density in different species (relatively high in the eel, bullfrog, and rat), were sometimes adjacent to acinar cells. At the electron microscopic level, positive nerve terminals were often demonstrated in close apposition to the outer membrane of acinar cells. The eel pancreas revealed an exceptional pattern of staining in neuronal cell bodies that were scattered in the interlobular connective tissue. Despite these anatomical differences, the omnipresence of this peptide suggests its essential role(s) in the pancreas. Accepted: 12 June 1997     

Galanin is co-localized with noradrenaline and neuropeptide Y in dog pancreas and celiac ganglion

Summary To visualize the localization and potential colocalization of noradrenaline and the putative pancreatic sympathetic neurotransmitters, galanin and neuropeptide Y (NPY), immunofluorescent staining for galanin, NPY and tyrosine hydroxylase (TH) was performed on sections of canine pancreas and celiac ganglion. In the pancreas, galanin-immuno-fluorescent nerve fibers were confirmed as densely and preferentially innervating the islets, whereas numerous NPY-positive nerve fibers were found in the exocrine parenchyma, the surrounding of the blood vessels and within the islets. Double-staining for the peptides and TH indicated that most galaninpositive nerve fibers were adrenergic, most NPY-positive nerve fibers were adrenergic, and many islet nerves contained both galanin and NPY, although some galaninpositive nerve fibers appeared to lack NPY. In the celiac ganglion, virtually all cell bodies were positive for both galanin and TH; a large subpopulation of these cells were also positive for NPY. Radioimmunoassay (RIA) of galanin in extracts of dog celiac ganglion revealed a very high content (256±33 pmol/g wet weight) of galanin-like immunoreactivity (GLIR), consistent with the dense staining observed. This GLIR behaved in a similar manner to synthetic porcine galanin in the RIA. In addition, the majority of the GLIR in ganglion extracts coeluted with the synthetic peptide upon gel filtration, although a minor peak of a larger apparent molecular weight was also observed, observations consistent with the presence of a precursor peptide. These findings suggest that galanin is a sympathetic post-ganglionic neurotransmitter in the canine endocrine pancreas and that NPY might serve a similar function.     

The presence and actions of NPY in the canine endocrine pancreas

Immunofluorescent staining for neuropeptide Y (NPY) in canine pancreatic tissue was performed together with an evaluation of the effects of synthetic NPY on the release of insulin (IRI), glucagon (IRG) and somatostatin (SLI) from the duodenal lobe of the canine pancreas in situ. NPY-like immunoreactivity was localized in perivascular nerve fibers throughout the acinar tissue. NPY-immunoreactive fibers were also demonstrated in the islets, usually surrounding blood vessels but also occasionally in fibers associated with endocrine cells, primarily at the periphery of islets. In addition, the ganglia dispersed in the pancreatic parenchyma were densely innervated by NPY-immunoreactive fibers, and these ganglia regularly contained cell bodies staining for NPY. Direct infusion of NPY into the pancreatic artery (p.a.) produced a dose-dependent decrease of pancreatic SLI output and of pancreatic venous blood flow. Low-dose p.a. infusion of NPY (50 pmol/min) had no effect on basal IRI or IRG output or on the islet response to glucose (5-g bolus, i.v.). High-dose p.a. infusion of NPY (500 pmol/min) transiently stimulated IRI output and modestly increased IRG output. However, the comparatively sparse innervation of canine islets with NPY-like immunoreactive fibers and the relatively minor effects of large doses of synthetic NPY on pancreatic hormone release lead us to conclude that this peptide is not an important neuromodulator of islet function in the dog.     

Tissue distribution and innervation pattern of peptide immunoreactivities in the rat pancreas.

