Islet Innervation Research Articles

Structure, interaction and nervous connectivity of beta cell primary cilia

Primary cilia are sensory organelles present in many cell types, partaking in various signaling processes. Primary cilia of pancreatic beta cells play pivotal roles in paracrine signaling and their dysfunction is linked to diabetes. Yet, the structural basis for their functions is unclear. We present three-dimensional reconstructions of beta cell primary cilia by electron and expansion microscopy. These cilia are spatially confined within deep ciliary pockets or narrow spaces between cells, lack motility components and display an unstructured axoneme organization. Furthermore, we observe a plethora of beta cell cilia-cilia and cilia-cell interactions with other islet and non-islet cells. Most remarkably, we have identified and characterized axo-ciliary synapses between beta cell cilia and the cholinergic islet innervation. These findings highlight the beta cell cilia’s role in islet connectivity, pointing at their function in integrating islet intrinsic and extrinsic signals and contribute to understanding their significance in health and diabetes.

Neuronal glucose sensing mechanisms and circuits in the control of insulin and glucagon secretion.

Glucose homeostasis is mainly under the control of the pancreatic islet hormones insulin and glucagon, which, respectively, stimulate glucose uptake and utilization by liver, fat, and muscle and glucose production by the liver. The balance between the secretions of these hormones is under the control of blood glucose concentrations. Indeed, pancreatic islet β-cells and α-cells can sense variations in glycemia and respond by an appropriate secretory response. However, the secretory activity of these cells is also under multiple additional metabolic, hormonal, and neuronal signals that combine to ensure the perfect control of glycemia over a lifetime. The central nervous system (CNS), which has an almost absolute requirement for glucose as a source of metabolic energy and thus a vital interest in ensuring that glycemic levels never fall below ∼5 mM, is equipped with populations of neurons responsive to changes in glucose concentrations. These neurons control pancreatic islet cell secretion activity in multiple ways: through both branches of the autonomic nervous system, through the hypothalamic-pituitary-adrenal axis, and by secreting vasopressin (AVP) in the blood at the level of the posterior pituitary. Here, we present the autonomic innervation of the pancreatic islets; the mechanisms of neuron activation by a rise or a fall in glucose concentration; how current viral tracing, chemogenetic, and optogenetic techniques allow integration of specific glucose sensing neurons in defined neuronal circuits that control endocrine pancreas function; and, finally, how genetic screens in mice can untangle the diversity of the hypothalamic mechanisms controlling the response to hypoglycemia.

OR12-05 Blunting Of Sympathetic Activation Of Pancreas As A Cause For Hypoglycemic Unawareness In Hypoglycemia Associated Autonomic Failure

