Islet Innervation Research Articles

Human pancreatic neuro-insular network in health and fatty infiltration.

Identification of a pancreatic neuro-insular network in mice suggests that a similar integration of islets and nerves may be present in the human pancreas. To characterise the neuro-insular network and the intra-pancreatic ganglia in a clinically related setting, we examined human pancreases in health and with fatty infiltration via 3-dimensional (3D) histology and compared the human pancreatic microenvironment with its counterpart in mice. Human pancreatic specimens from individuals with normal BMI, high BMI (≥25) and type 2 diabetes were used to investigate the neuro-insular network. Transparent specimens were prepared by tissue clearing for transmitted light and deep-tissue fluorescence imaging to simultaneously visualise infiltrated adipocytes, islets and neurovascular networks. High-definition images of human islets reveal that both the sympathetic and parasympathetic nerves enter the islet core and reside in the immediate microenvironment of islet cells. Around the islets, the neuro-insular network is visualised with 3D histology to identify the intra-pancreatic ganglia (peri-lobular and intra-parenchymal ganglia) and the islet-ganglionic association. In humans, but not in mice, pancreatic fatty infiltration (BMI dependent) features adipocytes infiltrating into the parenchyma and accumulating in the peri-lobular space, in which the peri-lobular ganglia also reside. We identified the formation of adipose-ganglionic complexes in the peri-lobular space and enlargement of ganglia around adipocytes. In the specimen from the individual with type 2 diabetes, an increase in the number of nerve projections from the intra-parenchymal ganglia is associated with severe fatty infiltration. We present new perspectives of human pancreas and islet innervation via 3D histology. Our results strongly suggest that fatty infiltration in the human pancreas creates a neurotrophic microenvironment and promotes remodelling of pancreatic innervation.

Differences between studying an islet β cell and studying whole pancreatic islets: immunological implications

The main role of the pancreatic islet β cell is to release the appropriate amount of insulin upon glucose stimulation. For this reason, islet transplantation has been advancing in the past few years as a therapeutic alternative for patients with diabetes, alongside the exciting field of manipulating β cell differentiation for the sake of β cell transplantation. However, do isolated β cells function the same as β cells within an intact islet? Within islets, β cells are surrounded by other cell types, including endocrine cells, endothelial cells and immune cells, a proximity which appears to be relevant for proper glucose homeostasis. Although insulin and glucose are the main regulators in this scenario, other factors, such as angiogenesis, local anti-inflammatory components and the activity profile of resident macrophages, have a profound effect on the function and fate of β cells. A paracrine interaction between β cells and α cell holds a dramatic effect on β cell function, which is additionally dependent on blood flow through the islet. Another important intercellular communication exists between β cells and endothelial cells, in this case a bidirectional interface. Moreover, β cell survival and proliferation is dependent on the potency of ECM proteins. Further parameters distinguish functionally between the isolated β cell and the intact islet, including the deposition of Zinc by β cells, synchronicity by electrical and calcium routes, the physical innervation of islets and more. In this review, we explore major parameters that relate to differences between the function of the isolated β cell and that of the β cell within an intact islet. These and some yet to be investigated aspects of β cell function should be included in the list of considerations when examining therapeutic targets for β cell–related pathologies and for the prospect of effective β cell replacement therapy.

Leptin Controls Parasympathetic Wiring of the Pancreas during Embryonic Life.

The autonomic nervous system plays a critical role in glucose metabolism through both its sympathetic and parasympathetic branches, but the mechanisms that underlie the development of the autonomic innervation of the pancreas remain poorly understood. Here, we report that cholinergic innervation of pancreatic islets develops during mid-gestation under the influence of leptin. Leptin-deficient mice display a greater cholinergic innervation of pancreatic islets beginning in embryonic life, and this increase persists into adulthood. Remarkably, a single intracerebroventricular injection of leptin in embryos caused a permanent reduction in parasympathetic innervation of pancreatic β cells and long-term impairments in glucose homeostasis. These developmental effects of leptin involve a direct inhibitory effect on the outgrowth of preganglionic axons from the hindbrain. These studies reveal an unanticipated regulatory role of leptin on the parasympathetic nervous system during embryonic development and may have important implications for our understanding of the early mechanisms that contribute to diabetes.

