ORCID Profile
0000-0003-1027-3662
Current Organisation
University of Oxford
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Publisher: eLife Sciences Publications, Ltd
Date: 13-05-2022
DOI: 10.7554/ELIFE.76262
Abstract: A developing understanding suggests that spatial compartmentalisation in pancreatic β cells is critical in controlling insulin secretion. To investigate the mechanisms, we have developed live-cell subcellular imaging methods using the mouse organotypic pancreatic slice. We demonstrate that the organotypic pancreatic slice, when compared with isolated islets, preserves intact β-cell structure, and enhances glucose-dependent Ca 2+ responses and insulin secretion. Using the slice technique, we have discovered the essential role of local activation of integrins and the downstream component, focal adhesion kinase (FAK), in regulating β cells. Integrins and FAK are exclusively activated at the β-cell capillary interface and using in situ and in vitro models we show their activation both positions presynaptic scaffold proteins, like ELKS and liprin, and regulates glucose-dependent Ca 2+ responses and insulin secretion. We conclude that FAK orchestrates the final steps of glucose-dependent insulin secretion within the restricted domain where β-cell contact the islet capillaries.
Publisher: The Company of Biologists
Date: 2020
DOI: 10.1242/JCS.236794
Abstract: F-actin dynamics are known to control insulin secretion but the point of intersection with the stimulus-secretion cascade is unknown. Here, using multiphoton imaging of β cells isolated from Lifeact-GFP transgenic mice, we show glucose stimulation does not cause global changes in subcortical F-actin. Instead, we observe spatially discrete and transient F-actin changes around each fusing granule. This F-actin remodelling is dependent on actin nucleation and is observed for granule fusion induced by either glucose or high potassium stimulation. Using GFP-labelled proteins we identify local enrichment of Arp3, dynamin and clathrin, all occurring after granule fusion, suggesting early recruitment of an endocytic complex to the fusing granules. Block of Arp2/3 activity with drugs or shRNA inhibit F-actin coating, traps granules at the cell membrane and reduces insulin secretion. Block of formin-mediated actin nucleation also blocks F-actin coating but has no effect on insulin secretion. We conclude that local Arp2/3 dependent actin nucleation at the sites of granule fusion plays an important role in post-fusion granule dynamics and in the regulation of insulin secretion.
Publisher: Frontiers Media SA
Date: 17-03-2022
DOI: 10.3389/FENDO.2022.842937
Abstract: We present a case of an obese 22-year-old man with activating GCK variant who had neonatal hypoglycemia, re-emerging with hypoglycemia later in life. We investigated him for asymptomatic hypoglycemia with a family history of hypoglycemia. Genetic testing yielded a novel GCK missense class 3 variant that was subsequently found in his mother, sister and nephew and reclassified as a class 4 likely pathogenic variant. Glucokinase enables phosphorylation of glucose, the rate-limiting step of glycolysis in the liver and pancreatic β cells. It plays a crucial role in the regulation of insulin secretion. Inactivating variants in GCK cause hyperglycemia and activating variants cause hypoglycemia. Spleen-preserving distal pancreatectomy revealed diffuse hyperplastic islets, nuclear pleomorphism and periductular islets. Glucose stimulated insulin secretion revealed increased insulin secretion in response to glucose. Cytoplasmic calcium, which triggers exocytosis of insulin-containing granules, revealed normal basal but increased glucose-stimulated level. Unbiased gene expression analysis using 10X single cell sequencing revealed upregulated INS and CKB genes and downregulated DLK1 and NPY genes in β-cells. Further studies are required to see if alteration in expression of these genes plays a role in the metabolic and histological phenotype associated with glucokinase pathogenic variant. There were more large islets in the patient’s pancreas than in control subjects but there was no difference in the proportion of β cells in the islets. His hypoglycemia was persistent after pancreatectomy, was refractory to diazoxide and improved with pasireotide. This case highlights the variable phenotype of GCK mutations. In-depth molecular analyses in the islets have revealed possible mechanisms for hyperplastic islets and insulin hypersecretion.
Publisher: Cold Spring Harbor Laboratory
Date: 23-12-2021
DOI: 10.1101/2021.12.21.473760
Abstract: A developing understanding suggests that spatial compartmentalisation of components the glucose stimulus-secretion pathway in pancreatic β cells are critical in controlling insulin secretion. To investigate the mechanisms, we have developed live-cell sub-cellular imaging methods using the organotypic pancreatic slice. We demonstrate that the organotypic pancreatic slice, when compared with isolated islets, preserves intact β cell structure, and enhances glucose dependent Ca 2+ responses and insulin secretion. Using the slice technique, we have discovered the essential role of local activation of integrins and the downstream component, focal adhesion kinase, in regulating β cells. Integrins and focal adhesion kinase are exclusively activated at the β cell capillary interface and using in situ and in vitro models we show their activation both positions presynaptic scaffold proteins, like ELKS and liprin, and regulates glucose dependent Ca 2+ responses and insulin secretion. We conclude that focal adhesion kinase orchestrates the final steps of glucose dependent insulin secretion within the restricted domain where β cells contact the islet capillaries.
Publisher: eLife Sciences Publications, Ltd
Date: 29-03-2022
Publisher: MDPI AG
Date: 08-09-2021
Abstract: Implant devices containing insulin-secreting β-cells hold great promise for the treatment of diabetes. Using in vitro cell culture, long-term function and viability are enhanced when β-cells are cultured with extracellular matrix (ECM) proteins. Here, our goal is to engineer a favorable environment within implant devices, where ECM proteins are stably immobilized on polymer scaffolds, to better support β-cell adhesion. Four different polymer candidates (low-density polyethylene (LDPE), polystyrene (PS), polyethersulfone (PES) and polysulfone (PSU)) were treated using plasma immersion ion implantation (PIII) to enable the covalent attachment of laminin on their surfaces. Surface characterisation analysis shows the increased hydrophilicity, polar groups and radical density on all polymers after the treatment. Among the four polymers, PIII-treated LDPE has the highest water contact angle and the lowest radical density which correlate well with the non-significant protein binding improvement observed after 2 months of storage. The study found that the radical density created by PIII treatment of aromatic polymers was higher than that created by the treatment of aliphatic polymers. The higher radical density significantly improves laminin attachment to aromatic polymers, making them better substrates for β-cell adhesion.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Jason Tong.