Β-cell Heterogeneity Research Articles

2077-P: Can Small ß-Cell Subpopulations Control Islet Dynamics? A Theoretical Study

Electrical activity of a pancreatic islet is globally activated under high glucose conditions and globally suppressed under low glucose conditions. This homogeneous behavior occurs due to the highly-coupled nature of the islet, despite heterogeneity of individual β-cells. Using hyperpolarization to silence β-cell activity, studies have shown that small subpopulations of highly functional β-cells can disproportionately control whole islet dynamics. Using a computational model of islet electrophysiology, we studied the theoretical basis of how small highly functional subpopulations of β-cells may regulate the global activity of islets. We modeled metabolic activity of β-cells as a continuous distribution and as a bimodal distribution. Then, in each case, we silenced the highest metabolically active cells and quantified the response of the islet. These highly metabolic cells (GKHigh) were silenced by hyperpolarization, similarly to experimental data. Only under a bimodal distribution, did a small (10%) population of GKHigh cells dramatically reduce islet activity, as experimentally demonstrated. Next, a simulation was run without GKHigh cells present in the islet. When GKHigh cells were absent, the islet remained globally active and had near normal duty cycle, suggesting that these cells are unnecessary for maintaining islet dynamics. Similarly, we investigated another functional subpopulation, β-cells with higher Ca2+ oscillation frequencies which could potentially act as pacemaker cells. When simulating the absence of 10% of these higher frequency cells, the global frequency of the islet was not significantly altered. These results indicate that small highly functional subpopulations are unable to drive islet function under normal conditions, but when targeted under hyperpolarizing conditions may cause dysfunction. Disclosure J.M. Dwulet: None. R.K. Benninger: None. Funding National Institutes of Health (R01DK106412, R01DK102950)

Characterizing pancreatic β-cell heterogeneity in the streptozotocin model by single-cell transcriptomic analysis

ObjectivesThe streptozotocin (STZ) model is widely used in diabetes research. However, the cellular and molecular states of pancreatic endocrine cells in this model remain unclear. This study explored the molecular characteristics of islet cells treated with STZ and re-evaluated β-cell dysfunction and regeneration in the STZ model. MethodsWe performed single-cell RNA sequencing of pancreatic endocrine cells from STZ-treated mice. High-quality sequencing data from 2,999 cells were used to identify clusters via Louvain clustering analysis. Principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), uniform manifold approximation and projection (UMAP), force-directed layout (FDL), and differential expression analysis were used to define the heterogeneity and transcriptomic changes in islet cells. In addition, qPCR and immunofluorescence staining were used to confirm findings from the sequencing data. ResultsUntreated β-cells were divided into two populations at the transcriptomic level, a large high-Glut2 expression (Glut2high) population and a small low-Glut2 expression (Glut2low) population. At the transcriptomic level, Glut2low β-cells in adult mice did not represent a developmentally immature state, although a fraction of genes associated with β-cell maturation and function were downregulated in Glut2low cells. After a single high-dose STZ treatment, most Glut2high cells were killed, but Glut2low cells survived and over time changed to a distinct cell state. We did not observe conversion of Glut2low to Glut2high β-cells up to 9 months after STZ treatment. In addition, we did not detect transcriptomic changes in the non-β endocrine cells or a direct trans-differentiation pathway from the α-cell lineage to the β-cell lineage in the STZ model. ConclusionsWe identified the heterogeneity of β-cells in both physiological and pathological conditions. However, we did not observe conversion of Glut2low to Glut2high β-cells, transcriptomic changes in the non-β endocrine cells, or direct trans-differentiation from the α-cell lineage to the β-cell lineage in the STZ model. Our results clearly define the states of islet cells treated with STZ and allow us to re-evaluate the STZ model widely used in diabetes studies.

Navigating the Depths and Avoiding the Shallows of Pancreatic Islet Cell Transcriptomes

Islet gene expression has been widely studied to better understand the transcriptional features that define a healthy β-cell. Transcriptomes of FACS-purified α-, β-, and δ-cells using bulk RNA-sequencing have facilitated our understanding of the complex network of cross talk between islet cells and its effects on β-cell function. However, these approaches were by design not intended to resolve heterogeneity between individual cells. Several recent studies used single-cell RNA sequencing (scRNA-Seq) to report considerable heterogeneity within mouse and human β-cells. In this Perspective, we assess how this newfound ability to assess gene expression at single-cell resolution has enhanced our understanding of β-cell heterogeneity. We conduct a comprehensive assessment of several single human β-cell transcriptome data sets and ask if the heterogeneity reported by these studies showed overlap and concurred with previously known examples of β-cell heterogeneity. We also illustrate the impact of the inevitable limitations of working at or below the limit of detection of gene expression at single cell resolution and their consequences for the quality of single–islet cell transcriptome data. Finally, we offer some guidance on when to opt for scRNA-Seq and when bulk sequencing approaches may be better suited.

