Publications by authors named "Alejandro Caicedo"

63 Publications

Targeting the Pancreatic α-Cell to Prevent Hypoglycemia in Type 1 Diabetes.

Diabetes 2021 12;70(12):2721-2732

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

Life-threatening hypoglycemia is a limiting factor in the management of type 1 diabetes. People with diabetes are prone to develop hypoglycemia because they lose physiological mechanisms that prevent plasma glucose levels from falling. Among these so-called counterregulatory responses, secretion of glucagon from pancreatic α-cells is preeminent. Glucagon, a hormone secreted in response to a lowering in glucose concentration, counteracts a further drop in glycemia by promoting gluconeogenesis and glycogenolysis in target tissues. In diabetes, however, α-cells do not respond appropriately to changes in glycemia and, thus, cannot mount a counterregulatory response. If the α-cell could be targeted therapeutically to restore its ability to prevent hypoglycemia, type 1 diabetes could be managed more efficiently and safely. Unfortunately, the mechanisms that allow the α-cell to respond to hypoglycemia have not been fully elucidated. We know even less about the pathophysiological mechanisms that cause α-cell dysfunction in diabetes. Based on published findings and unpublished observations, and taking into account its electrophysiological properties, we propose here a model of α-cell function that could explain its impairment in diabetes. Within this frame, we emphasize those elements that could be targeted pharmacologically with repurposed U.S. Food and Drug Administration-approved drugs to rescue α-cell function and restore glucose counterregulation in people with diabetes.
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http://dx.doi.org/10.2337/dbi20-0048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8660986PMC
December 2021

Optical Imaging of Pancreatic Innervation.

Front Endocrinol (Lausanne) 2021 27;12:663022. Epub 2021 Apr 27.

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

At the time of Ivan Pavlov, pancreatic innervation was studied by looking at pancreas secretions in response to electrical stimulation of nerves. Nowadays we have ways to visualize neuronal activity in real time thanks to advances in fluorescent reporters and imaging techniques. We also have very precise optogenetic and pharmacogenetic approaches that allow neuronal manipulations in a very specific manner. These technological advances have been extensively employed for studying the central nervous system and are just beginning to be incorporated for studying visceral innervation. Pancreatic innervation is complex, and the role it plays in physiology and pathophysiology of the organ is still not fully understood. In this review we highlight anatomical aspects of pancreatic innervation, techniques for pancreatic neuronal labeling, and approaches for imaging pancreatic innervation and .
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http://dx.doi.org/10.3389/fendo.2021.663022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8112238PMC
December 2021

Deciphering the Complex Communication Networks That Orchestrate Pancreatic Islet Function.

Diabetes 2021 01;70(1):17-26

Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL

Pancreatic islets are clusters of hormone-secreting endocrine cells that rely on intricate cell-cell communication mechanisms for proper function. The importance of multicellular cooperation in islet cell physiology was first noted nearly 30 years ago in seminal studies showing that hormone secretion from endocrine cell types is diminished when these cells are dispersed. These studies showed that reestablishing cellular contacts in so-called pseudoislets caused endocrine cells to regain hormone secretory function. This not only demonstrated that cooperation between islet cells is highly synergistic but also gave birth to the field of pancreatic islet organoids. Here we review recent advances related to the mechanisms of islet cell cross talk. We first describe new developments that revise current notions about purinergic and GABA signaling in islets. Then we comment on novel multicellular imaging studies that are revealing emergent properties of islet communication networks. We finish by highlighting and discussing recent synthetic approaches that use islet organoids of varied cellular composition to interrogate intraislet signaling mechanisms. This reverse engineering of islets not only will shed light on the mechanisms of intraislet signaling and define communication networks but also may guide efforts aimed at restoring islet function and β-cell mass in diabetes.
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http://dx.doi.org/10.2337/dbi19-0033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881851PMC
January 2021

Glucagon Resistance and Decreased Susceptibility to Diabetes in a Model of Chronic Hyperglucagonemia.

Diabetes 2021 02 25;70(2):477-491. Epub 2020 Nov 25.

