Publications by authors named "Rayner Rodriguez-Diaz"

26 Publications

  • Page 1 of 1

Pancreatic β-Cells Communicate With Vagal Sensory Neurons.

Gastroenterology 2021 02 26;160(3):875-888.e11. Epub 2020 Oct 26.

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Miller School of Medicine, University of Miami, Miami, Florida; Program in Neuroscience, Miller School of Medicine, University of Miami, Miami, Florida; Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, Florida; Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, Florida. Electronic address:

Background And Aims: Destroying visceral sensory nerves impacts pancreatic islet function, glucose metabolism, and diabetes onset, but how islet endocrine cells interact with sensory neurons has not been studied.

Methods: We characterized the anatomical pattern of pancreatic sensory innervation by combining viral tracing, immunohistochemistry, and reporter mouse models. To assess the functional interactions of β-cells with vagal sensory neurons, we recorded Ca responses in individual nodose neurons in vivo while selectively stimulating β-cells with chemogenetic and pharmacologic approaches.

Results: We found that pancreatic islets are innervated by vagal sensory axons expressing Phox2b, substance P, calcitonin-gene related peptide, and the serotonin receptor 5-HTR. Centrally, vagal neurons projecting to the pancreas terminate in the commissural nucleus of the solitary tract. Nodose neurons responded in vivo to chemogenetic stimulation of β-cells and to pancreas infusion with serotonin, but were not sensitive to insulin. Responses to chemogenetic and pharmacologic stimulation of β-cells were blocked by a 5-HTR antagonist and were enhanced by increasing serotonin levels in β-cells. We further confirmed directly in living pancreas slices that sensory terminals in the islet were sensitive to serotonin.

Conclusions: Our study establishes that pancreatic β-cells communicate with vagal sensory neurons, likely using serotonin signaling as a transduction mechanism. Serotonin is coreleased with insulin and may therefore convey information about the secretory state of β-cells via vagal afferent nerves.
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http://dx.doi.org/10.1053/j.gastro.2020.10.034DOI Listing
February 2021

Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell.

Nat Metab 2019 11 15;1(11):1110-1126. Epub 2019 Nov 15.

J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA.

Pancreatic beta cells synthesize and secrete the neurotransmitter γ-aminobutyric acid (GABA) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel (VRAC), functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from type 1 and type 2 diabetic patients, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.
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http://dx.doi.org/10.1038/s42255-019-0135-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236889PMC
November 2019

Secretory Functions of Macrophages in the Human Pancreatic Islet Are Regulated by Endogenous Purinergic Signaling.

Diabetes 2020 06 3;69(6):1206-1218. Epub 2020 Apr 3.

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL

Endocrine cells of the pancreatic islet interact with their microenvironment to maintain tissue homeostasis. Communication with local macrophages is particularly important in this context, but the homeostatic functions of human islet macrophages are not known. In this study, we show that the human islet contains macrophages in perivascular regions that are the main local source of the anti-inflammatory cytokine interleukin-10 (IL-10) and the metalloproteinase MMP9. Macrophage production and secretion of these homeostatic factors are controlled by endogenous purinergic signals. In obese and diabetic states, macrophage expression of purinergic receptors MMP9 and IL-10 is reduced. We propose that in those states, exacerbated β-cell activity due to increased insulin demand and increased cell death produce high levels of ATP that downregulate purinergic receptor expression. Loss of ATP sensing in macrophages may reduce their secretory capacity.
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http://dx.doi.org/10.2337/db19-0687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243286PMC
June 2020

A Nervous Breakdown that May Stop Autoimmune Diabetes.

Cell Metab 2020 02;31(2):215-216

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL 33136, USA; Program in Neuroscience, Miller School of Medicine, University of Miami, Miami, FL 33136, USA; Molecular Cell and Developmental Biology Program, Miller School of Medicine, University of Miami, Miami, FL 33136, USA; Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. Electronic address:

Neuromodulation is a promising new therapeutic avenue to treat chronic diseases. A paper that appeared recently in Nature Biotechnology (Guyot et al., 2019) now provides a roadmap for how to establish electrostimulation of nerves to delay or even prevent type 1 diabetes.
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http://dx.doi.org/10.1016/j.cmet.2020.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477910PMC
February 2020

The Local Paracrine Actions of the Pancreatic α-Cell.

