Paracrine Signaling by Pancreatic Islet Cilia.

The primary cilium is a sensory and signaling organelle present on most pancreatic islet endocrine cells, where it receives and interprets a wide range of intra-islet chemical cues including hormones, peptides, and neurotransmitters. The ciliary membrane possesses a molecular composition distinct from the plasma membrane, with enrichment of signaling mediators including G protein-coupled receptors (GPCRs), tyrosine kinase family receptors, membrane transporters and others. When activated, these membrane proteins interact with ion channels and adenylyl cyclases to trigger local Ca2+ and cAMP activity and transmit signals to the cell body. Here we review evidence supporting the emerging model in which primary cilia on pancreatic islet cells play a central role in the intra-islet communication network and discuss how changes in cilia-mediated paracrine function in islet cells might lead to diabetes.

Immuno-Scanning Electron Microscopy of Islet Primary Cilia.

The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that contain specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immuno-labeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the 3D ultrastructure of the cilium. Here we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT) 88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immuno-labeling, and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.

Beta cell primary cilia mediate somatostatin responsiveness via SSTR3.

Somatostatin is a paracrine modulator of insulin secretion and beta cell function with pleotropic effects on glucose homeostasis. The mechanism of somatostatin-mediated communication between delta and beta cells is not well-understood, which we address in this study via the ciliary somatostatin receptor 3 (SSTR3). Primary cilia are membrane organelles that act as signaling hubs in islets by virtue of their subcellular location and enrichment in signaling proteins such as G-protein coupled receptors (GPCRs). We show that SSTR3, a ciliary GPCR, mediates somatostatin suppression of insulin secretion in mouse islets. Quantitative analysis of calcium flux using a mouse model of genetically encoded beta cell-specific GCaMP6f calcium reporter shows that somatostatin signaling alters beta cell calcium flux after physiologic glucose stimulation, an effect that depends on endogenous SSTR3 expression and the presence of intact primary cilia on beta cells. Comparative in vitro studies using SSTR isoform antagonists demonstrate a role for SSTR3 in mediating somatostatin regulation of insulin secretion in mouse islets. Our findings support a model in which ciliary SSTR3 mediates a distinct pathway of delta-to-beta cell regulatory crosstalk and may serve as a target for paracrine modulation.

Scanning electron microscopy of human islet cilia.

Human islet primary cilia are vital glucose-regulating organelles whose structure remains uncharacterized. Scanning electron microscopy (SEM) is a useful technique for studying the surface morphology of membrane projections like cilia, but conventional sample preparation does not reveal the submembrane axonemal structure, which holds key implications for ciliary function. To overcome this challenge, we combined SEM with membrane-extraction techniques to examine primary cilia in native human islets. Our data show well-preserved cilia subdomains which demonstrate both expected and unexpected ultrastructural motifs. Morphometric features were quantified when possible, including axonemal length and diameter, microtubule conformations, and chirality. We further describe a ciliary ring, a structure that may be a specialization in human islets. Key findings are correlated with fluorescence microscopy and interpreted in the context of cilia function as a cellular sensor and communications locus in pancreatic islets.

Rediscovering Primary Cilia in Pancreatic Islets.

Primary cilia are microtubule-based sensory and signaling organelles on the surfaces of most eukaryotic cells. Despite their early description by microscopy studies, islet cilia had not been examined in the functional context until recent decades. In pancreatic islets as in other tissues, primary cilia facilitate crucial developmental and signaling pathways in response to extracellular stimuli. Many human developmental and genetic disorders are associated with ciliary dysfunction, some manifesting as obesity and diabetes. Understanding the basis for metabolic diseases in human ciliopathies has been aided by close examination of cilia action in pancreatic islets at cellular and molecular levels. In this article, we review the evidence for ciliary expression on islet cells, known roles of cilia in pancreas development and islet hormone secretion, and summarize metabolic manifestations of human ciliopathy syndromes. We discuss emerging data on primary cilia regulation of islet cell signaling and the structural basis of cilia-mediated cell crosstalk, and offer our interpretation on the role of cilia in glucose homeostasis and human diseases.