Abstract Disclosure: K. Thakkar: None. R. Hampton: None. M. Jimenez Gonzalez: None. D. Espinoza: None. S. Stanley: None. Introduction: Hypoglycemia activates the autonomic nervous system to suppress pancreatic insulin and increase glucagon release, restoring blood glucose levels to normal. These effects occur partly via sympathetic innervation of pancreatic islets. Recurrent hypoglycemia leads to a blunted autonomic response known as Hypoglycemia Associated Autonomic Failure (HAAF), which further exacerbates hypoglycemia. The mechanisms underlying HAAF are not well-understood, but changes in pancreatic innervation may contribute. Hypothesis: Decreased activity and remodeling of sympathetic innervation to the pancreas contribute to autonomic dysfunction with repeated hypoglycemia. Methods: To examine pancreatic sympathetic innervation with repeated hypoglycemia, C57Bl6 mice were treated with 0, 1, or 5 episodes (0, 1x, or 5x) of insulin-induced hypoglycemia. Pancreata, sympathetic celiac ganglia, and brains were harvested. Pancreatic tissue was immunostained for insulin, glucagon, and tyrosine hydroxylase (TH), a marker for sympathetic innervation. Samples were then cleared using iDISCO+, imaged, and analyzed using Imaris to examine the 3D distribution of innervation and islets. To determine if pancreatic sympathetic nerve function is altered in HAAF, we used viral delivery to express activating chemogenetic constructs specifically in pancreas-projecting sympathetic nerves. We assessed the effects of chemogenetic activation of pancreatic sympathetic innervation during insulin tolerance testing (ITT) in C57Bl6 mice with or without previous recurrent hypoglycemia. Results: 3D image analyses suggest acute hypoglycemia (1x) upregulates islet glucagon staining intensity. By comparison, recurrent hypoglycemia (5x) ​decreases pancreatic TH+ innervation and may blunt hypoglycemia-induced increases in islet glucagon staining intensity. Chemogenetic activation of pancreas sympathetic nerves in mice with repeated hypoglycemia increased blood glucose during an ITT while activation in mice without previous hypoglycemia did not significantly alter blood glucose. These data suggest reduced activity in pancreatic sympathetic innervation with recurrent hypoglycemia that can be restored by chemogenetic activation. Conclusions: Our data suggest repeated episodes of hypoglycemia may remodel the structure and function of peripheral pancreatic sympathetic innervation as compared to one episode of hypoglycemia. Restoring activity in pancreatic sympathetic innervation may partially reverse the severity of hypoglycemia in HAAF. Staining and analysis of celiac ganglia and sympathetic outflow circuits in the CNS will allow determination of the level at which blunting of sympathetic activation and/or remodeling of innervation occurs. Presentation: Friday, June 16, 2023

Deletion of Ascl1 in pancreatic β-cells improves insulin secretion, promotes parasympathetic innervation, and attenuates dedifferentiation during metabolic stress

ObjectiveASCL1, a pioneer transcription factor, is essential for neural cell differentiation and function. Previous studies have shown that Ascl1 expression is increased in pancreatic β-cells lacking functional KATP channels or after feeding of a high fat diet (HFD) suggesting that it may contribute to the metabolic stress response of β-cells. MethodsWe generated β-cell-specific Ascl1 knockout mice (Ascl1βKO) and assessed their glucose homeostasis, islet morphology and gene expression after feeding either a normal diet or HFD for 12 weeks, or in combination with a genetic disruption of Abcc8, an essential KATP channel component. ResultsAscl1 expression is increased in response to both a HFD and membrane depolarization and requires CREB-dependent Ca2+ signaling. No differences in glucose homeostasis or islet morphology were observed in Ascl1βKO mice fed a normal diet or in the absence of KATP channels. However, male Ascl1βKO mice fed a HFD exhibited decreased blood glucose levels, improved glucose tolerance, and increased β-cell proliferation. Bulk RNA-seq analysis of islets from Ascl1βKO mice from three studied conditions showed alterations in genes associated with the secretory function. HFD-fed Ascl1βKO mice showed the most extensive changes with increased expression of genes necessary for glucose sensing, insulin secretion and β-cell proliferation, and a decrease in genes associated with β-cell dysfunction, inflammation and dedifferentiation. HFD-fed Ascl1βKO mice also displayed increased expression of parasympathetic neural markers and cholinergic receptors that was accompanied by increased insulin secretion in response to acetylcholine and an increase in islet innervation. ConclusionsAscl1 expression is induced by stimuli that cause Ca2+-signaling to the nucleus and contributes in a multifactorial manner to the loss of β-cell function by promoting the expression of genes associated with cellular dedifferentiation, attenuating β-cells proliferation, suppressing acetylcholine sensitivity, and repressing parasympathetic innervation of islets. Thus, the removal of Ascl1 from β-cells improves their function in response to metabolic stress.

Obese Adipocytes Have Altered Redox Homeostasis with Metabolic Consequences.

