Periodic Reporting for period 1 - IMPACT-HEALTH (IMaging Pancreatic Alpha-cells Calcium Tied with HEterogeneous Analysis of Labeled Transcription factors with in situ Hybridization)
Berichtszeitraum: 2018-09-17 bis 2020-09-16
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.
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.
An oblique selective plane illumination microscope (oSPIM) has been built and characterized in the first part of the project. The working principle of this system is very similar to other light-sheet architectures.
The optimization of calcium imaging within intact islets with light-sheet microscopy represented a crucial aspect towards the success of this proposal. In the reported period, I managed to optimize the protocol for the imaging experiments, in order to maintain the same physiological condition of culture. By fine tuning of the property of the microscope, I could obtain three dimensional reconstructions of the entire population of alpha cells within a single islet, for multiple islets coming from different animals, and promising preliminary data were collected during the time of the project.
Moreover, I optimized a novel protocol for single molecule fluorescence in situ hybridization in intact islets. This has been a very challenging task, since islets are a very complex environment, and the relatively low signal-to-noise ratio of the single fluorescent mRNAs is hard to distinguish from the background created by scattering. Nevertheless, by changing the protocol and testing different conditions, I was able to overcome this issue, reporting smFISH experiments in intact murine islets using light-sheet microscopy. I tested different probes targeting several genes known to be involved in the regulation of glucagon secretion, such as PDX1, GPR40, GCK (glucokinase), GCG (glucagon), MAFB. GCG showed the best results, given the abundance of the gene in single alpha cells. Interestingly, the fluorescence signature of each cell within the islet showed heterogeneity in the expression of the gene, suggesting for the first time the evidence of the existence of subpopulations oh alpha cells within the intact islet, which supported the hypothesis of the project.
Unfortunately, the ongoing COVID pandemic and the subsequent lockdowns have prevented much of the correlative work that was supposed to be carried out to verify the relationship between heterogeneities in calcium activity and gene expression underlying regulation of glucagon secretion. This part is still work in progress.
At the same way, exploitation and dissemination have been limited to the first part of the project. Nevertheless, I obtained a travel award to participate in the 2019 Biophysical Society meeting, where I was able to present part of the work done in the construction and characterization of the optical architecture.
Later, in February 2019, I was able to travel to Washington University in St. Louis, where I presented my work at a seminar at the Cell Biology & Physiology department.
In march 2019, I presented my work in progress at the San Raffaele Scientific Institute retreat, where I met new colleagues and established new collaborations, especially with the Diabetes Research Institute groups.
Later, I presented my developing project at internal seminars in the institute and used social media to disseminate part of my work. Two publication stemming from the work done during the project are being prepared for submission. No website has been developed for the project.
The optimization of calcium imaging within intact islets with light-sheet microscopy represented a crucial aspect towards the success of this proposal. In the reported period, I managed to optimize the protocol for the imaging experiments, in order to maintain the same physiological condition of culture. By fine tuning of the property of the microscope, I could obtain three dimensional reconstructions of the entire population of alpha cells within a single islet, for multiple islets coming from different animals, and promising preliminary data were collected during the time of the project.
Moreover, I optimized a novel protocol for single molecule fluorescence in situ hybridization in intact islets. This has been a very challenging task, since islets are a very complex environment, and the relatively low signal-to-noise ratio of the single fluorescent mRNAs is hard to distinguish from the background created by scattering. Nevertheless, by changing the protocol and testing different conditions, I was able to overcome this issue, reporting smFISH experiments in intact murine islets using light-sheet microscopy. I tested different probes targeting several genes known to be involved in the regulation of glucagon secretion, such as PDX1, GPR40, GCK (glucokinase), GCG (glucagon), MAFB. GCG showed the best results, given the abundance of the gene in single alpha cells. Interestingly, the fluorescence signature of each cell within the islet showed heterogeneity in the expression of the gene, suggesting for the first time the evidence of the existence of subpopulations oh alpha cells within the intact islet, which supported the hypothesis of the project.
Unfortunately, the ongoing COVID pandemic and the subsequent lockdowns have prevented much of the correlative work that was supposed to be carried out to verify the relationship between heterogeneities in calcium activity and gene expression underlying regulation of glucagon secretion. This part is still work in progress.
At the same way, exploitation and dissemination have been limited to the first part of the project. Nevertheless, I obtained a travel award to participate in the 2019 Biophysical Society meeting, where I was able to present part of the work done in the construction and characterization of the optical architecture.
Later, in February 2019, I was able to travel to Washington University in St. Louis, where I presented my work at a seminar at the Cell Biology & Physiology department.
In march 2019, I presented my work in progress at the San Raffaele Scientific Institute retreat, where I met new colleagues and established new collaborations, especially with the Diabetes Research Institute groups.
Later, I presented my developing project at internal seminars in the institute and used social media to disseminate part of my work. Two publication stemming from the work done during the project are being prepared for submission. No website has been developed for the project.
The optimization of the smFISH protocol in intact islet has been particularly relevant since most of the reports in literature performing smFISH in pancreatic tissue have relied on cryo-prepared thin slices of tissue, which have the limit of showing only few slices of an islet.In this way, it was possible to examine different subpopulation of cells with a differential gene expression within the same intact islet.
By showing differential expression in glucagon mRNA in alpha cells within the same islet, I could provide evidence of the existence of subpopulations of these cells, thus supporting the possibility of a role for glucagon as an intra-islet modulator in the regulation of blood glucose levels.
By exploiting this imaging approach with other important genes for the regulation of glucagon secretion, it will be possible to find novel therapeutic targets for the treatment and the cure of diabetes, in ways independent from insulin replacement.
However, the current pandemic and the subsequent lockdowns have prevented this project to achieve complete results, but the preliminary data obtained so far could pave the way for novel combinations of imaging and biochemical approaches in order to tackle the heterogeneity of alpha cells and exploit it at the therapeutical level.
By showing differential expression in glucagon mRNA in alpha cells within the same islet, I could provide evidence of the existence of subpopulations of these cells, thus supporting the possibility of a role for glucagon as an intra-islet modulator in the regulation of blood glucose levels.
By exploiting this imaging approach with other important genes for the regulation of glucagon secretion, it will be possible to find novel therapeutic targets for the treatment and the cure of diabetes, in ways independent from insulin replacement.
However, the current pandemic and the subsequent lockdowns have prevented this project to achieve complete results, but the preliminary data obtained so far could pave the way for novel combinations of imaging and biochemical approaches in order to tackle the heterogeneity of alpha cells and exploit it at the therapeutical level.