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

Elevation of glucagon levels and increase in α-cell mass are associated with states of hyperglycemia in diabetes. Our previous studies have highlighted the role of nutrient signaling via mTOR complex 1 (mTORC1) regulation that controls glucagon secretion and α-cell mass. In the current studies we investigated the effects of activation of nutrient signaling by conditional deletion of the mTORC1 inhibitor, TSC2, in α-cells (αTSC2). We showed that activation of mTORC1 signaling is sufficient to induce chronic hyperglucagonemia as a result of α-cell proliferation, cell size, and mass expansion. Hyperglucagonemia in αTSC2 was associated with an increase in glucagon content and enhanced glucagon secretion. This model allowed us to identify the effects of chronic hyperglucagonemia on glucose homeostasis by inducing insulin secretion and resistance to glucagon in the liver. Liver glucagon resistance in αTSC2 mice was characterized by reduced expression of the glucagon receptor (GCGR), PEPCK, and genes involved in amino acid metabolism and urea production. Glucagon resistance in αTSC2 mice was associated with improved glucose levels in streptozotocin-induced β-cell destruction and high-fat diet-induced glucose intolerance. These studies demonstrate that chronic hyperglucagonemia can improve glucose homeostasis by inducing glucagon resistance in the liver.
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http://dx.doi.org/10.2337/db20-0440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881862PMC
February 2021

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

Beta cell dysfunction in diabetes: the islet microenvironment as an unusual suspect.

Diabetologia 2020 10 7;63(10):2076-2085. Epub 2020 Sep 7.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, 69978, Israel.

Cells in different tissues, including endocrine cells in the pancreas, live in complex microenvironments that are rich in cellular and acellular components. Intricate interactions with their microenvironment dictate most cellular properties, such as their function, structure and size, and maintain tissue homeostasis. Pancreatic islets are populated by endocrine, vascular and immune cells that are immersed in the extracellular matrix. While the intrinsic properties of beta cells have been vastly investigated, our understanding of their interactions with their surroundings has only recently begun to unveil. Here, we review current research on the interplay between the islet cellular and acellular components, and the role these components play in beta cell physiology and pathophysiology. Although beta cell failure is a key pathomechanism in diabetes, its causes are far from being fully elucidated. We, thus, propose deleterious alterations of the islet niche as potential underlying mechanisms contributing to beta cell failure. In sum, this review emphasises that the function of the pancreatic islet depends on all of its components. Graphical abstract.
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http://dx.doi.org/10.1007/s00125-020-05186-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655222PMC
October 2020

Long-term culture of human pancreatic slices as a model to study real-time islet regeneration.

Nat Commun 2020 06 29;11(1):3265. Epub 2020 Jun 29.

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

The culture of live pancreatic tissue slices is a powerful tool for the interrogation of physiology and pathology in an in vitro setting that retains near-intact cytoarchitecture. However, current culture conditions for human pancreatic slices (HPSs) have only been tested for short-term applications, which are not permissive for the long-term, longitudinal study of pancreatic endocrine regeneration. Using a culture system designed to mimic the physiological oxygenation of the pancreas, we demonstrate high viability and preserved endocrine and exocrine function in HPS for at least 10 days after sectioning. This extended lifespan allowed us to dynamically lineage trace and quantify the formation of insulin-producing cells in HPS from both non-diabetic and type 2 diabetic donors. This technology is expected to be of great impact for the conduct of real-time regeneration/developmental studies in the human pancreas.
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http://dx.doi.org/10.1038/s41467-020-17040-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324563PMC
June 2020

Blood Flow in the Pancreatic Islet: Not so Isolated Anymore.

Diabetes 2020 07;69(7):1336-1338

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

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http://dx.doi.org/10.2337/dbi20-0016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306118PMC
July 2020

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

Pancreas tissue slices from organ donors enable in situ analysis of type 1 diabetes pathogenesis.

JCI Insight 2020 04 23;5(8). Epub 2020 Apr 23.

Paul Langerhans Institute Dresden of the Helmholtz Zentrum München at the University Clinic Carl Gustav Carus of Technische Universität Dresden, Helmholtz Zentrum München, Neuherberg, Germany.