White and brown adipose tissues are organized to form a real organ, the adipose organ, in mice and humans. White adipocytes of obese animals and humans are hypertrophic. This condition is accompanied by a series of organelle alterations and stress of the endoplasmic reticulum. This stress is mainly due to reactive oxygen species activity and accumulation, lending to NLRP3 inflammasome activation. This last causes death of adipocytes by pyroptosis and the formation of large cellular debris that must be removed by macrophages. During their chronic scavenging activity, macrophages produce several secretory products that have collateral consequences, including interference with insulin receptor activity, causing insulin resistance. The latter is accompanied by an increased noradrenergic inhibitory innervation of Langerhans islets with de-differentiation of beta cells and type 2 diabetes. The whitening of brown adipocytes could explain the different critical death size of visceral adipocytes and offer an explanation for the worse clinical consequence of visceral fat accumulation. White to brown transdifferentiation has been proven in mice and humans. Considering the energy-dispersing activity of brown adipose tissue, transdifferentiation opens new therapeutic perspectives for obesity and related disorders.

Maternal low-calorie sweetener consumption rewires hypothalamic melanocortin circuits via a gut microbial co-metabolite pathway.

The prevalence of obesity and type 2 diabetes is growing at an alarming rate, including among pregnant women. Low-calorie sweeteners (LCSs) have increasingly been used as an alternative to sugar to deliver a sweet taste without the excessive caloric load. However, there is little evidence regarding their biological effects, particularly during development. Here, we used a mouse model of maternal LCS consumption to explore the impact of perinatal LCS exposure on the development of neural systems involved in metabolic regulation. We report that adult male, but not female, offspring from both aspartame- and rebaudioside A-exposed dams displayed increased adiposity and developed glucose intolerance. Moreover, maternal LCS consumption reorganized hypothalamic melanocortin circuits and disrupted parasympathetic innervation of pancreatic islets in male offspring. We then identified phenylacetylglycine (PAG) as a unique metabolite that was upregulated in the milk of LCS-fed dams and the serum of their pups. Furthermore, maternal PAG treatment recapitulated some of the key metabolic and neurodevelopmental abnormalities associated with maternal LCS consumption. Together, our data indicate that maternal LCS consumption has enduring consequences on the offspring's metabolism and neural development and that these effects are likely to be mediated through the gut microbial co-metabolite PAG.

Revisiting the Pathogenesis of Type 1 Diabetes: Importance of Neural Input to Pancreatic Islets and the Therapeutic Capability of Stem Cell Educator TM Therapy to Restore Their Integrity.

Type 1 diabetes (T1D) is an autoimmune disease with a shortage of islet β cells. To date, the etiology of T1D remains elusive. Increasing clinical evidence and animal studies demonstrate that autoimmune cells are directed against the nervous system of pancreatic islets, contributing to the development of T1D. Therefore, it highlights the necessity to explore novel clinical approaches to fundamentally correct the T1D autoimmunity not only focusing on islet β cells but also on protecting the islet nervous system. This allows the restoration of the integrity of islet innervation and the normal islet β-cell function. To address these issues, we developed a novel technology designated the Stem Cell Educator TM therapy, based on immune education by human cord-blood-derived multipotent stem cells (CB-SC). International amulticenter clinical trials demonstrated its clinical safety and efficacy to treat T1D and other autoimmune diseases. Stem Cell Educator TM therapy may have the potential to revolutionize the treatment of T1D, without the safety and ethical concerns associated with conventional immune and/or stem cell-based therapies.

Phosphoproteome reveals molecular mechanisms of aberrant rhythm in neurotransmitter-mediated islet hormone secretion in diabetic mice.