37-OR: Distinct Functional Beta-Cell Subpopulations in Intact Islets of Langerhans

Glucose-stimulated insulin secretion is driven by the coordinated Ca2+ response in pancreatic islets following glucose elevation. In mouse islets, spatial and temporal Ca2+ dynamics are disproportionally controlled by subpopulations of β-cells, as identified by optogenetics. It is unknown which factors underlie this functional heterogeneity of β-cells in intact islets. Here, we tested if distinct β-cell subpopulations may be identified based on Ca2+ response to glucose elevation, and if their Ca2+ uptake efficiency, metabolic activity and electrical coupling are significantly different. We used MIP-CreER GCaMP6s mouse model which expresses Ca2+-sensitive GFP specifically in β-cells. We performed simultaneous Ca2+ dynamics and metabolic activity (NADH) response to [2-11] mM glucose elevation, along with gap junction permeability measurements in individual islets. We observed three functional subpopulations of β-cells distinct from an islet average: cells with 1) the fastest response to glucose elevation (1st responders); 2) the fastest 2nd phase oscillatory Ca2+ response (wave-initiators); and 3) the non-coordinated Ca2+ oscillations (un-coordinated). While 1st responders and wave-initiators were in different sub-regions of the islet, both subpopulations accumulated more Ca2+ during initial Ca2+ elevation (p=0.0403, p=0.0101) compared to the islet-average. Wave-initiators were more metabolically active (p=0.0223). The un-coordinated cells accumulated less Ca2+ (p<0.001) and were less metabolically active (p=0.0296). Gap junction permeability was not significantly different between subpopulations. In conclusion, the 1st phase Ca2+ uptake and 2nd phase Ca2+ oscillations emerge in spatially- and metabolically-distinct subregions of the islet. This is likely due to increased metabolic activity of the β-cells in these regions which leads to greater membrane depolarization and thus, disproportionately enhances the islet Ca2+response. (NIH R01 DK102950 R01 DK106412) Disclosure V. Kravets: None. R.A. Piscopio: None. W. Schleicher: None. R.K. Benninger: None. Funding National Institutes of Health

1772-P: Insulin Granule Biogenesis at the Golgi Requires a Metabolically-Regulated, Non-centrosomal Subpopulation of Microtubules

Insulin undergoes biogenesis through the ER and Golgi, ultimately leading to packaging into vesicles that are secreted in response to glucose. The movement between these membranes and the process of budding from the Golgi are vital steps. Previous work has found a role for microtubules (MTs) in these processes in both pancreatic β-cells and other cell types. Here, we show that the last stages of this process are regulated by a specific subpopulation of MTs that are non-centrosomal. Their formation, or nucleation, at the Golgi membrane is regulated by a glucose signal-transduction pathway through cAMP and its effector EPAC2. This effect is especially pronounced during the first, rapid phase of insulin secretion, during which EPAC2 levels increase at the sites of their nucleation. Preventing new nucleation of this specific population dramatically affects the pipeline of insulin production, storage and release. There is an overall reduction of β-cell insulin content, and the remaining insulin becomes retained within the Golgi, likely because of stalling of insulin-granule budding. This diminished granule availability substantially effects the level of both basal and stimulated insulin secretion, though not the ratio between these levels during the first wave. Constant dynamic maintenance of this network is therefore critical for normal β-cell physiology. This is the first demonstration that the biogenesis of post-Golgi carriers, particularly large secretory granules, requires ongoing nucleation/replenishment of this network. We are further looking at the role of MTs in β-cell secretory activity in intact islets. We have observed that MTs promote β-cell heterogeneity by restricting secretion from a specific β-cell sub-population, where insulin release can be activated upon disruption of the entire MT network by the drug nocodazole. Disclosure K. Trogden: None. C.V. Wright: None. G. Gu: None. I. Kaverina: None. Funding National Institutes of Health (1F32DK117529-01A1)

Heterogeneity of the Human Pancreatic Islet.