In type 1 diabetes (T1D), autoimmune destruction of pancreatic β cells leads to insulin deficiency and loss of glycemic control. However, knowledge about human pancreas pathophysiology in T1D remains incomplete. To address this limitation, we established a pancreas tissue slice platform of donor organs with and without diabetes, facilitating the first live cell studies of human pancreas in T1D pathogenesis to our knowledge. We show that pancreas tissue slices from organ donors allow thorough assessment of processes critical for disease development, including insulin secretion, β cell physiology, endocrine cell morphology, and immune infiltration within the same donor organ. Using this approach, we compared detailed pathophysiological profiles for 4 pancreata from donors with T1D with 19 nondiabetic control donors. We demonstrate that β cell loss, β cell dysfunction, alterations of β cell physiology, and islet infiltration contributed differently to individual cases of T1D, allowing insight into pathophysiology and heterogeneity of T1D pathogenesis. Thus, our study demonstrates that organ donor pancreas tissue slices represent a promising and potentially novel approach in the search for successful prevention and reversal strategies of T1D.
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http://dx.doi.org/10.1172/jci.insight.134525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205437PMC
April 2020

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

Angiotensin-Receptor-Associated Protein Modulates Ca Signals in Photoreceptor and Mossy Fiber cells.

Sci Rep 2019 12 23;9(1):19622. Epub 2019 Dec 23.

Experimental Ophthalmology, Eye Hospital, University Medical Center Regensburg, 93053, Regensburg, Germany.

Fast, precise and sustained neurotransmission requires graded Ca signals at the presynaptic terminal. Neurotransmitter release depends on a complex interplay of Ca fluxes and Ca buffering in the presynaptic terminal that is not fully understood. Here, we show that the angiotensin-receptor-associated protein (ATRAP) localizes to synaptic terminals throughout the central nervous system. In the retinal photoreceptor synapse and the cerebellar mossy fiber-granule cell synapse, we find that ATRAP is involved in the generation of depolarization-evoked synaptic Ca transients. Compared to wild type, Ca imaging in acutely isolated preparations of the retina and the cerebellum from ATRAP knockout mice reveals a significant reduction of the sarcoendoplasmic reticulum (SR) Ca-ATPase (SERCA) activity. Thus, in addition to its conventional role in angiotensin signaling, ATRAP also modulates presynaptic Ca signaling within the central nervous system.
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http://dx.doi.org/10.1038/s41598-019-55380-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928155PMC
December 2019

Correction to: In vivo imaging of type 1 diabetes immunopathology using eye-transplanted islets in NOD mice.

Diabetologia 2019 Aug;62(8):1517

Diabetes Research Institute and Cell Transplant Center, University of Miami Miller School of Medicine, 1450 NW 10th Ave, Miami, FL, 33136, USA.

Unfortunately there is a mistake in the presentation of the affiliations in this paper.
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http://dx.doi.org/10.1007/s00125-019-4922-1DOI Listing
August 2019

In vivo imaging of type 1 diabetes immunopathology using eye-transplanted islets in NOD mice.

Diabetologia 2019 07 14;62(7):1237-1250. Epub 2019 May 14.

Diabetes Research Institute and Cell Transplant Center, University of Miami Miller School of Medicine, 1450 NW 10th Ave, Miami, FL, 33136, USA.

Aims/hypothesis: Autoimmune attack against the insulin-producing beta cells in the pancreatic islets results in type 1 diabetes. However, despite considerable research, details of the type 1 diabetes immunopathology in situ are not fully understood mainly because of difficult access to the pancreatic islets in vivo.

Methods: Here, we used direct non-invasive confocal imaging of islets transplanted in the anterior chamber of the eye (ACE) to investigate the anti-islet autoimmunity in NOD mice before, during and after diabetes onset. ACE-transplanted islets allowed longitudinal studies of the autoimmune attack against islets and revealed the infiltration kinetics and in situ motility dynamics of fluorescence-labelled autoreactive T cells during diabetes development. Ex vivo immunostaining was also used to compare immune cell infiltrations into islet grafts in the eye and kidney as well as in pancreatic islets of the same diabetic NOD mice.