The distribution of calcitonin gene-related peptide (CGRP), substance P/tachykinin (SP/TK), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and gastrin-releasing peptide (GRP) immunreactivities (IR) in the rat pancreas was investigated using radioimmunoassay and immunohistochemistry. CGRP, NPY and VIP tissue contents are much higher than GRP and SP/TK concentrations. Peptide-containing nerves are distributed to both the exocrine and endocrine pancreas. However, differences exist in terms of density and targets of innervation for each peptidergic system. In the acini and through the stroma, fibers IR for CGRP, NPY and VIP are greater than GRP- and SP/TK-containing processes. The vasculature is supplied by a prominent NPY, CGRP and, to a lesser extent, SP/TK innervation. VIP-IR is found occasionally, and GRP-IR is never detected, in fibers associated with blood vessels. Around ducts, CGRP- and NPY-positive neurites are greater than SP/TK- greater than or equal to VIP-IR fibers, whereas GRP-containing nerves are not visualized. In the islets, the density of peptidergic nerves is: VIP-, GRP- greater than or equal to CGRP-IR greater than NPY or SP/TK. In intrapancreatic ganglia. VIP- and, to a lesser extent, NPY-IRs are found in numerous neuronal cell bodies and in nerve fibers; GRP-IR is present in numerous nerve processes and in few cell bodies; CGRP- and SP/TK-IRs are detected only in fibers wrapping around unlabeled ganglion cells. The majority of CGRP-IR fibers contain SP/TK-IR. The existence of differential patterns of peptidergic nerves suggests that peptides exert their effects on pancreatic functions via different pathways.     

Distribution of immunoreactive neuropeptides in the pancreas of the bullfrog,Rana catesbeiana,demonstrated by immunofluorescence

Indirect double immunofluorescence labelling for eight neuropeptides in the pancreas of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of certain neuropeptides in the exocrine and endocrine pancreas. Immunoreactivity of substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), FMRFamide (FMRF), and galanin (GAL) was localized in nerve fibers distributed between the acini and around the duct system and vasculature of the exocrine pancreas. In these regions, CGRP-immunoreactive fibers were more numerous than those containing the other five peptides. Almost all SP fibers showed coexistence of SP with CGRP, and about one third of fibers also showed coexistence of SP with VIP, NPY, FMRF, and GAL. In the endocrine pancreas, SP, CGRP, VIP, and GAL were recognized in the nerve fibers around and within the islets of Langerhans, and VIP and GAL fibers were more numerous than SP and CGRP fibers. All CGRP fibers, and about half of the VIP and GAL fibers were immunoreactive for SP. NPY- and FMRF-immunoreactive cells were found at the periphery of the islets. These findings suggest that the exocrine and endocrine pancreatic functions of the bullfrog are under the control of peptidergic innervation.     

Localization of calcitonin gene-related peptide and islet amyloid polypeptide in the rat and mouse pancreas

Summary It was previously demonstrated that the two chemically related peptides calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP) both occur in the pancreas. We have now examined the cellular localization of CGRP and IAPP in the rat and the mouse pancreas. We found, in both the rat and the mouse pancreas, CGRP-immunoreactive nerve fibers throughout the parenchyma, including the islets, with particular association with blood vessels. CGRP-immunoreactive nerve fibers were regularly seen within the islets. In contrast, no IAPP-immunoreactive nerve fibers were demonstrated in this location. Furthermore, in rat islets, CGRP immunoreactivity was demonstrated in peripherally located cells, constituting a major subpopulation of the somatostatin cells. Such cells were lacking in the mouse islets. IAPP-like immunoreactivity was demonstrated in rat and mouse islet insulin cells, and, in the rat, also in a few non-insulin cells in the islet periphery. These cells seemed to be identical with somatostatin/CGRP-immunoreactive elements. In summary, the study shows (1) that CGRP, but not IAPP, is a pancreati neuropeptide both in the mouse and the rat; (2) that a subpopulation of rat somatostatin cells contain CGRP; (3) that mouse islet endocrine cells do not contain CGRP; (4) that insulin cells in both the rat and the mouse contain IAPP; and (5) that in the rat, a non-insulin cell population apparently composed of somatostatin cells stores immunoreactive IAPP. We conclude that CGRP is a pancreatic neuropeptide and IAPP is an islet endocrine peptide in both the rat and the mouse, whereas CGRP is an islet endocrine peptide in the rat.     

Localization of four polypeptide hormones in the saurian pancreas.