Diabetes 2020 04 27;69(4):550-558. Epub 2019 Dec 27.

Department of Medicine, University of Miami Miller School of Medicine, Miami, FL

Secretion of glucagon from the pancreatic α-cells is conventionally seen as the first and most important defense against hypoglycemia. Recent findings, however, show that α-cell signals stimulate insulin secretion from the neighboring β-cell. This article focuses on these seemingly counterintuitive local actions of α-cells and describes how they impact islet biology and glucose metabolism. It is mostly based on studies published in the last decade on the physiology of α-cells in human islets and incorporates results from rodents where appropriate. As this and the accompanying articles show, the emerging picture of α-cell function is one of increased complexity that needs to be considered when developing new therapies aimed at promoting islet function in the context of diabetes.
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http://dx.doi.org/10.2337/dbi19-0002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085245PMC
April 2020

Structural basis for delta cell paracrine regulation in pancreatic islets.

Nat Commun 2019 08 16;10(1):3700. Epub 2019 Aug 16.

Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, 636921, Singapore.

Little is known about the role of islet delta cells in regulating blood glucose homeostasis in vivo. Delta cells are important paracrine regulators of beta cell and alpha cell secretory activity, however the structural basis underlying this regulation has yet to be determined. Most delta cells are elongated and have a well-defined cell soma and a filopodia-like structure. Using in vivo optogenetics and high-speed Ca imaging, we show that these filopodia are dynamic structures that contain a secretory machinery, enabling the delta cell to reach a large number of beta cells within the islet. This provides for efficient regulation of beta cell activity and is modulated by endogenous IGF-1/VEGF-A signaling. In pre-diabetes, delta cells undergo morphological changes that may be a compensation to maintain paracrine regulation of the beta cell. Our data provides an integrated picture of how delta cells can modulate beta cell activity under physiological conditions.
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http://dx.doi.org/10.1038/s41467-019-11517-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697679PMC
August 2019

The Pericyte of the Pancreatic Islet Regulates Capillary Diameter and Local Blood Flow.

Cell Metab 2018 03;27(3):630-644.e4

Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA; Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, FL 33136, USA; Program in Neuroscience, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. Electronic address:

Efficient insulin secretion requires a well-functioning pancreatic islet microvasculature. The dense network of islet capillaries includes the islet pericyte, a cell that has barely been studied. Here we show that islet pericytes help control local blood flow by adjusting islet capillary diameter. Islet pericytes cover 40% of the microvasculature, are contractile, and are innervated by sympathetic axons. Sympathetic adrenergic input increases pericyte activity and reduces capillary diameter and local blood flow. By contrast, activating beta cells by increasing glucose concentration inhibits pericytes, dilates islet capillaries, and increases local blood flow. These effects on pericytes are mediated by endogenous adenosine, which is likely derived from ATP co-released with insulin. Pericyte coverage of islet capillaries drops drastically in type 2 diabetes, suggesting that, under diabetic conditions, islets lose this mechanism to control their own blood supply. This may lead to inadequate insulin release into the circulation, further deteriorating glycemic control.
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http://dx.doi.org/10.1016/j.cmet.2018.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876933PMC
March 2018

Paracrine Interactions within the Pancreatic Islet Determine the Glycemic Set Point.

Cell Metab 2018 03;27(3):549-558.e4

Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA; The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm 17177, Sweden; Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore; Pancreatic Islet Biology and Diabetes Consortium, Imperial College, London, UK. Electronic address:

Every animal species has a signature blood glucose level or glycemic set point. These set points are different, and the normal glycemic levels (normoglycemia) of one species would be life threatening for other species. Mouse normoglycemia can be considered diabetic for humans. The biological determinants of the glycemic set point remain unclear. Here we show that the pancreatic islet imposes its glycemic set point on the organism, making it the bona fide glucostat in the body. Moreover, and in contrast to rodent islets, glucagon input from the alpha cell to the insulin-secreting beta cell is necessary to fine-tune the distinctive human set point. These findings affect transplantation and regenerative approaches to treat diabetes because restoring normoglycemia may require more than replacing only the beta cells. Furthermore, therapeutic strategies using glucagon receptor antagonists as hypoglycemic agents need to be reassessed, as they may reset the overall glucostat in the organism.
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http://dx.doi.org/10.1016/j.cmet.2018.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5872154PMC
March 2018

Mouse pancreatic islet macrophages use locally released ATP to monitor beta cell activity.