Molecular phenotyping of single pancreatic islet leader beta cells by "Flash-Seq".

AIMS: Spatially-organized increases in cytosolic Ca2+ within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including "leaders" which initiate Ca2+ waves. Whether leader cells possess unique molecular features, or localisation, is unknown. MAIN METHODS: High speed confocal Ca2+ imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected "hub" cells. To explore transcriptomic differences between beta cell sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing ("Flash-Seq"). KEY FINDINGS: Distinct Ca2+ wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29 % of islets, though "hub" cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padj < 0.05) between leader and follower cells included genes involved in cilium biogenesis and transcriptional regulation. Providing some support for these findings, ADCY6 immunoreactivity tended to be higher in leader than follower cells, whereas cilia number and length tended to be lower in the former. Finally, leader cells were located significantly closer to delta, but not alpha, cells in Euclidian space than were follower cells. SIGNIFICANCE: The existence of both a discrete transcriptome and unique localisation implies a role for these features in defining the specialized function of leaders. These data also raise the possibility that localised signalling between delta and leader cells contributes to the initiation and propagation of islet Ca2+ waves.

Islet cilia and glucose homeostasis.

Diabetes is a growing pandemic affecting over ten percent of the U.S. population. Individuals with all types of diabetes exhibit glucose dysregulation due to altered function and coordination of pancreatic islets. Within the critical intercellular space in pancreatic islets, the primary cilium emerges as an important physical structure mediating cell-cell crosstalk and signal transduction. Many events leading to hormone secretion, including GPCR and second-messenger signaling, are spatiotemporally regulated at the level of the cilium. In this review, we summarize current knowledge of cilia action in islet hormone regulation and glucose homeostasis, focusing on newly implicated ciliary pathways that regulate insulin exocytosis and intercellular communication. We present evidence of key signaling proteins on islet cilia and discuss ways in which cilia might functionally connect islet endocrine cells with the non-endocrine compartments. These discussions aim to stimulate conversations regarding the extent of cilia-controlled glucose homeostasis in health and in metabolic diseases.

Fluorescence imaging of beta cell primary cilia.

Primary cilia are slender cell-surface organelles that project into the intercellular space. In pancreatic beta cells, primary cilia coordinate a variety of cell responses including GPCR signaling, calcium influx, and insulin secretion, along with likely many underappreciated roles in islet development and differentiation. To study cilia function in islet biology, direct visualization of primary cilia by microscopic methods is often a necessary first step. Ciliary abundance, distribution, and morphology are heterogeneous among islet cells and are best visualized by fluorescence microscopy, the tools for which are readily accessible to most researchers. Here we present a collection of fluorescence imaging methods that we have adopted and optimized for the observation of primary cilia in mouse and human islets. These include conventional confocal microscopy using fixed islets and pancreas sections, live-cell imaging with cilia-targeted biosensors and probes, cilia motion recordings, and quantitative analysis of primary cilia waveform in the ex vivo environment. We discuss practical considerations and limitations of our approaches as well as new tools on the horizon to facilitate the observation of primary cilia in pancreatic islets.

Islet primary cilia motility controls insulin secretion.

Primary cilia are specialized cell-surface organelles that mediate sensory perception and, in contrast to motile cilia and flagella, are thought to lack motility function. Here, we show that primary cilia in human and mouse pancreatic islets exhibit movement that is required for glucose-dependent insulin secretion. Islet primary cilia contain motor proteins conserved from those found in classic motile cilia, and their three-dimensional motion is dynein-driven and dependent on adenosine 5'-triphosphate and glucose metabolism. Inhibition of cilia motion blocks beta cell calcium influx and insulin secretion. Human beta cells have enriched ciliary gene expression, and motile cilia genes are altered in type 2 diabetes. Our findings redefine primary cilia as dynamic structures having both sensory and motile function and establish that pancreatic islet cilia movement plays a regulatory role in insulin secretion.

Cilia Action in Islets: Lessons From Mouse Models.