Glucagon, insulin, somatostatin, and pancreatic polypeptide have been localized in the anolian pancreas using peroxidase-antiperoxidase immunocytochemistry. The most abundant endocrine cell type contains glucagon. Insulin-containing cells are the next most numerous. Somatostatin-immunoreactive cells tend to be localized at the periphery of the islet cords. Pancreatic polypeptide-containing cells are a minor endocrine component scattered throughout the exocrine pancreas and occasionally within the islet areas. No staining was observed after application of antigastrin serum.     

Differential expression and localization of neuropeptide Y peptide in pancreatic islet of diabetic and high fat fed rats

Neuropeptide Y (NPY) inhibits insulin secretion. Increased numbers of pancreatic islet cells expressing NPY have been observed in type 1 diabetic rats. To understand the functional significance of NPY expression in islet cells, we investigated the effects of high fat feeding and diabetic conditions on the expression and location of NPY expressing cells in normal and diabetic rats. Twenty rats were maintained on either normal chow (ND) or a high fat dietary regimen (HFD) for 4 weeks. In half of each group, type 1 or type 2 diabetes (groups T1DM and T2DM, respectively) was induced by injection of streptozotocin. At 8 weeks rats were euthanized and the pancreases were processed for immunofluorescence labeling (NPY/insulin, NPY/glucagon, NPY/somatostatin, and NPY/pancreatic polypeptide). Compared with the ND group, HFD rats had significantly fewer alpha cells, but beta cells were similar, while T1DM and T2DM rats showed significant increases in the proportions of alpha, delta, and PP cells. Robust increases in NPY-positive islet cells were found in the HFD, T1DM, and T2DM rats compared with ND controls. In ND rats, 99.7% of the NPY-positive cells were PP cells. However, high fat feeding and diabetes resulted in significant increases in NPY-positive delta cells, with concomitant decreases in NPY-positive PP cells. In summary, high-fat feeding and diabetes resulted in changes in the hormonal composition of pancreatic islet and increased number of NPY-expressing islet cells. Under diabetic conditions NPY expression switched from predominantly a characteristic of PP cells to predominantly that of delta cells. This may be a factor in reduced pancreatic hormone secretion during diabetes.     

Distribution of neuropeptide Y and vasoactive intestinal polypeptide immunoreactive nerves in normal and transplanted pancreatic tissue

Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactive nerves were demonstrated in 21-day-old embryonic pancreatic tissue fragments transplanted into the anterior eye chamber of rats for 22, 45 and 109 days and in 60-day-old normal adult pancreas using immunohistochemical technique. In normal adult tissue, NPY-positive neurons lie close to the basal and lateral walls of the acinar cells. NPY-containing nerve fiber plexuses were found around blood vessels. VIP-immunopositive nerves were also discernible in the outer parts of the islets of Langerhans and on pancreatic ducts. In the transplants, it is not only the neural elements that survived but also the pancreatic ducts and the endocrine cells. VIP- and NPY-positive neurons were found in the stroma of the surviving pancreatic tissue. The distribution of these neural elements is similar to that of normal tissue in the surviving pancreatic ducts but different with regards to the acinar tissue. This study confirms that intrinsic nerves can survive and synthesize polypeptides even after 109 days of transplantation into the anterior eye chamber.     

Species differences in the immunoreactive patterns of calcitonin gene-related peptide in the pancreas

Summary In the pancreas, calcitonin gene-related peptide (CGRP) immunoreactivity has been described in nerve fibers and in distinct types of islet cells. This unique, apparently species-specific cell-type expression prompted the present investigation to clarify further the pattern of CGRP immunoreactivity in different mammalian species (i.e., different strains of rats, mice, guinea pigs, rabbits, cats, dogs, pigs, and humans) commonly used for functional and anatomical studies of the pancreas by means of immunohistochemistry using three different CGRP antibodies. In each species, CGRP-immunoreactive neurites innervate the exocrine and endocrine compartments, the vasculature, and the intrapancreatic ganglia, where they form dense networks encircling unstained cell bodies. The only exception is the pig pancreas, where the islets appear to be devoid of immunoreactive fibers. The overall density of immunoreactive pancreatic axons in different species is as follows: rat, mouse, and rabbit>guinea pigpig and cat> >dog and human. CGRP-immunoreactive endocrine cells appear to be restricted to the rat pancreas, where they form a subpopulation of somatostatin-containing D cells. In contrast, in mouse, guinea pig, cat, dog, and human pancreas, a homogeneous staining of the core of the islets, where insulin-producing B cells are located, was visualized in sections incubated with the rabbit CGRP antiserum at 4°C, but not at 37°C (an incubation temperature that does not affect the islet cell staining in the rat nor the fiber labeling in any species). Furthermore, the staining of islet B cells was not reproductible with all the CGRP antibodies used, all of which comparably stain nerve fibers in each species, and islet D cells in the rat. Immunoreactive islet cells were not visualized in pig and rabbit pancreas. These results are consistent with the hypothesis that the expression of CGRP in nerve fibers is a common feature of mammalian pancreas, whereas its expression in endocrine cells appears to be restricted to the D cells of the rat pancreas.     

