Biology subjects

Suba, K.

Publications and source records attributed to Suba, K..

4 recordsLinked to original sources

bioRxiv2022

Intra-islet glucagon signalling regulates pulsatile insulin secretion and glucose homeostasis

BackgroundType 2 diabetes (T2D) is characterised by the loss of pulsatile insulin secretion. We studied mice with {beta}-cell specific loss of the glucagon receptor (Gcgr fl/fl X Ins-1Cre), to investigate the role of intra-islet glucagon receptor signalling on pan-islet calcium oscillations and insulin pulsatility. MethodsFrequently sampled intravenous glucose tolerance tests were conducted on Gcgr {beta}-cell-/- and littermate controls. Crossing with GCaMP6f (STOP flox) animals further allowed for {beta}-cell specific expression of a fluorescent calcium indicator. These islets were functionally imaged in vitro and in vivo. Wild-type mice were transplanted with islets expressing GCaMP6f in {beta}-cells into the anterior eye chamber and placed on a high fat diet. Part of the cohort received a glucagon analogue (GCG-analogue) for 40 days and the control group were fed to achieve weight matching. Calcium imaging was performed regularly during the development of hyperglycaemia and in response to GCG-analogue treatment. ResultsGcgr {beta}-cell-/- mice exhibited impaired glucose tolerance following intraperitoneal glucose challenge (control 12.7mmol/L {+/-}0.6 vs. Gcgr {beta}-cell-/- 15.4mmol/L {+/-}0.0 at 15 min, p=0.002); fasting glycaemia was not different to controls. In vitro, Gcgr {beta}-cell-/- islets showed profound loss of synchronised calcium waves in response to glucose which was only partially rescued in vivo. First-phase insulin pulsatility on peripheral blood sampling (n=5) was significantly disordered in Gcgr {beta}-cell-/- mice (burst mass Gcgr {beta}-cell-/- 0.30 {+/-}0.03 versus 0.84 {+/-}0.23 for controls p=0.04). Diet induced obesity and hyperglycaemia resulted in a loss of co-ordinated [Ca2+]I waves in transplanted islets. This was reversed with GCG-analogue treatment, independently of weight-loss (n=8). ConclusionThese data provide novel evidence for the role of intra-islet GCGR signalling in sustaining synchronised calcium oscillations and support a possible therapeutic role for glucagonergic agents to restore the insulin pulsatility lost in T2D.

physiology↗

bioRxiv2022

Divergent acute versus prolonged in vivo GLP-1R responses in β-arrestin 2-deleted primary beta cells

The glucagon-like peptide-1 receptor (GLP-1R) is a major therapeutic target in type 2 diabetes (T2D) and obesity. Following activation, GLP-1Rs are rapidly desensitised by {beta}-arrestins, scaffolding proteins that terminate G protein interactions but also act as independent signalling mediators. While GLP-1R interacts with {beta}-arrestins 1 and 2, expression of the latter is greatly enhanced in beta cells, making this the most relevant isoform. Here, we have assessed in vivo glycaemic responses to the pharmacological GLP-1R agonist exendin-4 in adult beta cell-selective {beta}-arrestin 2 knockout (KO) mice. Lean female and high-fat, high-sucrose-fed KO mice of both sexes displayed worse acute responses versus control littermates, an effect that was inverted 6 hours post-agonist injection, resulting in prolonged in vivo cell-cell connectivity in KO islets implanted in mouse eyes. Similar effects were observed for the clinically relevant semaglutide and tirzepatide but not with exendin-phe1, an agonist biased away from {beta}-arrestin recruitment. Ex vivo acute cAMP was impaired, but overnight desensitisation was reduced in KO islets. The acute signalling defect was attributed to enhanced {beta}-arrestin 1 and phosphodiesterase (PDE) 4 activity in the absence of {beta}-arrestin 2, while the reduced desensitisation correlated with altered GLP-1R trafficking, involving impaired recycling and lysosomal targeting and increased trans-Golgi network (TGN) localisation and signalling, as well as reduced GLP-1R ubiquitination by the E3 ubiquitin ligase NEDD4. This study has unveiled fundamental aspects of the role of {beta}-arrestin 2 in regulating pharmacological GLP-1R responses with direct application to the rational design of improved GLP-1R-targeting therapeutics.

