analysis-beta-cell-function-using-single-cell-resolution-calcium

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

simultaneous-calcium-imaging-glucose-stimulation-living-zebrafish-to

Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo &#946;-Cell Function

The pancreatic &#946;-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in ...

TU Dresden

Paul Langerhans Institute Dresden of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus of TU Dresden

Centre for Regenerative Therapies TU Dresden

The pancreatic beta-cells control glucose homeostasis by secreting insulin in response to nutrient intake. The number of beta-cells is under tight metabolic control, as this number increases with higher nutrient intake. However, the signaling pathways matching nutrition with beta-cell mass plasticity remain poorly defined. By applying pharmacological and genetic manipulations, we show that reactive oxygen species (ROS) regulate dose-dependently beta-cell proliferation in vivo and in vitro. In particular, reducing ROS levels in beta-cells blocks their proliferation in response to nutrients. Using a non-invasive genetic sensor of intracellular hydrogen peroxide (HO), we reveal that glucose can directly increase the levels of HO. Furthermore, a moderate increase in HO levels can stimulate beta-cell proliferation. Interestingly, while high HO levels are inhibitory to beta-cell proliferation, they expand beta-cell mass in vivo by inducing rapid beta-cell neogenesis. Our study thus reveals a ROS-level-dependent mechanism linking nutrients with beta-cell mass plasticity. Hence, given the requirement of ROS for beta-cell mass expansion, antioxidant therapies should be applied with caution in diabetes.

Distinct Levels of Reactive Oxygen Species Coordinate Metabolic Activity with Beta-cell Mass Plasticity.

Traditionally, the development of novel therapeutics for metabolic diseases has relied mainly on high-throughput screening using biochemical or cell-based assays. While this approach represents a driving force in drug discovery, there is also a need to perform large-scale screens without disrupting inter-organ communication and tissue architecture, essential components for understanding the complexity of metabolic regulation and the identification of small molecules with appropriate biological activities in vivo. Hence, the zebrafish Danio rerio is gaining popularity in metabolic research and drug discovery, as this animal model allows screening of small molecules in the context of the whole-organism. Moreover, the zebrafish exhibits conserved function of the pancreas, liver and adipose tissue, which can be leveraged to identify novel targets in metabolic regulation, as well as to study the role of conserved genes associated with the risk of metabolic diseases in humans. Here we highlight recent advances in the identification of targets in metabolic regulation using the zebrafish as a model.

Catching new targets in metabolic disease with a zebrafish.

Islet inflammation and cytokine production are implicated in pancreatic β-cell dysfunction and diabetes pathogenesis. However, we lack therapeutics to protect the insulin-producing β-cells from inflammatory damage. Closing this clinical gap requires the establishment of new disease models of islet inflammation to facilitate screening efforts aimed at identifying new protective agents. Here, we have developed a genetic model of Interleukin-1β (Il-1β)-driven islet inflammation in zebrafish, a vertebrate that allows for non-invasive imaging of β-cells and  drug discovery. Live imaging of immune cells and β-cells in our model revealed dynamic migration, increased visitation and prolonged macrophage retention in the islet, together with robust activation of NF-κB signalling in β-cells. We find that Il-1β-mediated inflammation does not cause β-cell destruction but, rather, it impairs β-cell function and identity. , β-cells exhibit impaired glucose-stimulated calcium influx and reduced expression of genes involved in function and maturity. These defects are accompanied by α-cell expansion, glucose intolerance and hyperglycemia following a glucose challenge. Notably, we show that a medicinal plant derivative (wedelolactone) is capable of reducing the immune-cell infiltration while also ameliorating the hyperglycemic phenotype of our model. Importantly, these anti-diabetic properties in zebrafish are predictive of wedelolactone's efficacy in protecting rodent and human islets from cytokine-induced apoptosis. In summary, this new zebrafish model of diabetes opens a window to study the interactions between immune and β-cells , while also allowing the identification of therapeutic agents for protecting β-cells from inflammation.

Modelling pancreatic β-cell inflammation in zebrafish identifies the natural product wedelolactone for human islet protection.

During embryogenesis, beta-cells arise from the dorsal and ventral bud originating in the endoderm germ layer. As the animal develops to adulthood, the beta-cell mass dramatically increases. The expansion of the beta-cell population is driven by cell division among the embryonic beta-cells and supplanted by neogenesis from post-embryonic progenitors. Here, we describe a protocol for multicolor clonal analysis in zebrafish to define the contribution of individual embryonic beta-cells to the increase in cell numbers. This technique provides insights into the proliferative history of individual beta-cells in an islet. This insight helps in defining the replicative heterogeneity among individual beta-cells during development. Additionally, the ability to discriminate individual cells based on unique color signatures helps quantify the volume occupied by beta-cells and define the contribution of cellular size to the beta-cell mass.

Multicolor Labeling and Tracing of Pancreatic Beta-Cell Proliferation in Zebrafish.

Pancreatic β-cells form highly connected networks within isolated islets. Whether this behaviour pertains to the situation in vivo, after innervation and during continuous perfusion with blood, is unclear. In the present study, we used the recombinant Ca sensor GCaMP6 to assess glucose-regulated connectivity in living zebrafish Danio rerio, and in murine or human islets transplanted into the anterior eye chamber. In each setting, Ca waves emanated from temporally defined leader β-cells, and three-dimensional connectivity across the islet increased with glucose stimulation. Photoablation of zebrafish leader cells disrupted pan-islet signalling, identifying these as likely pacemakers. Correspondingly, in engrafted mouse islets, connectivity was sustained during prolonged glucose exposure, and super-connected 'hub' cells were identified. Granger causality analysis revealed a controlling role for temporally defined leaders, and transcriptomic analyses revealed a discrete hub cell fingerprint. We thus define a population of regulatory β-cells within coordinated islet networks in vivo. This population may drive Ca dynamics and pulsatile insulin secretion.

ArticleTotal : 2	Year
Publication title Cited by 9	2018
Publication title Cited by 1	2021

Article	Year
Distinct Levels of Reactive Oxygen Species Coordinate Metabolic Activity with Beta-cell Mass Plasticity. Scientific reports\| PubMed ID: 28652605	2017
Catching new targets in metabolic disease with a zebrafish. Current opinion in pharmacology\| PubMed ID: 28888214	2017
Modelling pancreatic β-cell inflammation in zebrafish identifies the natural product wedelolactone for human islet protection. Disease models & mechanisms\| PubMed ID: 30679186	2019
Multicolor Labeling and Tracing of Pancreatic Beta-Cell Proliferation in Zebrafish. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 32180193	2020
Leader β-cells coordinate Ca dynamics across pancreatic islets in vivo. Nature metabolism\| PubMed ID: 32694805	2019

Nikolay Ninov

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