Polypeptide YY- and neuropeptide Y-immunoreactive cells and nerves in the endocrine and exocrine pancreas of some vertebrates: An onto- and phylogenetic study

Summary The occurrence of polypeptide YY- and neuropeptide Y-immunoreactive cells and nerves in the pancreas of some species from all the eight main vertebrate groups (cyclostomes, cartilaginous fish, bony fish, amphibia, reptiles, birds, and mammals) was investigated. In addition, an ontogenetic study of these neurohormonal peptides was performed, using the rat pancreas. The distribution of these two peptides was compared with that of the structurally closely related pancreatic polypeptide.Polypeptide YY-immunoreactive cells were found to occur in the endocrine pancreas and neuropeptide Y-immunoreactivity was observed both in neurons and nerve fibres. The polypeptide YY-immunoreactive cells were limited to mammals and reptiles only. Neuropeptide Y-immunoreactive neurons and nerves were observed in reptiles, birds, and mammals only. One reptilian species (out of three) and one mammalian (out of six) failed to show any kind of immunoreactivity for the polypeptide or neuropeptide. Pancreatic polypeptide-immunoreactive cells were found in all the species examined except in the hagfish islet.In rat foetuses, polypeptide YY-immunoreactive cells and neuropeptide Y-immunoreactive nerve elements were first demonstrated at the seventeenth day of gestation, whereas pancreactic peptide-immunoreactive cells did not appear until postnatally, namely in two day-old rats. The polypeptide-containing cells, a new cell type in the endocrine pancreas, are rare. In contrast to the pancreatic peptide cells, they do not seem to have any kind of regional distribution.     

Cholinergic innervation of the pancreas in the sheep

Antibodies raised against vesicular acetylcholine transporter (VAChT) were applied to study the cholinergic innervation pattern of the pancreas of the sheep. To determine whether the cholinergic pancreatic neuronal elements contain tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) or substance P (SP) double immunocytochemistry was used. A moderate number of VAChT-immunoreactive (IR) nerve terminals were distributed between the acini, whereas only single cholinergic nerve fibres innervated the interlobular connective tissue. VAChT-positive nerve fibres supplying the endocrine pancreas were found only occasionally. The pancreatic blood vessels and ducts system were devoid of VAChT-containing nerve endings. All intrapancreatic neurons studied showed immunoreactivity to VAChT, but intrapancreatic ganglia were not innervated with cholinergic nerve fibres. The colocalization of VAChT and TH or VAChT and SP was detected in distinct populations of nerve fibres localized amongst the acini, but not within the islet nor in the connective tissue. Single VAChT-IR nerve terminals co-expressing NPY were distributed around the acini, islets as well as in the connective tissue septa. A moderate number of VAChT-IR/VIP-IR nerve endings were located in the exocrine pancreas, whereas the islets and connective tissue were innervated with VAChT/VIP-containing nerve fibres only occasionally. In the vast majority of VAChT-positive intrapancreatic perikarya the presence of TH was additionally found. A moderate number of VAChT-IR intrapancreatic perikarya co-expressed NPY, SP or VIP. The results of the present study demonstrate species-dependent cholinergic innervation pattern of the pancreas of the sheep. The co-localization of VAChT with the neuropeptides suggests the existence of functional interactions influencing the ovine pancreas (mainly exocrine) activity.     