Primary cilia as a signaling organelle have garnered recent attention as a regulator of pancreatic islet function. These rod-like sensors exist on all major islet endocrine cell types and transduce a variety of external cues, while dysregulation of cilia function contributes to the development of diabetes. The complex role of islet primary cilia has been examined using genetic deletion targeting various components of cilia. In this review, we summarize experimental models for the study of islet cilia and current understanding of mechanisms of cilia regulation of islet hormone secretion. Consensus from these studies shows that pancreatic cilia perturbation can cause both endocrine and exocrine defects that are relevant to human disease. We discuss future research directions that would further elucidate cilia action in distinct groups of islet cells, including paracrine and juxtacrine regulation, GPCR signaling, and endocrine-exocrine crosstalk.

Spontaneous restoration of functional ß-cell mass in obese SM/J mice.

Maintenance of functional ß-cell mass is critical to preventing diabetes, but the physiological mechanisms that cause ß-cell populations to thrive or fail in the context of obesity are unknown. High fat-fed SM/J mice spontaneously transition from hyperglycemic-obese to normoglycemic-obese with age, providing a unique opportunity to study ß-cell adaptation. Here, we characterize insulin homeostasis, islet morphology, and ß-cell function during SM/J's diabetic remission. As they resolve hyperglycemia, obese SM/J mice dramatically increase circulating and pancreatic insulin levels while improving insulin sensitivity. Immunostaining of pancreatic sections reveals that obese SM/J mice selectively increase ß-cell mass but not α-cell mass. Obese SM/J mice do not show elevated ß-cell mitotic index, but rather elevated α-cell mitotic index. Functional assessment of isolated islets reveals that obese SM/J mice increase glucose-stimulated insulin secretion, decrease basal insulin secretion, and increase islet insulin content. These results establish that ß-cell mass expansion and improved ß-cell function underlie the resolution of hyperglycemia, indicating that obese SM/J mice are a valuable tool for exploring how functional ß-cell mass can be recovered in the context of obesity.

Primary cilia control glucose homeostasis via islet paracrine interactions.

Pancreatic islets regulate glucose homeostasis through coordinated actions of hormone-secreting cells. What underlies the function of the islet as a unit is the close approximation and communication among heterogeneous cell populations, but the structural mediators of islet cellular cross talk remain incompletely characterized. We generated mice specifically lacking ß-cell primary cilia, a cellular organelle that has been implicated in regulating insulin secretion, and found that the ß-cell cilia are required for glucose sensing, calcium influx, insulin secretion, and cross regulation of α- and δ-cells. Protein expression profiling in islets confirms perturbation in these cellular processes and reveals additional targets of cilia-dependent signaling. At the organism level, the deletion of ß-cell cilia disrupts circulating hormone levels, impairs glucose homeostasis and fuel usage, and leads to the development of diabetes. Together, these findings demonstrate that primary cilia not only orchestrate ß-cell-intrinsic activity but also mediate cross talk both within the islet and from islets to other metabolic tissues, thus providing a unique role of cilia in nutrient metabolism and insight into the pathophysiology of diabetes.

Late-Onset T1DM and Older Age Predict Risk of Additional Autoimmune Disease.

OBJECTIVE: Type 1 diabetes (T1DM) is associated with other autoimmune diseases (AIDs), which may have serious health consequences. The epidemiology of AIDs in T1DM is not well defined in adults with T1DM. In this cross-sectional cohort study, we sought to characterize the incident ages and prevalence of AIDs in adults with T1DM across a wide age spectrum. RESEARCH DESIGN AND METHODS: A total of 1,212 adults seen at the Washington University Diabetes Center from 2011 to 2018 provided informed consent for the collection of their age, sex, race, and disease onset data. We performed paired association analyses based on age at onset of T1DM. Multivariate logistic regression was used to evaluate the independent effects of sex, race, T1DM age of onset, and T1DM duration on the prevalence of an additional AID. RESULTS: Mean ± SD age of T1DM onset was 21.2 ± 14.4 years. AID incidence and prevalence increased with age. Female sex strongly predicted AID risk. The most prevalent T1DM-associated AIDs were thyroid disease, collagen vascular diseases, and pernicious anemia. T1DM age of onset and T1DM duration predicted AID risk. Patients with late-onset T1DM after 30 years of age had higher risks of developing additional AIDs compared with patients with younger T1DM onset. CONCLUSIONS: The prevalence of AIDs in patients with T1DM increases with age and female sex. Later onset of T1DM is an independent and significant risk factor for developing additional AIDs. Individuals who are diagnosed with T1DM at older ages, particularly women, should be monitored for other autoimmune conditions.

