Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo β-Cell Function
Summary
Here, the study presents a protocol for calcium imaging and glucose stimulation of the pancreatic β-cells of the zebrafish in vivo.
Abstract
The pancreatic β-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in β-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, β-cells experience an influx of Ca2+. Thus, imaging the glucose-stimulated Ca2+ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying β-cell function. Previously, studies showed that isolated zebrafish islets expressing GCaMP6s exhibit significant Ca2+ activity upon stimulation with defined glucose concentrations. However, it is paramount to study how β-cells respond to glucose not in isolation, but in their native environment, where they are systemically connected, vascularized, and densely innervated. To this end, the study leveraged the optical transparency of the zebrafish larvae at early stages of development to illuminate β-cell activity in vivo. Here, a detailed protocol for Ca2+ imaging and glucose stimulation to investigate β-cell function in vivo is presented. This technique allows to monitor the coordinated Ca2+ dynamics in β-cells with single-cell resolution. Additionally, this method can be applied to work with any injectable solution such as small molecules or peptides. Altogether, the protocol illustrates the potential of the zebrafish model to investigate islet coordination in vivo and to characterize how environmental and genetic components might affect β-cell function.
Introduction
The pancreatic β-cells exhibit the unique capability for insulin secretion in response to glucose. After a carbohydrate-rich meal, the blood sugar increases and enters the β-cells, where it is quickly metabolized to produce ATP. The increase in the intracellular ratio of ATP/ADP leads to the closure of the ATP-dependent K+ channels, depolarizing the cell membrane and activating the voltage-dependent Ca2+ channels. The rapid increase in intracellular Ca2+ stimulates insulin-granule secretion by the β-cells.
Imaging of the islet cells within the intact pancreas in mice is demanding and requires exteriorization of the whole organ. A powerful alternative for non-invasive in vivo imaging is to transplant islets into the anterior chamber (AC) of the eye of mice1. Transplanted islets into the AC of mice mimics an in vivo environment, allowing longitudinal studies and Ca2+ imaging in vivo2,3. Nevertheless, the process of islet revascularization can take several weeks after islet transplantation4. Thus, preserving the original native environment, where the β-cells are vascularized and connected to the metabolic status of the organism, and achieving in vivo single-cell imaging resolution remain very challenging and time consuming.
To overcome these limitations, researchers have developed zebrafish transgenic lines expressing GECIs in β-cells, which allow for real-time visualization of Ca2+ influx in β-cells5,6,7,8,9. Using these tools, it was found that in cell culture, zebrafish β-cells show a conserved response to elevated glucose, similar to mouse and human islets. Moreover, the optical clarity of the zebrafish larvae has allowed to examine the function of β-cells in their native environment. Importantly, as early as 4 days post fertilization (dpf), the β-cells of the zebrafish primary islet express markers of maturity, such as the zebrafish orthologue of urocortin3 (ucn3l) and show in vitro responses to glucose in the physiological range6. In addition, the zebrafish islet is densely vascularized and innervated10. The genetic ablation of β-cells at this stage leads to glucose intolerance. Furthermore, the zebrafish shows conserved responses to glucose-lowering agents such as insulin and sulfonylureas, showing that the primary islet is a glucose responsive and systemically connected tissue controlling glucose levels. These special characteristics make the zebrafish a unique model to study islet β-cell activity in vivo11,12.
Theprotocol for Ca2+ imaging and simultaneous glucose injection directly in the circulation of zebrafish allows investigating the β-cell's glucose responsiveness in vivo. This protocol enables the injection of defined glucose concentrations in combination with high temporal and spatial resolution, which altogether reveal the coordinated response of β-cells to glucose and the presence of first-responder β-cells in vivo13. Moreover, the protocol can be adapted to any injectable solution such as chemical compounds or small peptides. Overall, this methodology illustrates the strength of the zebrafish model to investigate β-cell coordination in vivo and to characterize how environmental and genetic components might affect β-cells' function.
Protocol
Representative Results
Discussion
In this protocol, the Ca2+ dynamics of β-cells in their native microenvironment with single-cell resolution was explored. This is possible by stimulating the zebrafish β-cells with a glucose injection in the circulation while recording their Ca2+ dynamics using GCaMP6s.
The protocol provides three main advantages. First, researchers have demonstrated that zebrafish β-cells show a coordinated response to glucose stimulation in vivo…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Nikolay Ninov received funding from the Center for Regenerative Therapies Dresden at TU Dresden and the German Center for Diabetes Research (DZD), as well as research grants from the German Research Foundation (DFG) and the International Research Training Group (IRTG 2251), Immunological and Cellular Strategies in Metabolic Disease. We are grateful to the Light Microscopy Facility at the CRTD for the support in all the imaging techniques. We thank the Fish Facility at the CRTD for all the fish technical assistance and support.
