| 1. |
- Alenkvist, Ida, et al.
(författare)
-
Recruitment of Epac2A to Insulin Granule Docking Sites Regulates Priming for Exocytosis
- 2017
-
Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 66:10, s. 2610-2622
-
Tidskriftsartikel (refereegranskat)abstract
- Epac is a cAMP-activated guanine nucleotide exchange factor that mediates cAMP signaling in various types of cells, including -cells, where it is involved in the control of insulin secretion. Upon activation, the protein redistributes to the plasma membrane, but the underlying molecular mechanisms and functional consequences are unclear. Using quantitative high-resolution microscopy, we found that cAMP elevation caused rapid binding of Epac2A to the -cell plasma membrane, where it accumulated specifically at secretory granules and rendered them more prone to undergo exocytosis. cAMP-dependent membrane binding required the high-affinity cyclic nucleotide-binding (CNB) and Ras association domains, but not the disheveled-Egl-10-pleckstrin domain. Although the N-terminal low-affinity CNB domain (CNB-A) was dispensable for the translocation to the membrane, it was critical for directing Epac2A to the granule sites. Epac1, which lacks the CNB-A domain, was recruited to the plasma membrane but did not accumulate at granules. We conclude that Epac2A controls secretory granule release by binding to the exocytosis machinery, an effect that is enhanced by prior cAMP-dependent accumulation of the protein at the plasma membrane.
|
|
| 2. |
|
|
| 3. |
- Cortese, Giuliana, et al.
(författare)
-
Statistical Frailty Modeling for Quantitative Analysis of Exocytotic Events Recorded by Live Cell Imaging : Rapid Release of Insulin-Containing Granules Is Impaired in Human Diabetic beta-cells
- 2016
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:12
-
Tidskriftsartikel (refereegranskat)abstract
- Hormones and neurotransmitters are released when secretory granules or synaptic vesicles fuse with the cell membrane, a process denoted exocytosis. Modern imaging techniques, in particular total internal reflection fluorescence (TIRF) microscopy, allow the investigator to monitor secretory granules at the plasma membrane before and when they undergo exocytosis. However, rigorous statistical approaches for temporal analysis of such exocytosis data are still lacking. We propose here that statistical methods from time-to-event (also known as survival) analysis are well suited for the problem. These methods are typically used in clinical settings when individuals are followed over time to the occurrence of an event such as death, remission or conception. We model the rate of exocytosis in response to pulses of stimuli in insulin-secreting pancreatic beta-cell from healthy and diabetic human donors using piecewise-constant hazard modeling. To study heterogeneity in the granule population we exploit frailty modeling, which describe unobserved differences in the propensity to exocytosis. In particular, we insert a discrete frailty in our statistical model to account for the higher rate of exocytosis in an immediately releasable pool (IRP) of insulin-containing granules. Estimates of parameters are obtained from maximum-likelihood methods. Since granules within the same cell are correlated, i.e., the data are clustered, a modified likelihood function is used for log-likelihood ratio tests in order to perform valid inference. Our approach allows us for example to estimate the size of the IRP in the cells, and we find that the IRP is deficient in diabetic cells. This novel application of time-to-event analysis and frailty modeling should be useful also for the study of other well-defined temporal events at the cellular level.
|
|
| 4. |
- Gandasi, Nikhil, et al.