Diabetologia 2018 Jan 7;61(1):182-192. Epub 2017 Sep 7.

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, 1580 NW 10th Ave, Miami, FL, 33136, USA.

Aims/hypothesis: Tissue-resident macrophages sense the microenvironment and respond by producing signals that act locally to maintain a stable tissue state. It is now known that pancreatic islets contain their own unique resident macrophages, which have been shown to promote proliferation of the insulin-secreting beta cell. However, it is unclear how beta cells communicate with islet-resident macrophages. Here we hypothesised that islet macrophages sense changes in islet activity by detecting signals derived from beta cells.

Methods: To investigate how islet-resident macrophages respond to cues from the microenvironment, we generated mice expressing a genetically encoded Ca indicator in myeloid cells. We produced living pancreatic slices from these mice and used them to monitor macrophage responses to stimulation of acinar, neural and endocrine cells.

Results: Islet-resident macrophages expressed functional purinergic receptors, making them exquisite sensors of interstitial ATP levels. Indeed, islet-resident macrophages responded selectively to ATP released locally from beta cells that were physiologically activated with high levels of glucose. Because ATP is co-released with insulin and is exclusively secreted by beta cells, the activation of purinergic receptors on resident macrophages facilitates their awareness of beta cell secretory activity.

Conclusions/interpretation: Our results indicate that islet macrophages detect ATP as a proxy signal for the activation state of beta cells. Sensing beta cell activity may allow macrophages to adjust the secretion of factors to promote a stable islet composition and size.
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http://dx.doi.org/10.1007/s00125-017-4416-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868749PMC
January 2018

Pancreatic Islet Blood Flow Dynamics in Primates.

Cell Rep 2017 08;20(6):1490-1501

Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 637553, Singapore; Translational Pre-Clinical Model Platform, Singapore Eye Research Institute (SERI), Singapore General Hospital, Singapore 168751, Singapore; The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital L1, 171 76 Stockholm, Sweden; Imperial College London, London SW7 2AZ, UK; Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA. Electronic address:

Blood flow regulation in pancreatic islets is critical for function but poorly understood. Here, we establish an in vivo imaging platform in a non-human primate where islets transplanted autologously into the anterior chamber of the eye are monitored non-invasively and longitudinally at single-cell resolution. Engrafted islets were vascularized and innervated and maintained the cytoarchitecture of in situ islets in the pancreas. Blood flow velocity in the engrafted islets was not affected by increasing blood glucose levels and/or the GLP-1R agonist liraglutide. However, islet blood flow was dynamic in nature and fluctuated in various capillaries. This was associated with vasoconstriction events resembling a sphincter-like action, most likely regulated by adrenergic signaling. These observations suggest a mechanism in primate islets that diverts blood flow to cell regions with higher metabolic demand. The described imaging technology applied in non-human primate islets may contribute to a better understanding of human islet pathophysiology.
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http://dx.doi.org/10.1016/j.celrep.2017.07.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575201PMC
August 2017

Resealable, optically accessible, PDMS-free fluidic platform for ex vivo interrogation of pancreatic islets.

Lab Chip 2017 02;17(5):772-781

Department of Biomedical Engineering, Department of Pathology & Laboratory Medicine, University of Miami, Miami, FL 33136, USA. and Diabetes Research Institute, University of Miami, USA.