BackgroundAcetylcholine (ACh) and norepinephrine (NE) are representative neurotransmitters of parasympathetic and sympathetic nerves, respectively, that antagonize each other to coregulate internal body functions. This also includes the control of different kinds of hormone secretion from pancreatic islets. However, the molecular mechanisms have not been fully elucidated, and whether innervation in islets is abnormal in diabetes mellitus also remains unclear.Methods and resultsImmunofluorescence colocalization and islet perfusion were performed and the results demonstrated that ACh/NE and their receptors were highly expressed in islet and rapidly regulated different hormones secretion. Phosphorylation is considered an important posttranslational modification in islet innervation and it was identified by quantitative proteomic and phosphoproteomic analyses in this study. The phosphorylated islet proteins were found involved in many biological and pathological processes, such as synaptic signalling transduction, calcium channel opening and insulin signalling pathway. Then, the kinases were predicted by motif analysis and further screened and verified by kinase‐specific siRNAs in different islet cell lines (αTC1‐6, Min6 and TGP52). After functional verification, Ksr2 and Pkacb were considered the key kinases of ACh and NE in insulin secretion, and Cadps, Mlxipl and Pdcd4 were the substrates of these kinases measured by immunofluorescence co‐staining. Then, the decreased expression of receptors, kinases and substrates of ACh and NE were found in diabetic mice and the aberrant rhythm in insulin secretion could be improved by combined interventions on key receptors (M3 (pilocarpine) or α2a (guanfacine)) and kinases (Ksr2 or Pkacb).ConclusionsAbnormal innervation was closely associated with the degree of islet dysfunction in diabetic mice and the aberrant rhythm in insulin secretion could be ameliorated significantly after intervention with key receptors and kinases in the early stage of diabetes mellitus, which may provide a promising therapeutic strategy for diabetes mellitus in the future.

Unravelling innervation of pancreatic islets.

The central and peripheral nervous systems play critical roles in regulating pancreatic islet function and glucose metabolism. Over the last century, in vitro and in vivo studies along with examination of human pancreas samples have revealed the structure of islet innervation, investigated the contribution of sympathetic, parasympathetic and sensory neural pathways to glucose control, and begun to determine how the structure and function of pancreatic nerves are disrupted in metabolic disease. Now, state-of-the art techniques such as 3D imaging of pancreatic innervation and targeted in vivo neuromodulation provide further insights into the anatomy and physiological roles of islet innervation. Here, we provide a summary of the published work on the anatomy of pancreatic islet innervation, its roles, and evidence for disordered islet innervation in metabolic disease. Finally, we discuss the possibilities offered by new technologies to increase our knowledge of islet innervation and its contributions to metabolic regulation.

Pancreas-Brain Crosstalk.

The neural regulation of glucose homeostasis in normal and challenged conditions involves the modulation of pancreatic islet-cell function. Compromising the pancreas innervation causes islet autoimmunity in type 1 diabetes and islet cell dysfunction in type 2 diabetes. However, despite the richly innervated nature of the pancreas, islet innervation remains ill-defined. Here, we review the neuroanatomical and humoral basis of the cross-talk between the endocrine pancreas and autonomic and sensory neurons. Identifying the neurocircuitry and neurochemistry of the neuro-insular network would provide clues to neuromodulation-based approaches for the prevention and treatment of diabetes and obesity.

Islet sympathetic innervation and islet neuropathology in patients with type 1 diabetes

Dysregulation of glucagon secretion in type 1 diabetes (T1D) involves hypersecretion during postprandial states, but insufficient secretion during hypoglycemia. The sympathetic nervous system regulates glucagon secretion. To investigate islet sympathetic innervation in T1D, sympathetic tyrosine hydroxylase (TH) axons were analyzed in control non-diabetic organ donors, non-diabetic islet autoantibody-positive individuals (AAb), and age-matched persons with T1D. Islet TH axon numbers and density were significantly decreased in AAb compared to T1D with no significant differences observed in exocrine TH axon volume or lengths between groups. TH axons were in close approximation to islet α-cells in T1D individuals with long-standing diabetes. Islet RNA-sequencing and qRT-PCR analyses identified significant alterations in noradrenalin degradation, α-adrenergic signaling, cardiac β-adrenergic signaling, catecholamine biosynthesis, and additional neuropathology pathways. The close approximation of TH axons at islet α-cells supports a model for sympathetic efferent neurons directly regulating glucagon secretion. Sympathetic islet innervation and intrinsic adrenergic signaling pathways could be novel targets for improving glucagon secretion in T1D.