(författare)
-
GLP-1 metabolite GLP-1(9-36) is a systemic inhibitor of mouse and human pancreatic islet glucagon secretion
- 2024
-
Ingår i: DIABETOLOGIA. - 0012-186X .- 1432-0428. ; 67:3, s. 528-546
-
Tidskriftsartikel (refereegranskat)abstract
- Aims/hypothesis Diabetes mellitus is associated with impaired insulin secretion, often aggravated by oversecretion of glucagon. Therapeutic interventions should ideally correct both defects. Glucagon-like peptide 1 (GLP-1) has this capability but exactly how it exerts its glucagonostatic effect remains obscure. Following its release GLP-1 is rapidly degraded from GLP-1(7-36) to GLP-1(9-36). We hypothesised that the metabolite GLP-1(9-36) (previously believed to be biologically inactive) exerts a direct inhibitory effect on glucagon secretion and that this mechanism becomes impaired in diabetes.Methods We used a combination of glucagon secretion measurements in mouse and human islets (including islets from donors with type 2 diabetes), total internal reflection fluorescence microscopy imaging of secretory granule dynamics, recordings of cytoplasmic Ca2+ and measurements of protein kinase A activity, immunocytochemistry, in vivo physiology and GTP-binding protein dissociation studies to explore how GLP-1 exerts its inhibitory effect on glucagon secretion and the role of the metabolite GLP-1(9-36).Results GLP-1(7-36) inhibited glucagon secretion in isolated islets with an IC50 of 2.5 pmol/l. The effect was particularly strong at low glucose concentrations. The degradation product GLP-1(9-36) shared this capacity. GLP-1(9-36) retained its glucagonostatic effects after genetic/pharmacological inactivation of the GLP-1 receptor. GLP-1(9-36) also potently inhibited glucagon secretion evoked by beta-adrenergic stimulation, amino acids and membrane depolarisation. In islet alpha cells, GLP-1(9-36) led to inhibition of Ca2+ entry via voltage-gated Ca2+ channels sensitive to omega-agatoxin, with consequential pertussis-toxin-sensitive depletion of the docked pool of secretory granules, effects that were prevented by the glucagon receptor antagonists REMD2.59 and L-168049. The capacity of GLP-1(9-36) to inhibit glucagon secretion and reduce the number of docked granules was lost in alpha cells from human donors with type 2 diabetes. In vivo, high exogenous concentrations of GLP-1(9-36) (>100 pmol/l) resulted in a small (30%) lowering of circulating glucagon during insulin-induced hypoglycaemia. This effect was abolished by REMD2.59, which promptly increased circulating glucagon by >225% (adjusted for the change in plasma glucose) without affecting pancreatic glucagon content.Conclusions/interpretation We conclude that the GLP-1 metabolite GLP-1(9-36) is a systemic inhibitor of glucagon secretion. We propose that the increase in circulating glucagon observed following genetic/pharmacological inactivation of glucagon signalling in mice and in people with type 2 diabetes reflects the removal of GLP-1(9-36)'s glucagonostatic action.
|
|
| 5. |
- Gandasi, Nikhil R., et al.
(författare)
-
Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes
- 2017
-
Ingår i: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 127:6, s. 2353-2364
-
Tidskriftsartikel (refereegranskat)abstract
- Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic β cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human β cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in β cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II-III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D.
|
|
| 6. |
- Gandasi, Nikhil R, et al.
(författare)
-
Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site
- 2014
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Docking of secretory vesicles at the plasma membrane is a poorly understood prerequisite for exocytosis. Current models propose raft-like clusters containing syntaxin as docking receptor, but direct evidence for this is lacking. Here we provide quantitative measurements of several exocytosis proteins (syntaxin, SNAP25, munc18, munc13 and rab3) at the insulin granule release site and show that docking coincides with rapid de novo formation of syntaxin1/munc18 clusters at the nascent docking site. Formation of such clusters prevents undocking and is not observed during failed docking attempts. Overexpression of syntaxins' N-terminal Habc-domain competitively interferes with both cluster formation and successful docking. SNAP25 and munc13 are recruited to the docking site more than a minute later, consistent with munc13's reported role in granule priming rather than docking. We conclude that secretory vesicles dock by inducing syntaxin1/munc18 clustering in the target membrane, and find no evidence for preformed docking receptors.
|
|
| 7. |
- Gandasi, Nikhil R, et al.