We report the design and fabrication of a robust fluidic platform built out of inert plastic materials and micromachined features that promote optimized convective fluid transport. The platform is tested for perfusion interrogation of rodent and human pancreatic islets, dynamic secretion of hormones, concomitant live-cell imaging, and optogenetic stimulation of genetically engineered islets. A coupled quantitative fluid dynamics computational model of glucose stimulated insulin secretion and fluid dynamics was first utilized to design device geometries that are optimal for complete perfusion of three-dimensional islets, effective collection of secreted insulin, and minimization of system volumes and associated delays. Fluidic devices were then fabricated through rapid prototyping techniques, such as micromilling and laser engraving, as two interlocking parts from materials that are non-absorbent and inert. Finally, the assembly was tested for performance using both rodent and human islets with multiple assays conducted in parallel, such as dynamic perfusion, staining and optogenetics on standard microscopes, as well as for integration with commercial perfusion machines. The optimized design of convective fluid flows, use of bio-inert and non-absorbent materials, reversible assembly, manual access for loading and unloading of islets, and straightforward integration with commercial imaging and fluid handling systems proved to be critical for perfusion assay, and particularly suited for time-resolved optogenetics studies.
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http://dx.doi.org/10.1039/c6lc01504bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330806PMC
February 2017

The Different Faces of the Pancreatic Islet.

Adv Exp Med Biol 2016;938:11-24

Diabetes Research Institute/Department of Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA.

Type 1 diabetes (T1D) patients who receive pancreatic islet transplant experience significant improvement in their quality-of-life. This comes primarily through improved control of blood sugar levels, restored awareness of hypoglycemia, and prevention of serious and potentially life-threatening diabetes-associated complications, such as kidney failure, heart and vascular disease, stroke, nerve damage, and blindness. Therefore, beta cell replacement through transplantation of isolated islets is an important option in the treatment of T1D. However, lasting success of this promising therapy depends on durable survival and efficacy of the transplanted islets, which are directly influenced by the islet isolation procedures. Thus, isolating pancreatic islets with consistent and reliable quality is critical in the clinical application of islet transplantation.Quality of isolated islets is important in pre-clinical studies as well, as efforts to advance and improve clinical outcomes of islet transplant therapy have relied heavily on animal models ranging from rodents, to pigs, to nonhuman primates. As a result, pancreatic islets have been isolated from these and other species and used in a variety of in vitro or in vivo applications for this and other research purposes. Protocols for islet isolation have been somewhat similar across species, especially, in mammals. However, given the increasing evidence about the distinct structural and functional features of human and mouse islets, using similar methods of islet isolation may contribute to inconsistencies in the islet quality, immunogenicity, and experimental outcomes. This may also contribute to the discrepancies commonly observed between pre-clinical findings and clinical outcomes. Therefore, it is prudent to consider the particular features of pancreatic islets from different species when optimizing islet isolation protocols.In this chapter, we explore the structural and functional features of pancreatic islets from mice, pigs, nonhuman primates, and humans because of their prevalent use in nonclinical, preclinical, and clinical applications.
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http://dx.doi.org/10.1007/978-3-319-39824-2_2DOI Listing
June 2017

Liraglutide Compromises Pancreatic β Cell Function in a Humanized Mouse Model.

Cell Metab 2016 Mar 11;23(3):541-6. Epub 2016 Feb 11.

Diabetes Research Institute, University of Miami Miller School of Medicine, 1450 NW 10th Avenue, Miami, FL 33136, USA; The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital L1, Stockholm SE-17176, Sweden. Electronic address:

Incretin mimetics are frequently used in the treatment of type 2 diabetes because they potentiate β cell response to glucose. Clinical evidence showing short-term benefits of such therapeutics (e.g., liraglutide) is abundant; however, there have been several recent reports of unexpected complications in association with incretin mimetic therapy. Importantly, clinical evidence on the potential effects of such agents on the β cell and islet function during long-term, multiyear use remains lacking. We now show that prolonged daily liraglutide treatment of >200 days in humanized mice, transplanted with human pancreatic islets in the anterior chamber of the eye, is associated with compromised release of human insulin and deranged overall glucose homeostasis. These findings raise concern about the chronic potentiation of β cell function through incretin mimetic therapy in diabetes.
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http://dx.doi.org/10.1016/j.cmet.2016.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785083PMC
March 2016

Neural control of the endocrine pancreas.