Noradrenergic fibers are associated with beta-cell dedifferentiation and impaired beta-cell function in humans

Aims/hypothesisType 2 diabetes (T2D) is characterized by a progressive loss of beta-cell function, and the “disappearance” of beta-cells in T2D may also be caused by the process of beta -cell dedifferentiation. Since noradrenergic innervation inhibits insulin secretion and density of noradrenergic fibers is increased in type 2 diabetes mouse models, we aimed to study the relation between islet innervation, dedifferentiation and beta-cell function in humans. MethodsUsing immunohistochemistry and electron microscopy, we analyzed pancreata from organ donors and from patients undergoing pancreatic surgery. In the latter, a pre-surgical detailed metabolic characterization by oral glucose tolerance test (OGTT) and hyperglycemic clamp was performed before surgery, thus obtaining in vivo functional parameters of beta-cell function and insulin secretion. ResultsThe islets of diabetic subjects were 3 times more innervated than controls (0.91 ± 0.21 vs 0.32 ± 0.10, n.fibers/islet; p = 0.01), and directly correlated with the dedifferentiation score (r = 0.39; p = 0.03). In vivo functional parameters of insulin secretion, assessed by hyperglycemic clamp, negatively correlated with the increase in fibers [beta-cell Glucose Sensitivity (r = −0.84; p = 0.01), incremental second-phase insulin secretion (r = −0.84, p = 0.03) and arginine-stimulated insulin secretion (r = −0.76, p = 0.04)]. Moreover, we observed a progressive increase in fibers, paralleling worsening glucose tolerance (from NGT through IGT to T2D). Conclusions/interpretationNoradrenergic fibers are significantly increased in the islets of diabetic subjects and this positively correlates with beta-cell dedifferentiation score. The correlation between in vivo insulin secretion parameters and the density of pancreatic noradrenergic fibers suggests a significant involvement of these fibers in the pathogenesis of the disease, and indirectly, in the islet dedifferentiation process.

A 3D atlas of the dynamic and regional variation of pancreatic innervation in diabetes.

Understanding the detailed anatomy of the endocrine pancreas, its innervation, and the remodeling that occurs in diabetes can provide new insights into metabolic disease. Using tissue clearing and whole-organ imaging, we identified the 3D associations between islets and innervation. This technique provided detailed quantification of α and β cell volumes and pancreatic nerve fibers, their distribution and heterogeneity in healthy tissue, canonical mouse models of diabetes, and samples from normal and diabetic human pancreata. Innervation was highly enriched in the mouse endocrine pancreas, with regional differences. Islet nerve density was increased in nonobese diabetic mice, in mice treated with streptozotocin, and in pancreata of human donors with type 2 diabetes. Nerve contacts with β cells were preserved in diabetic mice and humans. In summary, our whole-organ assessment allows comprehensive examination of islet characteristics and their innervation and reveals dynamic regulation of islet innervation in diabetes.

