IMPACT-HEALTH · IMaging Pancreatic Alpha-cells Calcium Tied with HEterogeneous Analysis of Labeled Transcription factors with in situ Hybridization

IMaging Pancreatic Alpha-cells Calcium Tied with HEterogeneous Analysis of Labeled Transcription factors with in situ Hybridization

When blood glucose levels are elevated, insulin secretion from beta cells in the islet of Langerhans normally acts to restore the equilibrium. The action of insulin is countered by glucagon (which is secreted by alpha-cells) when glucose levels are low. Diabetes disrupts this balance, and while insulin defects are well-understood, aberrant glucagon secretion can also exacerbate the disease. Diabetes in Europe affects around 60 million people (as reported by the World Health Organization), and this number is only expected to grow in the future. For many decades, treatment of diabetes has relied mainly on insulin replacement (injections to patients), which allows people with the disease to live a regular life, but it does not represent a long-term cure. Since aberrant glucagon secretion has shown to exacerbate hyperglycemia and to lead to an increased risk of hypoglycemia, this study have proposed to extend the current knowledge about how glucagon secretion is regulated. To normalize glucagon secretion as a therapeutic strategy, it is crucial to have a better understanding of its regulatory mechanisms and the expression of specific genes regulating those mechanisms. The overall objective has been to exploit this differential gene expression, in order to target specific genes triggering various single-cells behaviors (for example minimizing or maximizing glucagon secretion in certain conditions) and discover novel therapeutic targets independent from insulin. Even if the ongoing pandemic and the subsequent lockdowns have prevented the full conclusion of the project, it was possible to achieve some important milestones, such as building and optimizing an oblique selective plane illumination microscope (oSPIM). This system is a light-sheet-based fluorescence microscope, an advanced optical microscopy technique that allows to watch the inside of cells with fine detail and fast speeds. Additionally, the system was made available to the experimental imaging center at the San Raffaele Scientific Institute, and many collaborators (Including people from the Diabetes Research Institute) have had the possibility of using this microscope for their research. Using this microscope, we were able to deeply characterize the calcium heterogeneities in alpha cells within intact islets of Langerhans. Moreover, we optimized a protocol to perform single molecule fluorescence in situ hybridization (smFISH, a technique used to detect specific sequences of RNA targets in single cells) in intact islets, and we could observe heterogeneities in the expression of the mature glucagon mRNA, which supported the hypothesis that there are indeed different subpopulations of alpha cells, at least at the post-transcriptional level.

Data: CORDIS, © European Union