cell biology↗

bioRxiv2020

Intravital imaging of islet Ca2+ dynamics reveals enhanced β cell connectivity after bariatric surgery in mice

Bariatric surgery improves both insulin sensitivity and secretion in type 2 diabetes. However, these changes are difficult to monitor directly and independently. In particular, the degree and the time course over which surgery impacts {beta} cell function, versus mass, have been difficult to establish. In this study, we investigated the effect of bariatric surgery on {beta} cell function in vivo by imaging Ca2+ dynamics prospectively and at the single cell level in islets engrafted into the anterior eye chamber. Islets expressing GCaMP6f selectively in the {beta} cell were transplanted into obese male hyperglycaemic mice that were then subjected to either vertical sleeve gastrectomy (VSG) or sham surgery. Imaged in vivo in the eye, VSG improved coordinated Ca2+ activity, with 90% of islets observed exhibiting enhanced Ca2+ wave activity ten weeks post-surgery, while islet wave activity in sham animals fell to zero discernible coordinated islet Ca2+ activity at the same time point. Correspondingly, VSG mice displayed significantly improved glucose tolerance and insulin secretion. Circulating fasting levels of GLP-1 were also increased after surgery, potentially contributing to improved {beta} cell performance. We thus demonstrate that bariatric surgery leads to time-dependent increases in individual {beta} cell function and intra-islet connectivity, together driving increased insulin secretion and diabetes remission, in a weight-loss independent fashion. Significance StatementUsed widely to treat obesity, bariatric surgery also relieves the symptoms of type 2 diabetes. The mechanisms involved in diabetes remission are still contested, with increased insulin sensitivity and elevated insulin secretion from pancreatic {beta} cells both implicated. Whilst the speed of remission - usually within a few days - argues for improvements in {beta} cell function rather than increases in mass, a direct demonstration of changes at the level of individual {beta} cells or islets has been elusive. Here, we combine vertical sleeve gastrectomy with intravital imaging of islets engrafted into the mouse anterior eye chamber to reveal that surgery causes a time-dependent improvement in glucose-induced Ca2+ dynamics and {beta} cell - {beta} cell connectivity, both of which likely underlie increased insulin release.

cell biology↗

bioRxiv2020

AVP-induced counter-regulatory glucagon is diminished in type 1 diabetes

Insulin-induced hypoglycemia is a major barrier to the treatment of type-1 diabetes (T1D). Accordingly, it is important that we understand the mechanisms regulating the circulating levels of glucagon - the bodys principal blood glucose-elevating hormone which is secreted from alpha-cells of the pancreatic islets. Varying glucose over the range of concentrations that occur physiologically between the fed and fuel-deprived states (from 8 to 4 mM) has no significant effect on glucagon secretion in isolated islets (in vitro) and yet associates with dramatic changes in plasma glucagon in vivo. The identity of the systemic factor(s) that stimulates glucagon secretion remains unknown. Here, we show that arginine-vasopressin (AVP), secreted from the posterior pituitary, stimulates glucagon secretion. Glucagon-secreting alpha-cells express high levels of the vasopressin 1b receptor gene (Avpr1b). Activation of AVP neurons in vivo increased circulating copeptin (the C-terminal segment of the AVP precursor peptide, a stable surrogate marker of AVP) and increased blood glucose; effects blocked by pharmacological antagonism of either the glucagon receptor or vasopressin 1b receptor. AVP also mediates the stimulatory effects of hypoglycemia produced by exogenous insulin and 2-deoxy-D-glucose on glucagon secretion. We show that the A1/C1 neurons of the medulla oblongata drive AVP neuron activation in response to insulin-induced hypoglycemia. Exogenous injection of AVP in vivo increased cytoplasmic Ca2+ in alpha-cells (implanted into the anterior chamber of the eye) and glucagon release. Hypoglycemia also increases circulating levels of AVP in humans and this hormone stimulates glucagon secretion from isolated human islets. In patients with T1D, hypoglycemia failed to increase both plasma copeptin and glucagon levels. These findings suggest that AVP is a physiological systemic regulator of glucagon secretion and that this mechanism becomes impaired in T1D.

cell biology↗