Pancreatic β-cells play a pivotal role in maintaining normoglycemia. Recent studies have revealed that the β-cell is not a homogeneous cell population but, rather, is heterogeneous in a number of properties such as electrical activity, gene expression, and cell surface markers. Identification of specific β-cell subpopulations altered in diabetic conditions would open a new avenue to develop targeted therapeutic interventions. As intense studies of β-cell heterogeneity are anticipated in the next decade, it is important that heterogeneity of the islet be recognized. Many studies in the past were undertaken with a small sample of islets, which might overlook important individual variance. In this study, by systematic analyses of the human islet in two and three dimensions, we demonstrate islet heterogeneity in size, number, architecture, cellular composition, and capillary density. There is no stereotypic human islet, and thus, a sufficient number of islets should be examined to ensure study reproducibility.

Evidence of a developmental origin for β-cell heterogeneity using a dual lineage-tracing technology.

The Cre/loxP system has been used extensively in mouse models with a limitation of one lineage at a time. Differences in function and other properties among populations of adult β-cells is termed β-cell heterogeneity, which was recently associated with diabetic phenotypes. Nevertheless, the presence of a developmentally derived β-cell heterogeneity is unclear. Here, we have developed a novel dual lineage-tracing technology, using a combination of two recombinase systems, Dre/RoxP and Cre/LoxP, to independently trace green fluorescent Pdx1-lineage cells and red fluorescent Ptf1a-lineage cells in the developing and adult mouse pancreas. We detected a few Pdx1+/Ptf1a- lineage cells in addition to the vast majority of Pdx1+/Ptf1a+ lineage cells in the pancreas. Moreover, Pdx1+/Ptf1a+ lineage β-cells had fewer Ki-67+ proliferating β-cells, and expressed higher mRNA levels of insulin, Glut2, Pdx1, MafA and Nkx6.1, but lower CCND1 and CDK4 levels, compared with Pdx1+/Ptf1a- lineage β-cells. Furthermore, more TSQ-high, SSC-high cells were detected in the Pdx1+Ptf1a+ lineage population than in the Pdx1+Ptf1a- lineage population. Together, these data suggest that differential activation of Ptf1a in the developing pancreas may correlate with this β-cell heterogeneity.

β-cell Heterogeneity: The Key to β-cell Replacement Therapy

β-cell Heterogeneity: The Key to β-cell Replacement Therapy

Islet Identity in Transplantation Procedures: The Intersection of Cellular Maturity and Function

Islet transplantation is a promising technique for millions of patients with diabetes, but is severely limited by a shortage of cadaveric donor islets, and more so because of stringent inclusion criteria for organ donation including donor metabolic function, age, and comorbidities. The impact of these diverse factors on islet health have led to a broad investigation of global influences on islet biology, not least of all, characterization of mature, functional cellular identity and maintenance of appropriate endocrine lineage. This review will discuss variables involved in donor islet inclusion characteristics and metabolic function, and related impact on islet transplant success; inherent and imposed models of islet and β-cell heterogeneity and plasticity; and the role of cellular dedifferentiation of islets and β-cells in the normal and pathophysiological states, including aging, diabetes subtypes, and islet transplantation. Examination of potential strategies for reduction of metabolic stress by endogenous and exogenous modes is further discussed.

Maintenance of ß-Cell Heterogeneity Is Required for Normal Islet Function

Aim: Recent studies have shown the existence of discrete β-cell subpopulations with differing levels of maturity and function. We sought here to understand how this diversity may influence islet function and insulin secretion using a ‘loss of heterogeneity‘ model. Material and Methods: Overexpression was achieved using an adenoviral polycistronic construct for Pdx1, MafA, Ngn3 and mCherry (Ad3-NPM). Pdx1 was silenced using short hairpin RNAs (shRNA). Gene and protein levels were detected by qPCR and immunohistochemistry (IHC), respectively. Ca2+ fluxes, ATP/ADP ratios and cAMP levels were analysed using dyes/biosensors and high-speed spinning disk microscopy. Glucose- (GSIS) and incretin-stimulated insulin secretion (ISIS) were measured using HTRF assays. Results: Ad3-NPM increased Pdx1 (12-fold vs. CT; P<0.01) and MafA levels (2-fold vs. CT) without affecting Ngn3. IHC showed that Pdx1 overexpression occurred predominantly in Pdx1low β-cells, forcing higher uniformity throughout the β-cell complement. The α/β-cell ratio did not change, indicating absence of transdifferentiation. Ca2+ responses to glucose were markedly blunted (ΔF=0.81 vs. 0.44 AU, CT vs. Ad3-NPM; P<0.01), and this was accompanied by a decrease in β-cell-β-cell cell connectivity and hub number (12.6 vs. 5.6 % hubs, CT vs. Ad3-NPM; P<0.05), the latter previously shown to be relatively immature cells with pacemaker features. ATP/ADP ratios and cAMP levels were unaffected. Glucose- and incretin-stimulated insulin secretion was impaired (% content secreted = 8.7 vs. 5.2 for GSIS and 56.4 vs. 29.02 for ISIS, CT vs. Ad3-NPM; both P<0.01). Similar results for Ca2+ dynamics, cellular connectivity and insulin secretion were obtained in islets treated with Pdx1 shRNA to increase the size of the Pdx1low population. Conclusion: Loss of β-cell heterogeneity, whether due to increased or decreased maturity, leads to islet failure and impaired insulin secretion. Thus, cellular diversity appears necessary for normal islet function. Disclosure D. Nasteska: None. G.A. Rutter: Research Support; Self; Servier. Q. Zhou: None. D. Hodson: None.