Regulation of islet glucagon secretion: Beyond calcium.

The islet of Langerhans plays a key role in glucose homeostasis through regulated secretion of the hormones insulin and glucagon. Islet research has focused on the insulin-secreting ß-cells, even though aberrant glucagon secretion from α-cells also contributes to the aetiology of diabetes. Despite its importance, the mechanisms controlling glucagon secretion remain controversial. Proper α-cell function requires the islet milieu, where ß- and δ-cells drive and constrain α-cell dynamics. The response of glucagon to glucose is similar between isolated islets and that measured in vivo, so it appears that the glucose dependence requires only islet-intrinsic factors and not input from blood flow or the nervous system. Elevated intracellular free Ca2+ is needed for α-cell exocytosis, but interpreting Ca2+ data is tricky since it is heterogeneous among α-cells at all physiological glucose levels. Total Ca2+ activity in α-cells increases slightly with glucose, so Ca2+ may serve a permissive, rather than regulatory, role in glucagon secretion. On the other hand, cAMP is a more promising candidate for controlling glucagon secretion and is itself driven by paracrine signalling from ß- and δ-cells. Another pathway, juxtacrine signalling through the α-cell EphA receptors, stimulated by ß-cell ephrin ligands, leads to a tonic inhibition of glucagon secretion. We discuss potential combinations of Ca2+ , cAMP, paracrine and juxtacrine factors in the regulation of glucagon secretion, focusing on recent data in the literature that might unify the field towards a quantitative understanding of α-cell function.

HIGH-RISK GASTRIC PATHOLOGY AND PREVALENT AUTOIMMUNE DISEASES IN PATIENTS WITH PERNICIOUS ANEMIA.

OBJECTIVE: Pernicious anemia (PA) develops from atrophic gastritis due to autoimmune destruction of parietal cells and results in achlorhydria, vitamin B12 and iron deficiencies, anemia, neurologic deficits, and premalignant and malignant stomach lesions. We report the presentation, diagnosis and gastric complications of PA in patients from an endocrinology practice. METHODS: Thirty-four patients (31 female, 3 male) with PA who underwent esophagogastroduodenoscopy (EGD) or gastrectomy were identified. Pertinent clinical, laboratory, and pathology findings were reviewed and summarized. RESULTS: The mean age of patients was 58.6 ± 14.2 years; the onset of PA was age 50.2 ± 15.3 years. Anemia reflected vitamin B12 and/or iron deficiencies. Parietal cell antibodies (PCA) were detected in 97% of patients, and intrinsic factor blocking antibody (IFBA) was found in 52%. Fasting gastrin and chromogranin A levels were elevated (1,518.0 ± 1,588.3 pg/mL, and 504.9.1 ± 1,524.9 ng/mL respectively). Autoimmune or immunologic diseases (AIDs) were present in 32/34 patients. Stomach pathology showed premalignant or malignant lesions in 26 patients, including gastric neuroendocrine tumors (GNETs) in 6 and adenocarcinoma in 1. One patient presented with neurologic symptoms and subacute combined degeneration of the posterior column of the spinal cord. CONCLUSION: PA should be suspected in patients with unexplained anemia or neurologic symptoms. The diagnosis of PA relies on fasting gastrin and gastric auto-antibody testing, in addition to hematologic evaluation. EGD with measurement of gastric pH and biopsies of the fundus and antrum identifies patients with achlorhydria, atrophic gastritis, and premalignant and malignant stomach lesions. EGD surveillance of patients with high-risk stomach lesions is recommended. ABBREVIATIONS: AID = autoimmune or immunologic disease; EGD = esophagogastroduodenoscopy; GNET = gastric neuroendocrine tumor; IFBA = intrinsic factor blocking antibody; PA = pernicious anemia; PCA = parietal cell antibody; T1D = type 1 diabetes.