Imaging of the islet neural network.

The islets of Langerhans receive signals from the circulation and nerves to modulate hormone secretion in response to physiological cues. Although the rich islet innervation has been documented in the literature dating as far back as Paul Langerhans' discovery of islets in the pancreas, it remains a challenging task for researchers to acquire detailed islet innervation patterns in health and disease due to the dispersed nature of the islet neurovascular network. In this article, we discuss the recent development of 3-dimensional (3D) islet neurohistology, in which transparent pancreatic specimens were prepared by optical clearing to visualize the islet microstructure, vasculature and innervation with deep-tissue microscopy. Mouse islets were used as an example to illustrate how to apply this 3D imaging approach to characterize (i) the islet parasympathetic innervation, (ii) the islet sympathetic innervation and its reinnervation after transplantation under the kidney capsule and (iii) the reactive cellular response of the Schwann cell network in islet injury. While presenting and characterizing the innervation patterns, we also discuss how to apply the signals derived from transmitted light microscopy, vessel painting and immunostaining of neural markers to verify the location and source of tissue information. In summary, the systematic development of tissue labelling, clearing and imaging methods to reveal the islet neuroanatomy offers insights to help study the neural-islet regulatory mechanisms and the role of neural tissue remodelling in the development of diabetes.

Consumption of a Western-style diet during pregnancy impairs offspring islet vascularization in a Japanese macaque model.

Children exposed to a maternal Western-style diet in utero have an increased risk of developing type 2 diabetes. Understanding the mechanisms and an investigation of possible interventions are critical to reversing this phenomenon. We examined the impact of maternal Western-style diet consumption on the development of islet vascularization and innervation, both of which are critical to normal islet function, in fetal and juvenile offspring. Furthermore, we assessed whether improved dietary intake or resveratrol supplementation could ameliorate the harmful consequences of Western-style diet consumption during pregnancy. Adult female Japanese macaques were maintained on a control or Western-style diet for 4-7 yr. One cohort of dams was switched back onto a control diet, whereas another cohort received resveratrol supplementation throughout gestation. Pregnancies were terminated in the early third trimester by C-section, or offspring were born naturally and sent to necropsy at 1 yr of age. Western-style diet consumption resulted in impaired fetal islet capillary density and sympathetic islet innervation. Furthermore, this reduction in vascularization persisted in the juvenile offspring. This effect is independent of changes in the expression of key angiogenic markers. Diet reversal normalized islet vascularization to control offspring levels, whereas resveratrol supplementation caused a significant increase in capillary density above controls. These data provide a novel mechanism by which maternal Western-style diet consumption leads to increased susceptibility to type 2 diabetes in the offspring. Importantly, an improved maternal diet may mitigate these harmful effects. However, until the long-term consequences of increased vascularization can be determined, resveratrol use during pregnancy is not advised.

Vascular endothelial growth factor coordinates islet innervation via vascular scaffolding

Neurovascular alignment is a common anatomical feature of organs, but the mechanisms leading to this arrangement are incompletely understood. Here, we show that vascular endothelial growth factor (VEGF) signaling profoundly affects both vascularization and innervation of the pancreatic islet. In mature islets, nerves are closely associated with capillaries, but the islet vascularization process during embryonic organogenesis significantly precedes islet innervation. Although a simple neuronal meshwork interconnects the developing islet clusters as they begin to form at E14.5, the substantial ingrowth of nerve fibers into islets occurs postnatally, when islet vascularization is already complete. Using genetic mouse models, we demonstrate that VEGF regulates islet innervation indirectly through its effects on intra-islet endothelial cells. Our data indicate that formation of a VEGF-directed, intra-islet vascular plexus is required for development of islet innervation, and that VEGF-induced islet hypervascularization leads to increased nerve fiber ingrowth. Transcriptome analysis of hypervascularized islets revealed an increased expression of extracellular matrix components and axon guidance molecules, with these transcripts being enriched in the islet-derived endothelial cell population. We propose a mechanism for coordinated neurovascular development within pancreatic islets, in which endocrine cell-derived VEGF directs the patterning of intra-islet capillaries during embryogenesis, forming a scaffold for the postnatal ingrowth of essential autonomic nerve fibers.

