Final Report Summary - HCSP IN BETA-CELLS (The highly calcium-sensitive pool of granules in biphasic insulin secretion: Experimental and theoretical investigations)
Diabetes prevalence unfortunately shows an increase, and it is estimated by the International Diabetes Federation that in the European region 8.4 % of the adult population (20 - 79 years) had diabetes in 2007, a number expected to increase to 9.8 % by 2025 (see http://www.eatlas.idf.org/index29fc.html(öffnet in neuem Fenster) for further details). The economic costs of the disease are considerable with, for example, Germany spending EUR 30 billion, the United Kingdom (UK) approximately 5.2 billion, and other countries generally using 5 - 11 % of their total healthcare budget, on diabetes (see http://ec.europa.eu/health/ph_information/dissemination/diseases/diabet8.pdf(öffnet in neuem Fenster) for details).For comparison, in the United States (US) approximately 8 % of the population (9.2 % of the adult population, see http://www.eatlas.idf.org/index3637.html(öffnet in neuem Fenster) for details) had diabetes and the costs were estimated to be USD 174 billion in 2007 (see http://www.diabetes.org/diabetesstatistics.jsp(öffnet in neuem Fenster) for details).
Diabetes is a result of insufficient release of the blood-sugar-lowering hormone insulin. Insulin secretion is biphasic in response to a sudden elevation in glucose levels with a first peak of secretion lasting 5 - 10 minutes followed by a sustained or rising second phase of secretion. The loss of the first phase is an early marker of type 2 diabetes, and the understanding of the regulation of biphasic insulin secretion is therefore of great clinical importance.
The present interdisciplinary project has investigated the cellular mechanisms behind biphasic insulin secretion from the pancreatic beta-cells, which are the only cells that produce and secrete insulin. Insulin is stored in secretory granules, and following a series of events following a glucose stimulus involving glucose metabolism and electrical activity, calcium flows into the cells through membrane channels and trigger exocytosis, a process where the granules fuse with the cell membrane, which allows the insulin molecules to be released to the extra-cellular space and enter the blood stream. It has been suggested that granules can be divided into different pools depending on their release-competence, and that biphasic insulin secretion results from the depletion of a readily releasable pool (RRP) of granules, with is then refilled more slowly from a reserve pool.
We have used the patch-clamp technique and mathematical modeling to study the process of calcium-regulated exocytosis, with particular attention to a putative immediately releasable pool (IRP) of granules, which has been suggested to be a subpool of the RRP located near calcium channels. Theoretical considerations suggested that the data underlying the suggestion of depletion of the IRP could alternatively be explained by inactivation of the calcium currents, and in particular that it is necessary to relate the amount of exocytosis to the amount of calcium flowing into the cell in order to discuss pool depletion (Pedersen, Biophys J., 2011).
These theoretical results were then used to study patch-clamp data using the appropriate statistical mixed-effects model. We reanalysed previously published results (Pedersen et al., Prog Biophys Mol Biol, 2011), as well as new data obtained during the project, both from insulin secretion cells (EASD oral presentation 2011, manuscript in preparation) and from their close cousins, the glucagon-secreting alpha-cells (Andersson et al., Pfl Arch, 2011).
We found no evidence of pool depletion corresponding to an IRP in either of these cases. We are currently modifying the experimental protocol in order to investigate this finding more closely. However, we found that insulin-secreting INS-1 832/13 cells contain a very small (2 - 3 granules) pool which is released in response to a minimal calcium influx. This pool is not influenced by the presence of high concentrations of the calcium buffer EGTA, in contrast to the vast amount of exocytosis. EGTA has little effect near calcium channels, which suggests that most of the RRP is located at some distance from the channels. We are currently modelling calcium diffusion and buffering in order to get more knowledge about the spatial location of the secretory granules.
Translating results from cell lines and animal studies to humans is complicated because of differences in e.g. electrophysiological properties. During the project, we have build mathematical models for exocytosis in human beta-cells (Pedersen et al., Prog Biophys Mol Biol, 2011) and the first published model of electrical activity in human beta-cells (Pedersen, Biophys J, 2010). Based on these studies we concluded that in human beta-cells the secretory granules are likely located further away from calcium channels than in their rodent counterparts, and that their electrophysiological properties allow electrical activity, which shows both similarities and differences compared to mouse beta-cells. This highlights the need for further studies on human donor cells. The models concerning human beta-cells are currently being coupled to models of in vivo human data operating on a different scale (Pedersen et al., IEEE Trans Biomed Eng, 2011).