Moyamoya syndrome causing stroke in young women with type 1 diabetes.

CONTEXT: Moyamoya syndrome is an idiopathic brain vasculopathy characterized by stenosis of major intracranial arteries. It often presents in patients with type 1 diabetes or thyroid disease and may have an autoimmune etiology. Moyamoya-related stroke poses a diagnostic challenge as initial symptoms and deficits vary greatly from classic ischemic stroke to encephalopathy, psychiatric, or seizure disorder. CASE DESCRIPTION: We report 4 patients with type 1 diabetes and other autoimmune diseases who developed moyamoya-related stroke at a young age. Despite having long-term diabetes, these patients exhibited no evidence of dyslipidemia or other typical risk factors for atherosclerosis which might contribute to premature stroke. Three of the four patients underwent revascularization surgery while one patient received conservative management. All patients had improved neurologic function after treatment, some with residual deficits. CONCLUSION: We highlight the importance of recognizing moyamoya syndrome in patients with pre-existing autoimmune diseases such as type 1 diabetes, as prompt diagnosis and treatment can have major impact on patient outcome and quality of life.

Autoimmune Diseases in Children and Adults With Type 1 Diabetes From the T1D Exchange Clinic Registry.

BACKGROUND AND AIMS: Type 1 diabetes (T1D) is associated with other autoimmune diseases (AIDs), but the prevalence and associated predictive factors for these comorbidities of T1D across all age groups have not been fully characterized. MATERIALS AND METHODS: Data obtained from 25 759 participants with T1D enrolled in the T1D Exchange Registry were used to analyze the types and frequency of AIDs as well as their relationships to gender, age, and race/ethnicity. Diagnoses of autoimmune diseases, represented as ordinal categories (0, 1, 2, 3, or more AIDs) were obtained from medical records of Exchange Registry participants. RESULTS: Among the 25 759 T1D Exchange participants, 50% were female, 82% non-Hispanic white, mean age was 23.0 ± 16.9 years and mean duration of diabetes was 11 years. Of these participants, 6876 (27%) were diagnosed with at least one AID. Frequency of two or more AIDs increased from 4.3% in participants aged younger than 13 years to 10.4% in those aged 50 years or older. The most common AIDs were thyroid (6097, 24%), gastrointestinal (1530, 6%), and collagen vascular diseases (432, 2%). Addison's disease was rare (75, 0.3%). The prevalence of one or more AIDs was increased in females and non-Hispanic whites and with older age. CONCLUSIONS: In the T1D Exchange Clinic Registry, a diagnosis of one or more AIDs in addition to T1D is common, particularly in women, non-Hispanic whites, and older individuals. Results of this study have implications for both primary care and endocrine practice and will allow clinicians to better anticipate and manage the additional AIDs that develop in patients with T1D.

Clitoral epidermoid cyst presenting as pseudoclitoromegaly of pregnancy.

Objective Acquired clitoromegaly is rare and may result from hormonal and nonhormonal causes, and evaluation of the pregnant patient with clitoromegaly invokes a specific set of differential diagnoses. Methods Case report. Results We describe the case of a young woman with pregnancy-associated clitoral enlargement whose hormonal evaluation proved negative. Further investigation concluded that an epidermoid cyst was the culprit of her pseudoclitoromegaly. The patient underwent successful surgical resection and has had no recurrence at her subsequent pregnancy. Conclusion We review the differential diagnosis of clitoromegaly, including hormonal and nonhormonal causes, with focus on the evaluation of pregnancy-associated clitoromegaly.

Novel SIRT1 mutation linked to autoimmune diabetes in humans.

Sirtuins are best known for their role in aging but also regulate many different biological processes. A study in this issue of Cell Metabolism (Biason-Lauber et al., 2013) now identifies a mutation in human SIRT1 causing a familial form of autoimmune diabetes.