The Incretin Response After Successful Islet Transplantation

The Incretin Response After Successful Islet Transplantation

Immunohistochemistry evidence for gastric ghrelin cells hypertrophy in obese patients

Ghrelin cells are endocrine cells of the gastrointestinal tract mainly present in the glands of the stomach. They produce the “hunger hormone” that promotes appetite and adiposity in animal and human models. In response to these anabolic effects, several elements have suggested the influence of ghrelin on the regulation of metabolic functions and the development of obesity-related disorders. However, its physiologic significance is quite complex and some of the effects of ghrelin are still debated in the literature. Recent evidence suggests that ghrelin influences glucose homeostasis through the modulation of insulin secretion and insulin receptor signalling. T2 diabetes is the most frequent metabolic disorder associated with obesity. To date, the most effective treatment of obesity is bariatric surgery and one of the most used techniques is sleeve gastrectomy. It consists in the resection of 25% of the stomach producing a neo anatomy in which this organ becomes a tube-like structure, lacking the ghrelinrich part. Forty-seven percent of T2 diabetic obese patients treated by sleeve gastrectomy rescue their diabetic condition just after surgery and before any weight reduction. This clinical evidence clearly connects this new morphology of the stomach with the positive metabolic improvement and suggests that ghrelin could play a key role in this striking effect. We studied the gastric mucosa of the fundus of sleeve gastrectomized T2 diabetic-obese patients (n.5) by immunohistochemistry with Anti- Ghrelin antibody to verify the ghrelin cell morphology, immunoreactivity and density. Anti- Chromogranin A antibody was used to detect the whole endocrine population of gastric glands. We used the normal part of the gastric fundus removed from patients (n.3) suffering for gastric cancer, as control. Ghrelin receptor has been shown on peripheral nerve fibers of the autonomous nervous system, and because a direct innervation of Langerhans islets by intestinal neurons have been shown in the past, we extended our immunohistochemistry analyses to neuronal antigens in order to study the nervous network of gastric mucosae in these surgical samples. Results show that ghrelin cells in T2 diabetic obese patients represented a consistent percentage (48.4% vs 38% in controls) of chromogranin A immunoreactive cell population. Furthermore, ghrelin cells were hypertrophic and intensely immunostained when compared with those found in gastric mucosae of controls. Neuronal specific enolase immunoreactivity was clearly enhanced in the gastric mucosae of obese patients. These data suggest that the stomach of T2 diabetic obese patients increase ghrelin content and that sleeve gastrectomy could have an important metabolic role by anatomical reduction of this hormone. Furthermore, our result suggest a possible paracrine effect on the peripheral neuronal population that could also be implicated in this complex and striking clinical effect of bariatric surgery.

Noninvasive in vivo model demonstrating the effects of autonomic innervation on pancreatic islet function

The autonomic nervous system is thought to modulate blood glucose homeostasis by regulating endocrine cell activity in the pancreatic islets of Langerhans. The role of islet innervation, however, has remained elusive because the direct effects of autonomic nervous input on islet cell physiology cannot be studied in the pancreas. Here, we used an in vivo model to study the role of islet nervous input in glucose homeostasis. We transplanted islets into the anterior chamber of the eye and found that islet grafts became densely innervated by the rich parasympathetic and sympathetic nervous supply of the iris. Parasympathetic innervation was imaged intravitally by using transgenic mice expressing GFP in cholinergic axons. To manipulate selectively the islet nervous input, we increased the ambient illumination to increase the parasympathetic input to the islet grafts via the pupillary light reflex. This reduced fasting glycemia and improved glucose tolerance. These effects could be blocked by topical application of the muscarinic antagonist atropine to the eye, indicating that local cholinergic innervation had a direct effect on islet function in vivo. By using this approach, we found that parasympathetic innervation influences islet function in C57BL/6 mice but not in 129X1 mice, which reflected differences in innervation densities and may explain major strain differences in glucose homeostasis. This study directly demonstrates that autonomic axons innervating the islet modulate glucose homeostasis.