(författare)
-
Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes
- 2018
-
Ingår i: Cell Metabolism. - : Elsevier BV. - 1550-4131 .- 1932-7420. ; 27:2, s. 470-478
-
Tidskriftsartikel (refereegranskat)abstract
- Glucose-stimulated insulin secretion is biphasic, with a rapid first phase and a slowly developing sustained second phase; both are disturbed in type 2 diabetes (T2D). Biphasic secretion results from vastly different release probabilities of individual insulin granules, but the morphological and molecular basis for this is unclear. Here, we show that human insulin secretion and exocytosis critically depend on the availability of membrane-docked granules and that T2D is associated with a strong reduction in granule docking. Glucose accelerated granule docking, and this effect was absent in T2D. Newly docked granules only slowly acquired release competence; this was regulated by major signaling pathways, but not glucose. Gene expression analysis indicated that key proteins involved in granule docking are downregulated in T2D, and overexpression of these proteins increased granule docking. The findings establish granule docking as an important glucose-dependent step in human insulin secretion that is dysregulated in T2D.
|
|
| 8. |
- Gandasi, Nikhil R.
(författare)
-
Molecular mechanisms of biphasic insulin secretion
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pancreatic beta-cells secrete insulin in response to increase in blood glucose concentration with a rapid first phase and slower, sustained second phase. This secretion pattern is similar in entire pancreas, isolated islets of Langerhans and single beta-cells and it is disrupted in type 2-diabetes. Insulin stored in secretory vesicles has to undergo preparatory steps upon translocation to the plasma membrane which include docking and priming before being released by exocytosis. A better understanding of the molecules involved in these steps is required to determine the rate limiting factors for sustained secretion. Here these processes were studied in real time using total internal reflection fluorescence microscopy, which enables observation of insulin granules localized at the plasma membrane. A pool of granules morphologically docked at the plasma membrane was found to be depleted upon repeated stimulations. Recovery of the docked pool of granules took tens of minutes and became rate limiting for sustained secretion. Shorter depolarization stimuli did not deplete the docked pool and allowed rapid recovery of releasable granules. When a new granule arrived at the plasma membrane, docking was initiated by de novo formation of syntaxin/munc18 clusters at the docking site. Two-thirds of the granules which arrived at the plasma membrane failed to recruit these proteins and hence failed to dock. Priming involved recruitment of several other proteins including munc13, SNAP25 and Cav1.2 channels. Exocytosing granules were in close proximity to Ca2+ influx sites with high degree of association with Cav1.2 channels. This is because of the association of these channels to exocytosis site through syntaxin and SNAP25. During exocytosis the assembled release machinery disintegrated and the proteins at the release site dispersed. Syntaxin dispersal was initiated already during fusion pore formation rather than after release during exocytosis. This was studied using a newly developed red fluorescent probe - NPY-tdmOrange2 which was the most reliable pH sensitive red granule marker to label insulin granules. Overall these data give new insights into the molecular mechanisms involved in biphasic insulin secretion. Disturbances in the secretion at the level of granule docking and fusion may contribute to the early manifestations of type-2 diabetes.
|
|
| 9. |
- Gandasi, Nikhil R., et al.
(författare)
-
Pancreatic Islet Biobanking Facilities in India: The Need of the Hour to Deal with Diabetes?
- 2023
-
Ingår i: Journal of the Indian Institute of Science. - : Springer. - 0970-4140 .- 0019-4964. ; 103, s. 381-385
-
Tidskriftsartikel (refereegranskat)abstract
- Endocrine pancreas regulates glucose homeostasis and prevents diabetes. Type-1 diabetes is characterized by destruction of the insulin secreting beta-cells within the endocrine pancreatic islets, resulting in lower insulin release. People with type-1 diabetes can be transplanted with pancreatic islets obtained from deceased donors which restores the beta-cell function. There are around 70 human islet isolation centers around the world which mostly collect endocrine pancreas from deceased donors. They assess the islet yield, functionality, viability, secretory capacity, and purity for transplantation and distribute this to donors. They also distribute a part of the pancreatic tissue for research, so that the cellular mechanisms in the human pancreatic tissue can be understood. This is crucial since human islet tissue has a unique cytoarchitecture compared to murine counterparts and therefore islet research with murine islets does not give complete picture of diabetes in humans. India is poised to take the mantle of the diabetes capital of the world in the near future. Despite this, there are no human islet isolation centers which can facilitate islet transplantation and diabetes research in India. This article highlights the glaring gap in the current infrastructure for diabetes care and provides critical insights into the role and potential of setting up islet tissue banks in the most populous country of the world.
|
|
| 10. |
|
|
