Disruption of the pancreatic vasculature in zebrafish affects islet architecture and function.

A dense local vascular network is crucial for pancreatic endocrine cells to sense metabolites and secrete hormones, and understanding the interactions between the vasculature and the islets may allow for therapeutic modulation in disease conditions. Using live imaging in two models of vascular disruption in zebrafish, we identified two distinct roles for the pancreatic vasculature. At larval stages, expression of a dominant negative version of Vegfaa (dnVegfaa) in ß-cells led to vascular and endocrine cell disruption with a minor impairment in ß-cell function. In contrast, expression of a soluble isoform of Vegf receptor 1 (sFlt1) in ß-cells blocked the formation of the pancreatic vasculature and drastically stunted glucose response, although islet architecture was not affected. Notably, these effects of dnVegfaa or sFlt1 were not observed in animals lacking vegfaa, vegfab, kdrl, kdr or flt1 function, indicating that they interfere with multiple ligands and/or receptors. In adults, disrupted islet architecture persisted in dnVegfaa-expressing animals, whereas sFlt1-expressing animals displayed large sheets of ß-cells along their pancreatic ducts, accompanied by impaired glucose tolerance in both models. Thus, our study reveals novel roles for the vasculature in patterning and function of the islet.

Intraislet SLIT-ROBO signaling is required for beta-cell survival and potentiates insulin secretion.

We previously cataloged putative autocrine/paracrine signaling loops in pancreatic islets, including factors best known for their roles in axon guidance. Emerging evidence points to nonneuronal roles for these factors, including the Slit-Roundabout receptor (Robo) family, in cell growth, migration, and survival. We found SLIT1 and SLIT3 in both beta cells and alpha cells, whereas SLIT2 was predominantly expressed in beta cells. ROBO1 and ROBO2 receptors were detected in beta and alpha cells. Remarkably, even modest knockdown of Slit production resulted in significant beta-cell death, demonstrating a critical autocrine/paracrine survival role for this pathway. Indeed, recombinant SLIT1, SLIT2, and SLIT3 decreased serum deprivation, cytokine, and thapsigargin-induced cell death under hyperglycemic conditions. SLIT treatment also induced a gradual release of endoplasmic reticulum luminal Ca(2+), suggesting a unique molecular mechanism capable of protecting beta cells from endoplasmic reticulum stress-induced apoptosis. SLIT treatment was also associated with rapid actin remodeling. SLITs potentiated glucose-stimulated insulin secretion and increased the frequency of glucose-induced Ca(2+) oscillations. These observations point to unexpected roles for local Slit secretion in the survival and function of pancreatic beta cells. Because diabetes results from a deficiency in functional beta-cell mass, these studies may contribute to therapeutic approaches for improving beta-cell survival and function.

A live-cell, high-content imaging survey of 206 endogenous factors across five stress conditions reveals context-dependent survival effects in mouse primary beta cells.

AIMS/HYPOTHESIS: Beta cell death is a hallmark of diabetes. It is not known whether specific cellular stresses associated with type 1 or type 2 diabetes require specific factors to protect pancreatic beta cells. No systematic comparison of endogenous soluble factors in the context of multiple pro-apoptotic conditions has been published. METHODS: Primary mouse islet cells were cultured in conditions mimicking five type 1 or type 2 diabetes-related stresses: basal 5 mmol/l glucose, cytokine cocktail (25 ng/ml TNF-α, 10 ng/ml IL-1ß, 10 ng/ml IFN-γ), 1 µmol/l thapsigargin, 1.5 mmol/l palmitate and 20 mmol/l glucose (all in the absence of serum). We surveyed the effects of a library of 206 endogenous factors (selected based on islet expression of their receptors) on islet cell survival through multi-parameter, live-cell imaging. RESULTS: Our survey pointed to survival factors exhibiting generalised protective effects across conditions meant to model different types of diabetes and stages of the diseases. For example, our survey and follow-up experiments suggested that OLFM1 is a novel protective factor for mouse and human beta cells across multiple conditions. Most strikingly, we also found specific protective survival factors for each model stress condition. For example, semaphorin4A (SEMA4A) was toxic to islet cells in the serum-free baseline and serum-free 20 mmol/l glucose conditions, but protective in the context of lipotoxicity. Rank product testing supported the consistency of our observations. CONCLUSIONS/INTERPRETATION: Collectively, our survey reveals previously unidentified islet cell survival factors and suggest their potential utility in individualised medicine.

Multi-parameter single-cell kinetic analysis reveals multiple modes of cell death in primary pancreatic ß-cells.