Islet nerves in focus—defining their neurobiological and clinical role

Although it is well established that the pancreatic islets are innervated by autonomic nerves, the detailed islet innervation pattern is still unclear. In this issue of Diabetologia (DOI: 10.1007/s00125-012-2699-6 ) novel details of the islet neuroanatomy and its plasticity in experimental diabetes are described. By using a 3-dimensional (3-D) imaging technique, it has been shown that, in islets from normal mice, sympathetic nerves mainly form a neurovascular complex in addition to innervating peripherally located islet alpha cells. There are also pronounced changes in islet neuroanatomy in experimental diabetes. These findings suggest novel neural-islet regulatory mechanisms as well as neural involvement in the development of diabetes, and therefore advance both basic and clinical knowledge of islet neurobiology.

Neurohormonal regulation of pancreatic secretion

Recent advances in the regulation of pancreatic secretion by neural and hormonal mechanisms are discussed in this review. It has been shown that the multidrug-resistance protein MRP4 may play a role in the efflux of cAMP from exocrine cells and neurokinin receptors are important in substance P-mediated inhibition of ductal bicarbonate secretion. Leptin attenuates glucagon secretion by downregulating glucagon gene expression, whereas ghrelin upregulates glucagon release by elevating intracellular calcium and phosphorylation of extracellular signal-regulated kinase (ERK). Cytokine interleukin 6 is secreted from muscles during exercise and induces the release of GLP-1 that stimulates insulin secretion. Osteocalcin and 17β-estradiol mediate their effects through G protein-coupled receptors, resulting in ERK phosphorylation and activation of protein kinase-dependent signaling pathways. Melatonin and ghrelin inhibit insulin secretion through inhibitory G proteins, whereas aldosterone may attenuate insulin secretion by increasing oxidative stress in islets cells. Finally, the pattern of innervation of human pancreatic islets has been examined and demonstrated to be very different from that in the mouse. Many different receptors and signaling pathways govern the complex biology of pancreatic secretion. Elucidation of these cellular mechanisms will aid in drug discovery and treatment as well as prevention of pancreatic diseases.

Innervation Patterns of Autonomic Axons in the Human Endocrine Pancreas

The autonomic nervous system regulates hormone secretion from the endocrine pancreas, the islets of Langerhans, thus impacting glucose metabolism. The parasympathetic and sympathetic nerves innervate the pancreatic islet, but the precise innervation patterns are unknown, particularly in human. Here we demonstrate that the innervation of human islets is different from that of mouse islets and does not conform to existing models of autonomic control of islet function. By visualizing axons in three dimensions and quantifying axonal densities and contacts within pancreatic islets, we found that, unlike mouse endocrine cells, human endocrine cells are sparsely contacted by autonomic axons. Few parasympathetic cholinergic axons penetrate the human islet, and the invading sympathetic fibers preferentially innervate smooth muscle cells of blood vessels located within the islet. Thus, rather than modulating endocrine cell function directly, sympathetic nerves may regulate hormone secretion in human islets by controlling local blood flow or by acting on islet regions located downstream.

Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming beta cell function in humans

Acetylcholine is a neurotransmitter that plays a major role in the function of the insulin secreting pancreatic beta cell1,2. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species1,3,4, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of the human islet provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. We anticipate these results to revise models about neural input and cholinergic signaling in the endocrine pancreas. Cholinergic signaling within the islet represents a potential therapeutic target in diabetes5, highlighting the relevance of this advance to future drug development.