Materials
| 35 mm diameter glass-bottom dishes | Mattek | P35G-1.5-14-C | We use this glass-bottom dish to mount the zebrafish larvae and perform confocal microscopy |
| Blue-Green filter cube | ZEISS | 489038-9901-000 | Filter Set 38 HE |
| Confocal Microscope | ZEISS | LSM 980 | |
| D-(+)-Glucose | Sigma-Aldrich | G7528 | |
| Excel (2016) | https://www.office.com/ | ||
| Femtojet | Eppendorf | 5252000013 | This equipment is a pneumatic micropump, which allows precise volume delivery and is accompanied by a capillary holder. We use the micropump Femtojet (injection pressure between 500-1000 hPa; compensation pressure = 0 hPa; and delivery time = 1 second). |
| Femtotips, glass capillaries ready to be used. | Eppendorf | 5242952008 | This are ready to use glass capillaries that can substitute the pulled-capillaries. |
| FIJI, using ImageJ Version: 1.51c | https://fiji.sc/ | ||
| Fluorescence lamp | ZEISS | 423013-9010-000 | Illuminator HXP 120 V |
| Glass capillaries 3.5" | Drummonds Scientific Company | 3-000-203-G/X | We use these glass capillaries to prepare the injection capillaries by pulling them with a capillary -puller |
| Injectman | Eppendorf | 5192000019 | This equipment allows for 3D manipulation of the capillary holder |
| Low melting agarose | Biozym | Art. -Nr.: 840101 | We use the agarose to mount the zebrafish onto the glass-bottom dish |
| Microloader tip for glass microcapillaries 0.5 – 20 µL, 100 mm | Eppendorf | 5242956003 | Long tips for loading the glass capillaries with the solutions |
| Micro-tweezers | Dumont Swiss made | 0102-4-PO | We use the micro-tweezers to move the zebrafish larvae during the mounting and to cut off the tip of the glass capillary (eg. Dumont, size 4). |
| Mineral oil | Sigma-Aldrich | M5904 | We use the mineral oil to calibrate the drop size to inject |
| P-1000 Next Generation Pipette Puller | Science Products | P-1000 | We use this capillary puller to prepare the glass capillary using the Drumond capillaries. We use the P-1000 capillary puller with the following parameters: Heat: 650, Pull: 20, Vel: 160, Time: 200, Pressure: 500. |
| PTU (1-phenyl-2-thiourea ) | Sigma-Aldrich | P7629 | We use this compound to inhibit pigmentation during zebrafish development |
| Red Filter Cube | ZEISS | 000000-1114-462 | Filter set 45 HQ TexasRed |
| Stereo microscope | ZEISS | 495015-0001-000 | SteREO Discovery.V8 |
| Syringe filters, sterile. Pore size 0.2 µm | Pall Corporation | 4612 | We use these to filter all the solutions and prevent the capillary needle clothing |
| Transgenic Zebrafish line:Tg(ins:cdt1-mCherry;cryaa:CFP); Tg(ins:GCaMP6s;cryaa:mCherry) | |||
| tricaine methanesulfonate (MS222) | Sigma-Aldrich | E10521 | We use this compound to anesthetize the fish larvae |
References
- Speier, ., et al. Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye. Nature Protocol. 3 (8), 1278-1286 (2008).
- Jacob, S., et al. In vivo Ca(2+) dynamics in single pancreatic beta cells. Federation of American Society of Experimental Biology Journal. 34 (1), 945-959 (2020).
- Chen, C., et al. Alterations in beta-cell calcium dynamics and efficacy outweigh islet mass adaptation in compensation of insulin resistance and prediabetes onset. Diabetes. 65 (9), 2676-2685 (2016).
- Cohrs, C. M., et al. Vessel network architecture of adult human islets promotes distinct cell-cell interactions in situ and is altered after transplantation. Endocrinology. 158 (5), 1373-1385 (2017).
- Janjuha, S., Pal Singh, S., Ninov, N. Analysis of beta-cell function using single-cell resolution calcium imaging in zebrafish islets. Journal of Visualized Experiments: JoVE. (137), (2018).
- Singh, S. P., et al. Different developmental histories of beta-cells generate functional and proliferative heterogeneity during islet growth. Nature Communications. 8 (1), 664 (2017).
- Lorincz, R., et al. In vivo monitoring of intracellular Ca2+ dynamics in the pancreatic beta-cells of zebrafish embryos. Islets. 10 (6), 221-238 (2018).
- Mullapudi, S. T., et al. Disruption of the pancreatic vasculature in zebrafish affects islet architecture and function. Development. 146 (21), (2019).
- Zhao, J., et al. In vivo imaging of beta-cell function reveals glucose-mediated heterogeneity of beta-cell functional development. eLife. 8, 41540 (2019).
- Yang, Y. H. C., Kawakami, K., Stainier, D. Y. A new mode of pancreatic islet innervation revealed by live imaging in zebrafish. eLife. 7, 34519 (2018).
- Eames, S. C., Philipson, L. H., Prince, V. E., Kinkel, M. D. Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis. Zebrafish. 7 (2), 205-213 (2010).
- Nath, A. K., et al. PTPMT1 inhibition lowers glucose through succinate dehydrogenase phosphorylation. Cell Reports. 10 (5), 694-701 (2015).
- Salem, V., et al. Leader β-cells coordinate Ca2+ dynamics across pancreatic islets in vivo. Nature Metabolism. 1 (6), 615-629 (2019).
- Ninov, N., et al. Metabolic regulation of cellular plasticity in the pancreas. Current Biology. 23 (13), 1242-1250 (2013).
- Nüsslein-Volhard, C., Dahm, R. . Zebrafish: A Practical Approach. , (2002).
- Isogai, S., Horiguchi, M., Weinstein, B. M. The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Developmental Biology. 230 (2), 278-301 (2001).
- Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nature Methods. 9 (7), 676-682 (2012).
- Delgadillo-Silva, L. F., et al. Modelling pancreatic beta-cell inflammation in zebrafish identifies the natural product wedelolactone for human islet protection. Disease Models & Mechanisms. 12 (1), 036004 (2019).
- Emfinger, C. H., et al. Beta-cell excitability and excitability-driven diabetes in adult zebrafish islets. Physiological Reports. 7 (11), 14101 (2019).
- Zhang, M., Goforth, P., Bertram, R., Sherman, A., Satin, L. The Ca2+ dynamics of isolated mouse beta-cells and islets: implications for mathematical models. Biophysical Journal. 84 (5), 2852-2870 (2003).
.