SUN-654 Dynamic and Regional Variation of Pancreatic Innervation in Diabetes

Background: The pancreas is a highly heterogeneous organ, with regional anatomical, developmental and functional differences. The endocrine pancreas is densely innervated, and neural signals play a significant role in glucose regulation by modulating pancreatic hormone release. However, relatively little is known about the anatomical relationships between islets and nerves across the whole pancreas. Since thin filamentous structures, such as nerves, are difficult to quantify and trace over large volumes using thin section histology, there is a need for high resolution imaging and rendering of intact pancreatic tissue in 3D. Aim: To use optical clearing, whole organ imaging, and 3D rendering to quantify islets and innervation across the whole pancreas in healthy mice, in two mouse models of diabetes, and in pancreatic samples from nondiabetic and diabetic human donors. Methods: Whole-mount staining and clearing was performed using iDISCO+ to quantify innervation, defined by the neuronal marker NF200, and beta cells in pancreata from C57Bl/6 mice, non-obese diabetic (NOD) mice, streptozotocin (STZ)-treated mice, and in pancreatic samples from nondiabetic and diabetic human donors. Z-stacked optical sections were acquired with an Ultramicroscope II at 4x or 12x magnification. Imaris was used to create digital surfaces covering the NF200+ innervation and islets to automatically determine innervation density and islet/nerve interactions. Results: Beta cell volumes were 1-4% in the human pancreas, and 1-2% in the healthy mouse pancreas, with regional variations in islet volume and insulin intensity. There were also significant differences in islet biology between the diabetes models. Innervation of the endocrine pancreas was significantly enriched compared to the surrounding exocrine pancreas, with regional variation. Islets were closely associated with pancreatic innervation and decreased in size with increasing distance from nerves in both mouse and human pancreatic tissue. Innervated islets were relatively preserved in models of diabetes. Finally, islet innervation and expression of neural markers were higher in human samples from diabetic patients and in mouse models of diabetes, with temporal and regional differences. Conclusions: 3D imaging and unbiased analysis across the whole pancreas provides comprehensive measurement of pancreatic nerve volumes and distribution. It allows detailed analysis of the anatomical relationship between nerves and islets, and reveals a close association that is maintained across species. The relative enrichment of innervated islets in diabetes and dynamic changes in islet innervation during the development of diabetes suggest further work is needed to examine the role of pancreatic nerves in preserving and protecting beta cells.

SAT-LB104 Maternal Non-Nutritive Sweeteners Consumption Perturbs Development of Melanocortin Circuits Causing Long-Term Metabolic Alterations in the Offspring

Maternal Non-Nutritive Sweeteners Consumption Perturbs Development of Melanocortin Circuits Causing Long-Term Metabolic Alterations in the Offspring Soyoung Park1, Alice Jang1, Josephine Lowy1, Sebastien G Bouret2 1Developmental Neuroscience & Diabetes and Obesity Programs, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, USA. 2 Inserm, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Jean-Pierre Aubert Research Centre, UMR-S 1172, Lille, 59000, France.With the growing prevalence of obesity and diabetes, including among pregnant women, non-nutritive sweeteners (NNS) have been widely used as an alternative to sugar in the attempt to deliver a sweet taste without excessive caloric load. However, there is little evidence regarding their biological effects particularly during critical periods of growth and development. In this study, we aimed to investigate whether maternal NNS consumption causes long-term neurodevelopmental and metabolic perturbations in the offspring. Adult female wild-type mice were exposed to either water (control), aspartame (0.03%), or rebaudioside A (0.02%) throughout pregnancy and lactation. Adult male, but not female, offspring from both aspartame- and rebaudioside A-exposed dams displayed increased adiposity and developed glucose intolerance. Moreover, maternal NNS consumption impaired pro-opiomelanocortin axonal projections to the paraventricular nucleus of the hypothalamus as well as reduced parasympathetic innervation of pancreatic islets. Taken together, our data indicate that maternal NNS consumption causes neurodevelopmental abnormalities in the offspring that may contribute to lifelong metabolic dysregulations.

2140-P: Pancreatic Islet and Exocrine Tissue Innervation Is Not Altered in Type 1 Diabetes