Best Pract Res Clin Endocrinol Metab 2014 Oct 20;28(5):745-56. Epub 2014 May 20.

Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA; Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA; Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, FL 33136, USA; Program in Neuroscience, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. Electronic address:

The autonomic nervous system affects glucose metabolism partly through its connection to the pancreatic islet. Since its discovery by Paul Langerhans, the precise innervation patterns of the islet has remained elusive, mainly because of technical limitations. Using 3-dimensional reconstructions of axonal terminal fields, recent studies have determined the innervation patterns of mouse and human islets. In contrast to the mouse islet, endocrine cells within the human islet are sparsely contacted by autonomic axons. Instead, the invading sympathetic axons preferentially innervate smooth muscle cells of blood vessels. This innervation pattern suggests that, rather than acting directly on endocrine cells, sympathetic nerves may control hormone secretion by modulating blood flow in human islets. In addition to autonomic efferent axons, islets also receive sensory innervation. These axons transmit sensory information to the brain but also have the ability to locally release neuroactive substances that have been suggested to promote diabetes pathogenesis. We discuss recent findings on islet innervation, the connections of the islet with the brain, and the role islet innervation plays during the progression of diabetes.
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http://dx.doi.org/10.1016/j.beem.2014.05.002DOI Listing
October 2014

Control of insulin secretion by cholinergic signaling in the human pancreatic islet.

Diabetes 2014 Aug 21;63(8):2714-26. Epub 2014 Mar 21.

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FLDiabetes Research Institute, University of Miami Miller School of Medicine, Miami, FLDepartment of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, FLProgram in Neuroscience, Miller School of Medicine, University of Miami, Miami, FL

Acetylcholine regulates hormone secretion from the pancreatic islet and is thus crucial for glucose homeostasis. Little is known, however, about acetylcholine (cholinergic) signaling in the human islet. We recently reported that in the human islet, acetylcholine is primarily a paracrine signal released from α-cells rather than primarily a neural signal as in rodent islets. In this study, we demonstrate that the effects acetylcholine produces in the human islet are different and more complex than expected from studies conducted on cell lines and rodent islets. We found that endogenous acetylcholine not only stimulates the insulin-secreting β-cell via the muscarinic acetylcholine receptors M3 and M5, but also the somatostatin-secreting δ-cell via M1 receptors. Because somatostatin is a strong inhibitor of insulin secretion, we hypothesized that cholinergic input to the δ-cell indirectly regulates β-cell function. Indeed, when all muscarinic signaling was blocked, somatostatin secretion decreased and insulin secretion unexpectedly increased, suggesting a reduced inhibitory input to β-cells. Endogenous cholinergic signaling therefore provides direct stimulatory and indirect inhibitory input to β-cells to regulate insulin secretion from the human islet.
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http://dx.doi.org/10.2337/db13-1371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113066PMC
August 2014

Neurotransmitters act as paracrine signals to regulate insulin secretion from the human pancreatic islet.

J Physiol 2014 Aug 3;592(16):3413-7. Epub 2014 Mar 3.

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, 33136, USA

In this symposium review we discuss the role of neurotransmitters as paracrine signals that regulate pancreatic islet function. A large number of neurotransmitters and their receptors has been identified in the islet, but relatively little is known about their involvement in islet biology. Interestingly, neurotransmitters initially thought to be present in autonomic axons innervating the islet are also present in endocrine cells of the human islet. These neurotransmitters can thus be released as paracrine signals to help control hormone release. Here we propose that the role of neurotransmitters may extend beyond controlling endocrine cell function to work as signals modulating vascular flow and immune responses within the islet.
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http://dx.doi.org/10.1113/jphysiol.2013.269910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4229339PMC
August 2014

Novel approaches to studying the role of innervation in the biology of pancreatic islets.

Endocrinol Metab Clin North Am 2013 Mar 20;42(1):39-56. Epub 2012 Dec 20.

Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.