New Understanding of β-Cell Heterogeneity and In Situ Islet Function.

Insulin-secreting β-cells are heterogeneous in their regulation of hormone release. While long known, recent technological advances and new markers have allowed the identification of novel subpopulations, improving our understanding of the molecular basis for heterogeneity. This includes specific subpopulations with distinct functional characteristics, developmental programs, abilities to proliferate in response to metabolic or developmental cues, and resistance to immune-mediated damage. Importantly, these subpopulations change in disease or aging, including in human disease. Although discovering new β-cell subpopulations has substantially advanced our understanding of islet biology, a point of caution is that these characteristics have often necessarily been identified in single β-cells dissociated from the islet. β-Cells in the islet show extensive communication with each other via gap junctions and with other cell types via diffusible chemical messengers. As such, how these different subpopulations contribute to in situ islet function, including during plasticity, is not well understood. We will discuss recent findings revealing functional β-cell subpopulations in the intact islet, the underlying basis for these identified subpopulations, and how these subpopulations may influence in situ islet function. Furthermore, we will discuss the outlook for emerging technologies to gain further insight into the role of subpopulations in in situ islet function.

β-Cells are not uniform after all-Novel insights into molecular heterogeneity of insulin-secreting cells.

While the β-cells of the endocrine pancreas are defined as cells with high levels of insulin production and tight stimulus-secretion coupling, the existence of functional heterogeneity among them has been known for decades. Recent advances in molecular technologies, in particular single-cell profiling on both the protein and messenger RNA level, have uncovered that β-cells exist in several antigenically and molecularly definable states. Using antibodies to cell surface markers or multidimensional clustering of β-cells using more than 20 protein markers by mass cytometry, 4 distinct groups of β-cells could be differentiated. However, whether these states represent permanent cell lineages or are readily interconvertible from one group to another remains to be determined. Nevertheless, future analysis of the pathogenesis of type 1 and type 2 diabetes will certainly benefit from a growing appreciation of β-cell heterogeneity. Here, we aim to summarize concisely the recent advances in the field and their possible impact on our understanding of β-cell physiology and pathophysiology.

Evidence for Loss in Identity, De-Differentiation, and Trans-Differentiation of Islet β-Cells in Type 2 Diabetes

The two main types of diabetes mellitus have distinct etiologies, yet a similar outcome: loss of islet β-cell function that is solely responsible for the secretion of the insulin hormone to reduce elevated plasma glucose toward euglycemic levels. Type 1 diabetes (T1D) has traditionally been characterized by autoimmune-mediated β-cell death leading to insulin-dependence, whereas type 2 diabetes (T2D) has hallmarks of peripheral insulin resistance, β-cell dysfunction, and cell death. However, a growing body of evidence suggests that, especially during T2D, key components of β-cell failure involves: (1) loss of cell identity, specifically proteins associated with mature cell function (e.g., insulin and transcription factors like MAFA, PDX1, and NKX6.1), as well as (2) de-differentiation, defined by regression to a progenitor or stem cell-like state. New technologies have allowed the field to compare islet cell characteristics from normal human donors to those under pathophysiological conditions by single cell RNA-Sequencing and through epigenetic analysis. This has revealed a remarkable level of heterogeneity among histologically defined “insulin-positive” β-cells. These results not only suggest that these β-cell subsets have different responses to insulin secretagogues, but that defining their unique gene expression and epigenetic modification profiles will offer opportunities to develop cellular therapeutics to enrich/maintain certain subsets for correcting pathological glucose levels. In this review, we will summarize the recent literature describing how β-cell heterogeneity and plasticity may be influenced in T2D, and various possible avenues of therapeutic intervention.

Heterogeneity of β-cells.

Commentary on Bader et al., appeared in Nature 2016; 535: 430-434.

Impact of islet architecture on β-cell heterogeneity, plasticity and function.