Improved Second Phase Insulin Secretion and Preserved Insulin Sensitivity After Islet Transplantation

Improved Second Phase Insulin Secretion and Preserved Insulin Sensitivity After Islet Transplantation

The islet-acinar axis of the pancreas: more than just insulin

Although the role of the islets in the regulation of acinar cell function seemed a mystery to investigators who observed their dispersion among pancreatic acini, over time an appreciation for this intricate and unique structural arrangement has developed. The last three decades have witnessed a steadily growing understanding of the interrelationship of the endocrine and the exocrine pancreas. The islet innervation and vascular anatomy have been more fully characterized and provide an appropriate background for our current understanding. The interrelationship between the endocrine and exocrine pancreas is mediated by islet-derived hormones such as insulin and somatostatin, other humoral factors including pancreastatin and ghrelin, and also neurotransmitters (nitric oxide, peptide YY, substance P, and galanin) released by the nerves innervating the pancreas. Although considerable progress has been achieved, further work is required to fully delineate the complex interplay of the numerous mechanisms involved. This review aims to provide a comprehensive update of the current literature available, bringing together data gleaned from studies addressing the actions of individual hormones, humoral factors, and neurotransmitters on the regulation of amylase secretion from the acinar cell. This comprehensive view of the islet-acinar axis of the pancreas while acknowledging the dominant role played by insulin and somatostatin on exocrine secretion sheds light on the influence of the various neuropeptides on amylase secretion.

MRI as a tool to monitor islet transplantation

The development of new methods for noninvasive imaging is an area of biotechnology that is of great relevance for the diagnosis and characterization of diabetes mellitus. Noninvasive imaging can be used to study the dynamics of beta-cell mass and function; beta-cell death; vascularity, innervation and autoimmune attack of pancreatic islets; and the efficacy of islet transplantation to remedy beta-cell loss in patients with diabetes mellitus. In this Review, we focus on the application of MRI for monitoring islet transplantation and on the potential causes of islet graft failure, which are still poorly understood. Questions that have been addressed by MRI studies encompass graft longevity, and the effects of immune rejection, glucose toxic effects, and the transplanted islets' purity on graft fate. We also highlight novel technologies for simultaneous imaging and delivery of experimental therapies that aim to extend the lifespan and functionality of islet grafts. On the basis of this evidence, MRI represents a valuable platform for a thorough investigation of beta-cell function in the context of islet transplantation. State-of-the-art multimodality approaches, such as PET-MRI, can extend our current capabilities and help answer the critical questions that currently inhibit the prevention and cure of diabetes mellitus.

Extracellular Matrix in Pancreatic Islets: Relevance to Scaffold Design and Transplantation

Intrahepatic islet transplantation provides a potentially more benign alternative to pancreatic transplantation. However, islet transplants are associated with limited engraftment potential. This inefficiency is likely at least partially attributable to the isolation process, which removes islets from their native environment. Isolation not only disrupts the internal vascularization and innervation of islets, but also fundamentally changes interactions between islet cells and macromolecules of the extracellular matrix (ECM). Signaling interactions between islet cells and ECM are known to regulate multiple aspects of islet physiology, including survival, proliferation, and insulin secretion. Although it is highly likely that disruptions to these interactions during isolation significantly affect transplant outcomes, the true implications of these conditions are not well understood. The following article reviews current understandings and uncertainties in islet-ECM interactions and explains their potential impact on posttransplant engraftment. Topics covered include matrix and receptor compositions in native islets, effects of isolation and culture on islet-ECM interactions, and potential for postisolation restoration of islet-ECM interactions. Greater understanding in these areas may help to reduce isolation and transplantation stresses and improve islet engraftment.