Evolution of neuropeptide Y and its related peptides

1. The neuropeptide Y (NPY) family of peptides includes also the gut endocrine peptide YY (PYY), tetrapod pancreatic polypeptide (PP), and fish pancreatic peptide-tyrosine (PY). All peptides are 36 amino acids long.2. Sequences from many types of vertebrates show that NPY has remained extremely well conserved throughout vertebrate evolution with 92% identity between mammals and cartilaginous fishes.3. PYY has 97–100% identity between cartilaginous fishes and bony fishes, but is less conserved in amphibians and mammals (83% identity between amphibians and sharks and 75% identity between mammals and sharks).4. NPY and PYY share 70–80% identity in most species.5. Both NPY and PYY were present in the early vertebrate ancestor because both peptides have been found in lampreys.6. The tissue distribution appears to have been largely conserved between phyla, except that PYY has more widespread neuronal expression in lower vertebrates.7. Pancreatic polypeptide has diverged considerably among tetrapods leaving only 50% identity between mammals, birdsJreptiles and frogs.8. Several lines of evidence suggest that the PP gene arose by duplication of the PYY gene, probably in the early evolution of the tetrapods.9. The pancreatic peptide PY found in anglerfish and daddy sculpin may have resulted from an independent duplication of the PYY gene.10. The relationships of the recently described mollusc and worm peptides NPF and PYF with the NPY family still appear unclear.     

Notch-responsive cells initiate the secondary transition in larval zebrafish pancreas

Zebrafish provide a highly versatile model in which to study vertebrate development. Many recent studies have elucidated early events in the organogenesis of the zebrafish pancreas; however, several aspects of early endocrine pancreas formation in the zebrafish are not homologous to the mammalian system. To better identify mechanisms of islet formation in the zebrafish, with true homology to those observed in mammals, we have temporally and spatially characterized zebrafish secondary islet formation. As is the case in the mouse, we show that Notch inhibition leads to precocious differentiation of endocrine tissues. Furthermore, we have used transgenic fish expressing fluorescent markers under the control of a Notch-responsive element to observe the precursors of these induced endocrine cells. These pancreatic Notch-responsive cells represent a novel population of putative progenitors that are associated with larval pancreatic ductal epithelium, suggesting functional homology between secondary islet formation in zebrafish and the secondary transition in mammals. We also show that Notch-responsive cells persist in the adult pancreas and possess the classical characteristics of centroacinar cells, a cell type believed to be a multipotent progenitor cell in adult mammalian pancreas.     

Joint migration of gonadotropin-releasing hormone (GnRH) and neuropeptide Y (NPY) neurons from olfactory placode to central nervous system

The olfactory epithelium in vertebrates generates the olfactory sensory neurons and several migratory cell types. Prominent among the latter are the gonadotropin-releasing hormone (GnRH) neurons that differentiate within the olfactory epithelium during embryogenesis and migrate along the olfactory nerve to the central nervous system. We initiated studies to characterize additional neuronal phenotypes of olfactory epithelial derivation. Neuropeptide Y (NPY) neurons are functionally related to the reproductive axis, modulating the release of GnRH and directly enhancing GnRH-induced luteinizing hormone (LH) secretion from gonadotrophs. We demonstrate that a population of migratory NPY neurons originates within the olfactory epithelium of the chick. At stage 25, NPY-positive fibers, but not cells, were detected in the epithelium and the nerve. By stages 28–34, NPY neurons and processes were present in the olfactory epithelium, olfactory nerve, and at the junction of the olfactory nerve and forebrain. In these regions the number of NPY neurons increased until stage 30 and then declined as development progressed. Electron microscopic immunocytochemistry confirmed the neuronal phenotype of the NPY-positive cells. The origin and migratory nature of some of these NPY cells was confirmed by double-label immunocytochemical detection of NPY and GnRH. A large percentage of the NPY-cells coexpressed the GnRH peptide. Between stages 28 and 34 single- and double-labeled NPY and GnRH neurons were found side by side along the GnRH migratory route emanating from the nasal epithelium, along the olfactory nerve, and into the ventral forebrain. These data suggest that an NPY population originates in the olfactory epithelium and migrates into the central nervous system during embryogenesis. By stage 42, no NPY/GnRH double-labeled cells were detected. © 1996 John Wiley & Sons, Inc.     