Programmed ß-cell death plays an important role in both type 1 and type 2 diabetes. Most of what is known about the mechanisms of ß-cell death comes from single time-point, single parameter measurements of bulk populations of mixed cells. Such approaches are inadequate for determining the true extent of the heterogeneity in death mechanisms. Here, we characterized the timing and order of molecular events associated with cell death in single ß-cells under multiple diabetic stress conditions, including hyperglycemia, cytokine exposure, nutrient deprivation and endoplasmic reticulum (ER) stress. We simultaneously measured the kinetics of six distinct cell death mechanisms by using a caspase-3 sensor and three vital dyes, together with brightfield imaging. We identified several cell death modes where the order of events that usually define apoptosis were not observed. This we termed 'partial apoptosis'. Remarkably, complete classical apoptosis, defined as cells with plasma membrane blebbing, caspase-3 activity, nuclear condensation and membrane annexin V labeling prior to loss of plasma membrane integrity, was found in only half of the cytokine-treated primary ß-cells and never in cells stressed by serum removal. By contrast, in the MIN6 cell line, death occurred almost exclusively through complete classical apoptosis. Ambient glucose modulated the cell death mode and kinetics in primary ß-cells. Taken together, our data define the kinetic progression of ß-cell death mechanisms under different conditions and illustrate the heterogeneity and plasticity of cell death modes in ß-cells. We conclude that apoptosis is not the primary mode of adult primary ß-cell death.

Effects of insulin on human pancreatic cancer progression modeled in vitro.

BACKGROUND: Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined. METHODS: Here, we examined the effects of a range of insulin doses on signalling, proliferation and survival in three human cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells, the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells were treated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTT assays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting. Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to the survival of each cell model. RESULTS: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling. CONCLUSIONS: Our data suggest that excessive insulin signalling may contribute to proliferation and survival in human immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult human pancreatic ductal cells. These data suggest that signalling pathways involved in cell survival may be rewired during pancreatic cancer progression.

Innervation modulates the functional connectivity between pancreatic endocrine cells.

The importance of pancreatic endocrine cell activity modulation by autonomic innervation has been debated. To investigate this question, we established an in vivo imaging model that also allows chronic and acute neuromodulation with genetic and optogenetic tools. Using the GCaMP6s biosensor together with endocrine cell fluorescent reporters, we imaged calcium dynamics simultaneously in multiple pancreatic islet cell types in live animals in control states and upon changes in innervation. We find that by 4 days post fertilization in zebrafish, a stage when islet architecture is reminiscent of that in adult rodents, prominent activity coupling between beta cells is present in basal glucose conditions. Furthermore, we show that both chronic and acute loss of nerve activity result in diminished beta-beta and alpha-beta activity coupling. Pancreatic nerves are in contact with all islet cell types, but predominantly with beta and delta cells. Surprisingly, a subset of delta cells with detectable peri-islet neural activity coupling had significantly higher homotypic coupling with other delta cells suggesting that some delta cells receive innervation that coordinates their output. Overall, these data show that innervation plays a vital role in the maintenance of homotypic and heterotypic cellular connectivity in pancreatic islets, a process critical for islet function.

Brain-Body Control of Glucose Homeostasis-Insights From Model Organisms.

Tight regulation of blood glucose is essential for long term health. Blood glucose levels are defended by the correct function of, and communication between, internal organs including the gastrointestinal tract, pancreas, liver, and brain. Critically, the brain is sensitive to acute changes in blood glucose level and can modulate peripheral processes to defend against these deviations. In this mini-review we highlight select key findings showcasing the utility, strengths, and limitations of model organisms to study brain-body interactions that sense and control blood glucose levels. First, we discuss the large platform of genetic tools available to investigators studying mice and how this field may yet reveal new modes of communication between peripheral organs and the brain. Second, we discuss how rats, by virtue of their size, have unique advantages for the study of CNS control of glucose homeostasis and note that they may more closely model some aspects of human (patho)physiology. Third, we discuss the nascent field of studying the CNS control of blood glucose in the zebrafish which permits ease of genetic modification, large-scale measurements of neural activity and live imaging in addition to high-throughput screening. Finally, we briefly discuss glucose homeostasis in drosophila, which have a distinct physiology and glucoregulatory systems to vertebrates.

Bax and Bak jointly control survival and dampen the early unfolded protein response in pancreatic ß-cells under glucolipotoxic stress.