Results: We found similar immune infiltration in native pancreatic and ACE-transplanted islets, which established the ACE-transplanted islets as reliable reporters of the autoimmune response. Longitudinal studies in ACE-transplanted islets identified in vivo hallmarks of islet inflammation that concurred with early immune infiltration of the islets and preceded their collapse and hyperglycaemia onset. A model incorporating data on ACE-transplanted islet degranulation and swelling allowed early prediction of the autoimmune attack in the pancreas and prompted treatments to intercept type 1 diabetes.

Conclusions/interpretation: The current findings highlight the value of ACE-transplanted islets in studying early type 1 diabetes pathogenesis in vivo and underscore the need for timely intervention to halt disease progression.
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http://dx.doi.org/10.1007/s00125-019-4879-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561836PMC
July 2019

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy.

J Vis Exp 2018 10 2(140). Epub 2018 Oct 2.

Department of Medicine, University of Miami.

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes´ dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.
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http://dx.doi.org/10.3791/58236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235380PMC
October 2018

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

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets.

J Vis Exp 2017 Sep 13(127). Epub 2017 Sep 13.

Department of Medicine, University of Miami;

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 µm of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).
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http://dx.doi.org/10.3791/56089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695231PMC
September 2017

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

Regulator of G-protein signaling Gβ5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion.

FASEB J 2017 11 7;31(11):4734-4744. Epub 2017 Jul 7.

Department of Molecular and Cellular Pharmacology, Leonard M. Miller School of Medicine, University of Miami, Miami, Florida, USA; and

In pancreatic β cells, muscarinic cholinergic receptor M3 (M3R) stimulates glucose-induced secretion of insulin. Regulator of G-protein signaling (RGS) proteins are critical modulators of GPCR activity, yet their role in β cells remains largely unknown. R7 subfamily RGS proteins are stabilized by the G-protein subunit Gβ5, such that the knockout of the gene results in degradation of all R7 subunits. We found that knockout in mice or in the insulin-secreting MIN6 cell line almost completely eliminates insulinotropic activity of M3R. Moreover, overexpression of Gβ5-RGS7 strongly promotes M3R-stimulated insulin secretion. Examination of this noncanonical mechanism in MIN6 cells showed that cAMP, diacylglycerol, or Ca levels were not significantly affected. There was no reduction in the amplitude of free Ca responses in islets from the mice, but the frequency of Ca oscillations induced by cholinergic agonist was lowered by more than 30%. Ablation of impaired M3R-stimulated phosphorylation of ERK1/2. Stimulation of the ERK pathway in cells by epidermal growth factor restored M3R-stimulated insulin release to near normal levels. Identification of the novel role of Gβ5-R7 in insulin secretion may lead to a new therapeutic approach for improving pancreatic β-cell function.-Wang, Q., Pronin, A. N., Levay, K., Almaca, J., Fornoni, A., Caicedo, A., Slepak, V. Z. Regulator of G-protein signaling Gβ5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion.
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http://dx.doi.org/10.1096/fj.201700197RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5636707PMC
November 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

β-arrestin-2 is an essential regulator of pancreatic β-cell function under physiological and pathophysiological conditions.

Nat Commun 2017 02 1;8:14295. Epub 2017 Feb 1.

Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA.

β-arrestins are critical signalling molecules that regulate many fundamental physiological functions including the maintenance of euglycemia and peripheral insulin sensitivity. Here we show that inactivation of the β-arrestin-2 gene, barr2, in β-cells of adult mice greatly impairs insulin release and glucose tolerance in mice fed with a calorie-rich diet. Both glucose and KCl-induced insulin secretion and calcium responses were profoundly reduced in β-arrestin-2 (barr2) deficient β-cells. In human β-cells, barr2 knockdown abolished glucose-induced insulin secretion. We also show that the presence of barr2 is essential for proper CAMKII function in β-cells. Importantly, overexpression of barr2 in β-cells greatly ameliorates the metabolic deficits displayed by mice consuming a high-fat diet. Thus, our data identify barr2 as an important regulator of β-cell function, which may serve as a new target to improve β-cell function.
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http://dx.doi.org/10.1038/ncomms14295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296650PMC
February 2017

Human Beta Cells Produce and Release Serotonin to Inhibit Glucagon Secretion from Alpha Cells.