Immunocytochemical study of tyrosine hydroxylase and dopamine β-hydroxylase immunoreactivities in the rat pancreas

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine -hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory acitivity of the acinar cells, duct cells and endocrine cells.     

Variation in plasma glucose and pancreatic β cells in the turtle, Lissemys punctata (order: Chelonia; family: Trionychidae)

The present study relates to the determination of the plasma glucose level and volumetric analysis of β cells in pancreatic islets of the soft‐shelled turtle Lissemys punctata during different phases of its reproductive cycle. Reproductive events play a vital role in influencing the plasma glucose level and β‐cell behaviour in the pancreatic islets. The colour of the pancreas is either yellowish or pinkish, depending on endocrine activity. Islets are present throughout the gland and range from individual cells to small or large clumps, depending on the seasonal cycle. Splenic islets are dense with more blood capillaries and nerve innervations irrespective of sex and season. The endocrine cell mass forms irregular patches without connective tissue capsule. β cells occupy the inner region of the islets, being surrounded by other cell types. Lissemys punctata exhibits higher β‐cell activity during hibernation. Most insulin‐secreting cells acquire a larger size during the regressive period. An analysis indicates that β cells outnumber the non‐β endocrine cell mass in both number and per cent volume. There is negative correlation between islet mass and animal weight. Between the periods of reproductive cycles, a difference exists with respect to fasting plasma glucose and β‐cell volume.     

CART knock out mice have impaired insulin secretion and glucose intolerance, altered beta cell morphology and increased body weight

CART peptides are anorexigenic and are widely expressed in the central and peripheral nervous systems, as well as in endocrine cells in the pituitary, adrenal medulla and the pancreatic islets. To study the role of CART in islet function, we used CART null mutant mice (CART KO mice) and examined insulin secretion in vivo and in vitro, and expression of islet hormones and markers of beta-cell function using immunocytochemistry. We also studied CART expression in the normal pancreas. In addition, body weight development and food intake were documented. We found that in the normal mouse pancreas, CART was expressed in numerous pancreatic nerve fibers, both in the exocrine and endocrine portion of the gland. CART was also expressed in nerve cell bodies in the ganglia. Double immunostaining revealed expression in parasympathetic (vasoactive intestinal polypeptide (VIP)-containing) and in fewer sensory fibers (calcitonin gene-related peptide (CGRP)-containing). Although the expression of islet hormones appeared normal, CART KO islets displayed age dependent reduction of pancreatic duodenal homeobox 1 (PDX-1) and glucose transporter-2 (GLUT-2) immunoreactivity, indicating beta-cell dysfunction. Consistent with this, CART KO mice displayed impaired glucose-stimulated insulin secretion both in vivo after an intravenous glucose challenge and in vitro following incubation of isolated islets in the presence of glucose. The impaired insulin secretion in vivo was associated with impaired glucose elimination, and was apparent already in young mice with no difference in body weight. In addition, CART KO mice displayed increased body weight at the age of 40 weeks, without any difference in food intake. We conclude that CART is required for maintaining normal islet function in mice.     

Neuropeptide Y: Direct and indirect action on insulin secretion in the rat

Neuropeptide Y (NPY) was tested for an ability to directly influence the release of insulin using an in vitro isolated rat pancreatic islet system. NPY, at doses ranging from 100 pg/ml to 1 μg/ml, had no significant effect on the basal release (5.5 mM glucose) of insulin. However, NPY treatment resulted in a significant, dose-dependent (1 ng/ml to 1 μg/ml) inhibition of glucose-stimulated (11 mM) insulin release. When tested in a perfused rat pancreas preparation in situ, NPY administration led to a marked inhibition of both basal and stimulated insulin release followed by a postinhibitory rebound which exceeded the control insulin levels by 3-fold. In contrast, the intracerebroventricular (ICV) microinjection of NPY (5 μg) produced a significant but delayed (30 min) elevation of circulating insulin. It is therefore suggested that the direct action of NPY on insulin release is inhibitory while the central action of NPY indirectly results in an increase in plasma insulin. Thus, NPY may be added to the growing list of peptidergic agents which may affect the endocrine pancreas by acting as neurotransmitters and/or neuromodulators.