ER stress and apoptosis contribute to the loss of pancreatic ß-cells under pro-diabetic conditions of glucolipotoxicity. Although activation of canonical intrinsic apoptosis is known to require pro-apoptotic Bcl-2 family proteins Bax and Bak, their individual and combined involvement in glucolipotoxic ß-cell death are not known. It has also remained an open question if Bax and Bak in ß-cells have non-apoptotic roles in mitochondrial function and ER stress signaling, as suggested in other cell types. Using mice with individual or combined ß-cell deletion of Bax and Bak, we demonstrated that glucolipotoxic ß-cell death in vitro occurs by both non-apoptotic and apoptotic mechanisms, and the apoptosis could be triggered by either Bax or Bak alone. In contrast, they had non-redundant roles in mediating staurosporine-induced apoptosis. We further established that Bax and Bak do not affect normal glucose-stimulated ß-cell Ca2+ responses, insulin secretion, or in vivo glucose tolerance. Finally, our experiments revealed that combined deletion of Bax and Bak amplified the unfolded protein response in islets during the early stages of chemical- or glucolipotoxicity-induced ER stress. These findings shed new light on roles of the core apoptosis machinery in ß-cell survival and stress signals of importance for the pathobiology of diabetes.

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

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

Whole-Organism Chemical Screening Identifies Modulators of Pancreatic ß-Cell Function.

ß-Cell loss and dysfunction play a critical role in the progression of type 1 and type 2 diabetes. Identifying new molecules and/or molecular pathways that improve ß-cell function and/or increase ß-cell mass should significantly contribute to the development of new therapies for diabetes. Using the zebrafish model, we screened 4,640 small molecules to identify modulators of ß-cell function. This in vivo strategy identified 84 stimulators of insulin expression, which simultaneously reduced glucose levels. The insulin promoter activation kinetics for 32 of these stimulators were consistent with a direct mode of action. A subset of insulin stimulators, including the antidiabetic drug pioglitazone, induced the coordinated upregulation of gluconeogenic pck1 expression, suggesting functional response to increased insulin action in peripheral tissues. Notably, Kv1.3 inhibitors increased ß-cell mass in larval zebrafish and stimulated ß-cell function in adult zebrafish and in the streptozotocin-induced hyperglycemic mouse model. In addition, our data indicate that cytoplasmic Kv1.3 regulates ß-cell function. Thus, using whole-organism screening, we have identified new small-molecule modulators of ß-cell function and glucose metabolism.

Screening for insulin-independent pathways that modulate glucose homeostasis identifies androgen receptor antagonists.

Pathways modulating glucose homeostasis independently of insulin would open new avenues to combat insulin resistance and diabetes. Here, we report the establishment, characterization, and use of a vertebrate 'insulin-free' model to identify insulin-independent modulators of glucose metabolism. insulin knockout zebrafish recapitulate core characteristics of diabetes and survive only up to larval stages. Utilizing a highly efficient endoderm transplant technique, we generated viable chimeric adults that provide the large numbers of insulin mutant larvae required for our screening platform. Using glucose as a disease-relevant readout, we screened 2233 molecules and identified three that consistently reduced glucose levels in insulin mutants. Most significantly, we uncovered an insulin-independent beneficial role for androgen receptor antagonism in hyperglycemia, mostly by reducing fasting glucose levels. Our study proposes therapeutic roles for androgen signaling in diabetes and, more broadly, offers a novel in vivo model for rapid screening and decoupling of insulin-dependent and -independent mechanisms.

Multiparameter screening reveals a role for Na+ channels in cytokine-induced ß-cell death.

Pancreatic ß-cell death plays a role in both type 1 and type 2 diabetes, but clinical treatments that specifically target ß-cell survival have not yet been developed. We have recently developed live-cell imaging-based, high-throughput screening methods capable of identifying factors that modulate pancreatic ß-cell death, with the hope of finding drugs that can intervene in this process. In the present study, we used a high-content screen and the Prestwick Chemical Library of small molecules to identify drugs that block cell death resulting from exposure to a cocktail of cytotoxic cytokines (25 ng/mL TNF-α, 10 ng/mL IL-1ß, and 10 ng/mL IFN-γ). Data analysis with self-organizing maps revealed that 19 drugs had profiles similar to that of the no cytokine condition, indicating protection. Carbamazepine, an antiepileptic Na(+) channel inhibitor, was particularly interesting because Na(+) channels are not generally considered targets for antiapoptotic therapy in diabetes and because the function of these channels in ß-cells has not been well studied. We analyzed the expression and characteristics of Na(+) currents in mature ß-cells from MIP-GFP mice. We confirmed the dose-dependent protective effects of carbamazepine and another use-dependent Na(+) channel blocker in cytokine-treated mouse islet cells. Carbamazepine down-regulated the proapoptotic and endoplasmic reticulum stress signaling induced by cytokines. Together, these studies point to Na(+) channels as a novel therapeutic target in diabetes.

Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.

Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.