Although β-cell heterogeneity was discovered more than 50 years ago, the underlying principles have been explored only during the past decade. Islet-cell heterogeneity arises during pancreatic development and might reflect the existence of distinct populations of progenitor cells and the developmental pathways of endocrine cells. Heterogeneity can also be acquired in the postnatal period owing to β-cell plasticity or changes in islet architecture. Furthermore, β-cell neogenesis, replication and dedifferentiation represent alternative sources of β-cell heterogeneity. In addition to a physiological role, β-cell heterogeneity influences the development of diabetes mellitus and its response to treatment. Identifying phenotypic and functional markers to discriminate distinct β-cell subpopulations and the mechanisms underpinning their regulation is warranted to advance current knowledge of β-cell function and to design novel regenerative strategies that target subpopulations of β cells. In this context, the Wnt/planar cell polarity (PCP) effector molecule Flattop can distinguish two unique β-cell subpopulations with specific transcriptional signatures, functional properties and differential responses to environmental stimuli. In vivo targeting of these β-cell subpopulations might, therefore, represent an alternative strategy for the future treatment of diabetes mellitus.

Physiology: Pancreatic β-cell heterogeneity revisited.

Two analyses of insulin-producing β-cells reveal differences in what has long been considered a homogeneous population. These differences might reflect changes during maturation or ageing, or distinct cell lineages. See Letter p.430 Pancreatic β-cells are functionally heterogeneous, and differences in their characteristics may be important in the context of insulin-dependent diabetes. Heiko Lickert and colleagues have found a marker, the Fltp reporter gene, that is expressed in mature β-cell populations but not in proliferative β-cells. Fltp expression is triggered by Wnt signalling and polarization of β-cells as they organize into three-dimensional islets and the authors show that this change in architecture is sufficient to induce mouse and human β-cell maturation.

Identification of proliferative and mature β-cells in the islets of Langerhans.

Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of β-cells. Pancreatic β-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature β-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger β-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for β-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional β-cell heterogeneity and induce β-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional β-cell mass in diabetic patients.

Statistical approaches and software for clustering islet cell functional heterogeneity

ABSTRACTWorldwide efforts are underway to replace or repair lost or dysfunctional pancreatic β-cells to cure diabetes. However, it is unclear what the final product of these efforts should be, as β-cells are thought to be heterogeneous. To enable the analysis of β-cell heterogeneity in an unbiased and quantitative way, we developed model-free and model-based statistical clustering approaches, and created new software called TraceCluster. Using an example data set, we illustrate the utility of these approaches by clustering dynamic intracellular Ca2+ responses to high glucose in ∼300 simultaneously imaged single islet cells. Using feature extraction from the Ca2+ traces on this reference data set, we identified 2 distinct populations of cells with β-like responses to glucose. To the best of our knowledge, this report represents the first unbiased cluster-based analysis of human β-cell functional heterogeneity of simultaneous recordings. We hope that the approaches and tools described here will be helpful for those studying heterogeneity in primary islet cells, as well as excitable cells derived from embryonic stem cells or induced pluripotent cells.

Islet-cell biology in 2015: Understanding secretion, ageing and death in β cells.

The pancreatic islets of Langerhans have been intensively investigated for many years, largely because of their central role in the pathogenesis of type 1 and type 2 diabetes mellitus. Notable advances in 2015 related to glucose-stimulated insulin secretion in β cells, β-cell death and the role of epigenetics in β-cell heterogeneity.

Intrinsic Islet Heterogeneity and Gap Junction Coupling Determine Spatiotemporal Ca2+ Wave Dynamics

Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca2+]i). Associated with each synchronized oscillation is a propagating calcium wave mediated by Connexin36 (Cx36) gap junctions. A computational islet model predicted that waves emerge due to heterogeneity in β-cell function throughout the islet. To test this, we applied defined patterns of glucose stimulation across the islet using a microfluidic device and measured how these perturbations affect calcium wave propagation. We further investigated how gap junction coupling regulates spatiotemporal [Ca2+]i dynamics in the face of heterogeneous glucose stimulation. Calcium waves were found to originate in regions of the islet having elevated excitability, and this heterogeneity is an intrinsic property of islet β-cells. The extent of [Ca2+]i elevation across the islet in the presence of heterogeneity is gap-junction dependent, which reveals a glucose dependence of gap junction coupling. To better describe these observations, we had to modify the computational islet model to consider the electrochemical gradient between neighboring β-cells. These results reveal how the spatiotemporal [Ca2+]i dynamics of the islet depend on β-cell heterogeneity and cell-cell coupling, and are important for understanding the regulation of coordinated insulin release across the islet.