Cell Rep 2016 12;17(12):3281-3291

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

In the pancreatic islet, serotonin is an autocrine signal increasing beta cell mass during metabolic challenges such as those associated with pregnancy or high-fat diet. It is still unclear whether serotonin is relevant for regular islet physiology and hormone secretion. Here, we show that human beta cells produce and secrete serotonin when stimulated with increases in glucose concentration. Serotonin secretion from beta cells decreases cyclic AMP (cAMP) levels in neighboring alpha cells via 5-HT receptors and inhibits glucagon secretion. Without serotonergic input, alpha cells lose their ability to regulate glucagon secretion in response to changes in glucose concentration, suggesting that diminished serotonergic control of alpha cells can cause glucose blindness and the uncontrolled glucagon secretion associated with diabetes. Supporting this model, pharmacological activation of 5-HT receptors reduces glucagon secretion and has hypoglycemic effects in diabetic mice. Thus, modulation of serotonin signaling in the islet represents a drug intervention opportunity.
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http://dx.doi.org/10.1016/j.celrep.2016.11.072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217294PMC
December 2016

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

Spatial and temporal coordination of insulin granule exocytosis in intact human pancreatic islets.

Diabetologia 2015 Dec 16;58(12):2810-8. Epub 2015 Sep 16.

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: Insulin secretion is widely studied because it plays a central role in glucose homeostasis and diabetes. Processes from insulin granule fusion in beta cells to in vivo insulin secretion have been elucidated, but data at the cellular level do not fully account for several aspects of the macroscopic secretory pattern. Here we investigated how individual secretory events are coordinated spatially and temporally within intact human islets.

Methods: We used the fluorescent probe neuropeptide Y (NPY)-pHluorin to visualise insulin granule secretion in isolated intact human islets.

Results: We found that individual beta cells respond to increases in glucose concentration by releasing insulin granules in very discrete bursts with periods consistent with in vivo pulsatile insulin secretion. In successive secretory bursts during prolonged exposure to high glucose levels, secretory events progressively localised to preferential release sites, coinciding with the transition to second phase insulin secretion. Granule secretion was very synchronised in neighbouring beta cells, forming discrete regional clusters of activity.

Conclusions/interpretation: These results reveal how individual secretory events are coordinated to produce pulsatile insulin secretion from human islets.
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http://dx.doi.org/10.1007/s00125-015-3747-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6132229PMC
December 2015

Young capillary vessels rejuvenate aged pancreatic islets.

Proc Natl Acad Sci U S A 2014 Dec 17;111(49):17612-7. Epub 2014 Nov 17.

Center for Plant Aging Research, Institute for Basic Science, and Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology, Daegu 711-873, Republic of Korea;

Pancreatic islets secrete hormones that play a key role in regulating blood glucose levels (glycemia). Age-dependent impairment of islet function and concomitant dysregulation of glycemia are major health threats in aged populations. However, the major causes of the age-dependent decline of islet function are still disputed. Here we demonstrate that aging of pancreatic islets in mice and humans is notably associated with inflammation and fibrosis of islet blood vessels but does not affect glucose sensing and the insulin secretory capacity of islet beta cells. Accordingly, when transplanted into the anterior chamber of the eye of young mice with diabetes, islets from old mice are revascularized with healthy blood vessels, show strong islet cell proliferation, and fully restore control of glycemia. Our results indicate that beta cell function does not decline with age and suggest that islet function is threatened by an age-dependent impairment of islet vascular function. Strategies to mitigate age-dependent dysregulation in glycemia should therefore target systemic and/or local inflammation and fibrosis of the aged islet vasculature.
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http://dx.doi.org/10.1073/pnas.1414053111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267392PMC
December 2014
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