The autonomic nervous system helps regulate glucose homeostasis by acting on pancreatic islets of Langerhans. Despite decades of research on the innervation of the pancreatic islet, the mechanisms used by the autonomic nervous input to influence islet cell biology have not been elucidated. This article discusses how these barriers can be overcome to study the role of the autonomic innervation of the pancreatic islet in glucose metabolism. It describes recent advances in microscopy and novel approaches to studying the effects of nervous input that may help clarify how autonomic axons regulate islet biology.
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http://dx.doi.org/10.1016/j.ecl.2012.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3576136PMC
March 2013

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

Proc Natl Acad Sci U S A 2012 Dec 10;109(52):21456-61. Epub 2012 Dec 10.

Diabetes Research Institute, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

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.
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http://dx.doi.org/10.1073/pnas.1211659110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535593PMC
December 2012

Real-time detection of acetylcholine release from the human endocrine pancreas.

Nat Protoc 2012 May 3;7(6):1015-23. Epub 2012 May 3.

Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, Florida, USA.

Neurons, sensory cells and endocrine cells secrete neurotransmitters and hormones to communicate with other cells and to coordinate organ and system function. Validation that a substance is used as an extracellular signaling molecule by a given cell requires a direct demonstration of its secretion. In this protocol we describe the use of biosensor cells to detect neurotransmitter release from endocrine cells in real-time. Chinese hamster ovary cells expressing the muscarinic acetylcholine (ACh) receptor M3 were used as ACh biosensors to record ACh release from human pancreatic islets. We show how ACh biosensors loaded with the Ca(2+) indicator Fura-2 and pressed against isolated human pancreatic islets allow the detection of ACh release. The biosensor approach is simple; the Ca(2+) signal generated in the biosensor cell reflects the presence (release) of a neurotransmitter. The technique is versatile because biosensor cells expressing a variety of receptors can be used in many applications. The protocol takes ∼3 h.
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http://dx.doi.org/10.1038/nprot.2012.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538842PMC
May 2012

Donor islet endothelial cells in pancreatic islet revascularization.

Diabetes 2011 Oct 26;60(10):2571-7. Epub 2011 Aug 26.

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden.

Objective: Freshly isolated pancreatic islets contain, in contrast to cultured islets, intraislet endothelial cells (ECs), which can contribute to the formation of functional blood vessels after transplantation. We have characterized how donor islet endothelial cells (DIECs) may contribute to the revascularization rate, vascular density, and endocrine graft function after transplantation of freshly isolated and cultured islets.

Research Design And Methods: Freshly isolated and cultured islets were transplanted under the kidney capsule and into the anterior chamber of the eye. Intravital laser scanning microscopy was used to monitor the revascularization process and DIECs in intact grafts. The grafts' metabolic function was examined by reversal of diabetes, and the ultrastructural morphology by transmission electron microscopy.

Results: DIECs significantly contributed to the vasculature of fresh islet grafts, assessed up to 5 months after transplantation, but were hardly detected in cultured islet grafts. Early participation of DIECs in the revascularization process correlated with a higher revascularization rate of freshly isolated islets compared with cultured islets. However, after complete revascularization, the vascular density was similar in the two groups, and host ECs gained morphological features resembling the endogenous islet vasculature. Surprisingly, grafts originating from cultured islets reversed diabetes more rapidly than those originating from fresh islets.

Conclusions: In summary, DIECs contributed to the revascularization of fresh, but not cultured, islets by participating in early processes of vessel formation and persisting in the vasculature over long periods of time. However, the DIECs did not increase the vascular density or improve the endocrine function of the grafts.
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http://dx.doi.org/10.2337/db10-1711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178280PMC
October 2011

High-resolution, noninvasive longitudinal live imaging of immune responses.

Proc Natl Acad Sci U S A 2011 Aug 18;108(31):12863-8. Epub 2011 Jul 18.

Diabetes Research Institute, Department of Biomedical Engineering, University of Miami, Miami, FL 33136, USA.