Impaired counterregulatory response of glucagon to hypoglycemia is a common complication of type 1 diabetes (T1D). One proposed contributor to abnormal glucagon secretion is decreased sympathetic innervation in T1D islets. To systematically assess the innervation of human pancreatic tissues, we examined samples from donors with recent-onset T1D (<10 years, age 13-45 years, n = 4), longstanding T1D (>10 years, age 27-63 years, n = 3), and nondiabetic controls (age 10-52 years, n = 4). Islet and exocrine tissue innervation was visualized by pan-neuronal marker tubulin β-3 and analyzed by a 2-D morphometry and 3-D rendering. By quantifying the nerve fiber length (1.9±0.4 nm/μm2) and density (646±94 fibers/mm2), we found that innervation in control human islets was nearly 20-fold less than previously reported in mouse islets. In contrast to mouse, human exocrine tissue was far more innervated than islets with fiber length of 6.8±0.4 nm/μm2(p<0.05), and density of 1336±50 fibers/mm2(p<0.05). The 3-D analysis showed that nerve fibers follow extracellular matrix (ECM) formed around acini and from there, they extend further into islets along the ECM made by islet vasculature. Although T1D islets were primarily composed of α cells, the islet area was similar to controls (control: 15.7±1.5 mm2, recent-onset T1D: 16.5±1.4 mm2, long-standing T1D: 15.3±1.5 mm2; p>0.05), and so was the 3-D arrangement of nerve fibers in exocrine tissue and islets. Regardless of T1D duration, the islet fiber length (recent-onset: 1.5±0.2 nm/μm2; long-standing: 1.0±0.2 nm/μm2) and density (recent-onset: 623±61 fibers/mm2; longstanding: 486±49 fibers/mm2), as well as exocrine fiber length (recent-onset: 5.2±.5 nm/μm2; longstanding: 5.8±0.4 nm/μm2) and density (recent-onset: 1119±77 fibers/mm2; longstanding: 1537±72 fibers/mm2) did not differ from controls (p>0.05). These data indicate that neuronal patterning of human pancreas is significantly different from that of mouse and islet innervation does not appear to be altered in T1D. Disclosure T.M. Richardson: None. R. Haliyur: None. R. Aramandla: None. R. Bottino: Research Support; Self; Imagine Pharma. A.C. Powers: None. M. Brissova: None.

A new mode of pancreatic islet innervation revealed by live imaging in zebrafish.

Pancreatic islets are innervated by autonomic and sensory nerves that influence their function. Analyzing the innervation process should provide insight into the nerve-endocrine interactions and their roles in development and disease. Here, using in vivo time-lapse imaging and genetic analyses in zebrafish, we determined the events leading to islet innervation. Comparable neural density in the absence of vasculature indicates that it is dispensable for early pancreatic innervation. Neural crest cells are in close contact with endocrine cells early in development. We find these cells give rise to neurons that extend axons toward the islet as they surprisingly migrate away. Specific ablation of these neurons partly prevents other neurons from migrating away from the islet resulting in diminished innervation. Thus, our studies establish the zebrafish as a model to interrogate mechanisms of organ innervation, and reveal a novel mode of innervation whereby neurons establish connections with their targets before migrating away.

Deletion of the insulin receptor in sensory neurons increases pancreatic insulin levels

Insulin is known to have neurotrophic properties and loss of insulin support to sensory neurons may contribute to peripheral diabetic neuropathy (PDN). Here, genetically-modified mice were generated in which peripheral sensory neurons lacked the insulin receptor (SNIRKO mice) to determine whether disrupted sensory neuron insulin signaling plays a crucial role in the development of PDN and whether SNIRKO mice develop symptoms of PDN due to reduced insulin neurotrophic support. Our results revealed that SNIRKO mice were euglycemic and never displayed significant changes in a wide range of sensorimotor behaviors, nerve conduction velocity or intraepidermal nerve fiber density. However, SNIRKO mice displayed elevated serum insulin levels, glucose intolerance, and increased insulin content in the islets of Langerhans of the pancreas. These results contribute to the growing idea that sensory innervation of pancreatic islets is key to regulating islet function and that a negative feedback loop of sensory neuron insulin signaling keeps this regulation in balance. Our results suggest that a loss of insulin receptors in sensory neurons does not lead to peripheral nerve dysfunction. The SNIRKO mice will be a powerful tool to investigate sensory neuron insulin signaling and may give a unique insight into the role that sensory neurons play in modifying islet physiology.

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network.

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to <20 µm as formalin fixed paraffin section on glass slides or <500 µm for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples >500 µm using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth >150 µm with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to <20 µm as formalin fixed paraffin section on glass slides or <500 µm for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples >500 µm using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth >150 µm with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.