Reconstructing the Pancreas: Restoration of Normoglycemia, Exocrine Function, and Islet Innervation by Islet Transplantation to the Pancreas

Impaired function in transplanted islets may be ascribed in part to disturbed reinnervation. The objectives of this study were to determine whether islet transplantation to the pancreas in the presence of nerve growth factor (NGF) would restore islet innervation and endocrine and exocrine pancreatic function. Streptozotocin-diabetic Lewis rats received 800 syngeneic islets beneath the pancreatic capsule in the presence or absence of NGF (20 ng/d for 14 days). Fasting blood glucose was measured for 3 months. The pancreata were isolated and perfused in situ. Pancreatic juice was collected for amylase determination. The sympathetic trunks were isolated and stimulated electrically. The tissues were immunostained for nerve markers. All islet recipients remained euglycemic (4.2 ± 0.6 mmol/L glucose). Ductal amylase concentrations were restored to near normal levels in contrast to diabetic controls (normal rat 98 ± 8 U/L, islet transplant 78.4 ± 9 U/L, diabetic control 14.5 ± 8 U/L). NGF enhanced the innervation of transplanted islets in contrast to control islet transplants. Sympathetic adrenergic innervation was significantly increased by NGF (tyrosine hydroxylase [ P < .001] and neuropeptide Y [ P < .05]). No differences in parasympathetic innervation were observed (vesicular acetylcholine transporter). Electrical stimulation of the sympathetic trunks in the presence of 4 μmol/L phentolamine and 5 μmol/L atropine resulted in increased insulin secretion in NGF-treated islet transplants (164%) compared with control transplants (30%). The combination of growth factors and the pancreatic site may allow the use of fewer islets than conventional islet transplant sites and promote more normal transplanted islet function by the enhancement of islet reinnervation.

Pancreatic innervation in mouse development and β-cell regeneration

Pancreatic innervation is being viewed with increasing interest with respect to pancreatic disease. At the same time, relatively little is currently known about innervation dynamics during development and disease. The present study employs confocal microscopy to analyze the growth and development of sympathetic and sensory neurons and astroglia during pancreatic organogenesis and maturation. Our research reveals that islet innervation is closely linked to the process of islet maturation—neural cell bodies undergo intrapancreatic migration/shuffling in tandem with endocrine cells, and close neuro-endocrine contacts are established quite early in pancreatic development. In addition, we have assayed the effects of large-scale β-cell loss and repopulation on the maintenance of islet innervation with respect to particular neuron types. We demonstrate that depletion of the β-cell population in the rat insulin promoter (RIP)-cmyc ER mouse line has cell-type-specific effects on postganglionic sympathetic neurons and pancreatic astroglia. This study contributes to a greater understanding of how cooperating physiological systems develop together and coordinate their functions, and also helps to elucidate how permutation of one organ system through stress or disease can specifically affect parallel systems in an organism.

Parasympathetic Innervation and Function of Endocrine Pancreas Requires the Glial Cell Line–Derived Factor Family Receptor α2 (GFRα2)

Vagal parasympathetic input to the islets of Langerhans is a regulator of islet hormone secretion, but factors promoting parasympathetic islet innervation are unknown. Neurturin signaling via glial cell line-derived neurotrophic factor family receptor alpha2 (GFRalpha2) has been demonstrated to be essential for the development of subsets of parasympathetic and enteric neurons. Here, we show that the parasympathetic nerve fibers and glial cells within and around the islets express GFRalpha2 and that islet parasympathetic innervation in GFRalpha2 knockout (KO) mice is reduced profoundly. In wild-type mice, neuroglucopenic stress produced a robust increase in plasma levels of islet hormones. In the GFRalpha2-KO mice, however, pancreatic polypeptide and insulin responses were completely lost and glucagon response was markedly impaired. Islet morphology and sympathetic innervation, as well as basal secretions of the islet hormones, were unaffected. Moreover, a glucose tolerance test failed to reveal differences between the genotypes, indicating that direct glucose-stimulated insulin secretion was not affected by GFRalpha2 deficiency. These results show that GFRalpha2 signaling is needed for development of the parasympathetic islet innervation that is critical for vagally induced hormone secretion. The GFRalpha2-KO mouse represents a useful model to study the role of parasympathetic innervation of the endocrine pancreas in glucose homeostasis.