Intravital imaging emerged as an indispensible tool in biological research, and a variety of imaging techniques have been developed to noninvasively monitor tissues in vivo. However, most of the current techniques lack the resolution to study events at the single-cell level. Although intravital multiphoton microscopy has addressed this limitation, the need for repeated noninvasive access to the same tissue in longitudinal in vivo studies remains largely unmet. We now report on a previously unexplored approach to study immune responses after transplantation of pancreatic islets into the anterior chamber of the mouse eye. This approach enabled (i) longitudinal, noninvasive imaging of transplanted tissues in vivo; (ii) in vivo cytolabeling to assess cellular phenotype and viability in situ; (iii) local intervention by topical application or intraocular injection; and (iv) real-time tracking of infiltrating immune cells in the target tissue.
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http://dx.doi.org/10.1073/pnas.1105002108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150878PMC
August 2011

Innervation patterns of autonomic axons in the human endocrine pancreas.

Cell Metab 2011 Jul;14(1):45-54

Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.

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.
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http://dx.doi.org/10.1016/j.cmet.2011.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135265PMC
July 2011

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

Nat Med 2011 Jun 19;17(7):888-92. Epub 2011 Jun 19.

Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, Florida, USA.

Acetylcholine is a neurotransmitter that has a major role in the function of the insulin-secreting pancreatic beta cell. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to the case in mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of human islets 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. Cholinergic signaling within islets represents a potential therapeutic target in diabetes, highlighting the relevance of this advance to future drug development.
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http://dx.doi.org/10.1038/nm.2371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3132226PMC
June 2011

ATP-gated P2X3 receptors constitute a positive autocrine signal for insulin release in the human pancreatic beta cell.

Proc Natl Acad Sci U S A 2010 Apr 22;107(14):6465-70. Epub 2010 Mar 22.

Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.

Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human beta cell's sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i), and hormone release in vitro, we show that human beta cells express ionotropic ATP receptors of the P2X(3) type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X(3) receptors in the beta-cell plasma membrane, resulting in increased [Ca(2+)](i) and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the beta cell's secretory machinery. This may explain how the human pancreatic beta cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo.
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http://dx.doi.org/10.1073/pnas.0908935107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851966PMC
April 2010

Characterization of pancreatic ductal cells in human islet preparations.

Lab Invest 2008 Nov 8;88(11):1167-77. Epub 2008 Sep 8.

Cell Transplant Center, Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL 33136, USA.

Substantial amounts of nonendocrine cells are implanted as part of human islet grafts, and a possible influence of nonendocrine cells on clinical islet transplantation outcome has been postulated. There are currently no product release criteria specific for nonendocrine cells due to lack of available methods. The aims of this study were to develop a method for the evaluation of pancreatic ductal cells (PDCs) for clinical islet transplantation and to characterize them regarding phenotype, viability, and function. We assessed 161 human islet preparations using laser scanning cytometry (LSC/iCys) for phenotypic analysis of nonendocrine cells and flow cytometry (FACS) for PDC viability. PDC and beta-cells obtained from different density fractions during the islet cell purification were compared in terms of viability. Furthermore, we examined PDC ability to produce proinflammatory cytokines/chemokines, vascular endothelial growth factor (VEGF) and tissue factor (TF) relevant to islet graft outcome. Phenotypic analysis by LSC/iCys indicated that single staining for CK19 or CA19-9 was not enough for identifying PDCs, and that double staining for amylase and CK19 or CA19-9 allowed for quantitative evaluation of acinar cells and PDC content in human islet preparation. PDC showed a significantly higher viability than beta-cells (PDC vs beta-cell: 75.5+/-13.9 and 62.7+/-18.7%; P<0.0001). Although beta-cell viability was independent of its density, that of PDCs was higher as the density from which they were recovered increased. There was no correlation between PDCs and beta-cell viability (R(2)=0.0078). PDCs sorted from high-density fractions produced significantly higher amounts of proinflammatory mediators and VEGF, but not TF. We conclude that PDCs isolated from different fractions had different viability and functions. The precise characterization and assessment of these cells in addition to beta-cells in human islet cell products may be of assistance in understanding their contribution to islet engraftment and in developing strategies to enhance islet graft function.
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http://dx.doi.org/10.1038/labinvest.2008.87DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3793849PMC
November 2008
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