Besides the study of electrophysiology and exocytosis in beta-cells, we have also used calcium imaging of intact human and mouse pancreatic islets, in order to understand whether different ion channels are involved in the activation of beta-cells, and to measure calcium dynamics in response to glucose.
In conclusion, the project has given new insight in the pool of secretory granules believed to underlie the first phase of insulin secretion. Moreover, it has provided new important tools for the correct analysis of exocytosis data from both insulin-secreting and other cell types, as well as mathematical models of human beta-cells, which should be valuable for further investigations of defective insulin secretion, and possibly for the development of pharmacological treatments of diabetes.
Diabetes is a result of insufficient release of the blood-sugar-lowering hormone insulin. Insulin secretion is biphasic in response to a sudden elevation in glucose levels with a first peak of secretion lasting 5 - 10 minutes followed by a sustained or rising second phase of secretion. The loss of the first phase is an early marker of type 2 diabetes, and the understanding of the regulation of biphasic insulin secretion is therefore of great clinical importance.
The present interdisciplinary project has investigated the cellular mechanisms behind biphasic insulin secretion from the pancreatic beta-cells, which are the only cells that produce and secrete insulin. Insulin is stored in secretory granules, and following a series of events following a glucose stimulus involving glucose metabolism and electrical activity, calcium flows into the cells through membrane channels and trigger exocytosis, a process where the granules fuse with the cell membrane, which allows the insulin molecules to be released to the extra-cellular space and enter the blood stream. It has been suggested that granules can be divided into different pools depending on their release-competence, and that biphasic insulin secretion results from the depletion of a readily releasable pool (RRP) of granules, with is then refilled more slowly from a reserve pool.
We have used the patch-clamp technique and mathematical modeling to study the process of calcium-regulated exocytosis, with particular attention to a putative immediately releasable pool (IRP) of granules, which has been suggested to be a subpool of the RRP located near calcium channels. Theoretical considerations suggested that the data underlying the suggestion of depletion of the IRP could alternatively be explained by inactivation of the calcium currents, and in particular that it is necessary to relate the amount of exocytosis to the amount of calcium flowing into the cell in order to discuss pool depletion (Pedersen, Biophys J., 2011).
These theoretical results were then used to study patch-clamp data using the appropriate statistical mixed-effects model. We reanalysed previously published results (Pedersen et al., Prog Biophys Mol Biol, 2011), as well as new data obtained during the project, both from insulin secretion cells (EASD oral presentation 2011, manuscript in preparation) and from their close cousins, the glucagon-secreting alpha-cells (Andersson et al., Pfl Arch, 2011).
We found no evidence of pool depletion corresponding to an IRP in either of these cases. We are currently modifying the experimental protocol in order to investigate this finding more closely. However, we found that insulin-secreting INS-1 832/13 cells contain a very small (2 - 3 granules) pool which is released in response to a minimal calcium influx. This pool is not influenced by the presence of high concentrations of the calcium buffer EGTA, in contrast to the vast amount of exocytosis. EGTA has little effect near calcium channels, which suggests that most of the RRP is located at some distance from the channels. We are currently modelling calcium diffusion and buffering in order to get more knowledge about the spatial location of the secretory granules.
Translating results from cell lines and animal studies to humans is complicated because of differences in e.g. electrophysiological properties. During the project, we have build mathematical models for exocytosis in human beta-cells (Pedersen et al., Prog Biophys Mol Biol, 2011) and the first published model of electrical activity in human beta-cells (Pedersen, Biophys J, 2010). Based on these studies we concluded that in human beta-cells the secretory granules are likely located further away from calcium channels than in their rodent counterparts, and that their electrophysiological properties allow electrical activity, which shows both similarities and differences compared to mouse beta-cells. This highlights the need for further studies on human donor cells. The models concerning human beta-cells are currently being coupled to models of in vivo human data operating on a different scale (Pedersen et al., IEEE Trans Biomed Eng, 2011).
Besides the study of electrophysiology and exocytosis in beta-cells, we have also used calcium imaging of intact human and mouse pancreatic islets, in order to understand whether different ion channels are involved in the activation of beta-cells, and to measure calcium dynamics in response to glucose.
In conclusion, the project has given new insight in the pool of secretory granules believed to underlie the first phase of insulin secretion. Moreover, it has provided new important tools for the correct analysis of exocytosis data from both insulin-secreting and other cell types, as well as mathematical models of human beta-cells, which should be valuable for further investigations of defective insulin secretion, and possibly for the development of pharmacological treatments of diabetes.