Protein phosphorylation in electrically permeabilized islets of Langerhans. Effects of Ca2+, cyclic AMP, a phorbol ester and noradrenaline

Jones, Peter M.; Salmon, D. Michael; Howell, S. L.

doi:10.1042/bj2540397

Cited by 161 publications

(108 citation statements)

References 51 publications

(56 reference statements)

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“…Briefly, isolated mouse or human islets were transferred to chambers containing 1 μm pore-size nylon filters and perifused at 37°C and a flow rate of 0.5 ml/min with a physiological salt solution [22] containing 2 mmol/l or 20 mmol/l glucose in the absence or presence of recombinant mouse or human CCL5. Perifusate samples were collected at 2 min intervals for the duration of the experiments and the secreted insulin measured by radioimmunoassay [25].…”

Section: Methodsmentioning

confidence: 99%

The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis

et al. 2013

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Aims/hypothesis Chemokine (C-C motif) ligand 5 (CCL5) acts at C-C chemokine receptors (CCRs) to promote immune cell recruitment to sites of inflammation, but is also an agonist at G-protein-coupled receptor 75 (GPR75), which has very limited homology with CCRs. GPR75 is coupled to Gq to elevate intracellular calcium, so we investigated whether islets express this receptor and whether its activation by CCL5 increases beta cell calcium levels and insulin secretion. Methods Islet CCL5 receptor mRNA expression was measured by quantitative RT-PCR and GPR75 was detected in islets by western blotting and immunohistochemistry. In some experiments GPR75 was downregulated by transient transfection with small interfering RNA. Real-time changes in intracellular calcium were determined by single-cell microfluorimetry. Dynamic insulin secretion from perifused islets was quantified by radioimmunoassay. Glucose homeostasis in lean and obese mice was determined by measuring glucose and insulin tolerance, and insulin secretion in vivo. Results Mouse and human islets express GPR75 and its ligand CCL5. Exogenous CCL5 reversibly increased intracellular calcium in beta cells via GPR75, this phenomenon being dependent on phospholipase C activation and calcium influx. CCL5 also stimulated insulin secretion from mouse and human islets in vitro, and improved glucose tolerance in lean mice and in a mouse model of hyperglycaemia and insulin resistance (ob/ob ). The improvement in glucose tolerance was associated with enhanced insulin secretion in vivo, without changes in insulin sensitivity. Conclusions/interpretation Although CCL5 is implicated in the pathogenesis of diabetes through activation of CCRs, it has beneficial effects on beta cells through GPR75 activation.

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Section: Methodsmentioning

confidence: 99%

The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis

et al. 2013

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“…The insulin content of perifusion and static incubation samples was determined using an in-house radioimmunoassay [11].…”

Section: Methodsmentioning

confidence: 99%

Kisspeptin stimulation of insulin secretion: mechanisms of action in mouse islets and rats

et al. 2009

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Aims/hypothesis Kisspeptin is a novel peptide identified as an endogenous ligand of the G-protein-coupled receptor 54 (GPR-54), which plays a crucial role in puberty and reproductive function. High levels of GPR-54 and kisspeptin have been reported in the pancreas and we have previously shown that kisspeptin potentiates glucose-induced insulin release from isolated islets, although the mechanisms underlying this effect were unclear. Methods Insulin secretion from isolated mouse islets was measured to characterise the effects of kisspeptin. The effects of kisspeptin on both p42/44 mitogen-activated protein kinase (MAPK) phosphorylation and intracellular Ca 2+ ([Ca 2+ ] i ) in mouse islets were also investigated. Furthermore, kisspeptin was administered to rats in vivo and effects on plasma insulin levels measured. Results In the current study, kisspeptin induced a concentration-dependent potentiation of glucose-induced (20 mmol/l) insulin secretion from mouse islets, with maximal effects at 1 µmol/l, but had no effect on insulin secretion at a substimulatory concentration of glucose (2 mmol/l). Activation of GPR-54 by kisspeptin also caused reversible increases in [Ca 2+ ] i in Fura-2 loaded dispersed islet cells.The kisspeptin-induced potentiation of glucose-induced insulin secretion was completely abolished by inhibitors of phospholipase C and p42/44 MAPK, but not by inhibitors of protein kinase C or p38 MAPK. Intravenous administration of kisspeptin into conscious, unrestrained rats caused an increase in circulating insulin levels, whilst central administration of kisspeptin had no effect, indicating a peripheral site of action. Conclusions/interpretation These observations suggest that neither typical protein kinase C isoforms nor p38 MAPK are involved in the potentiation of glucose-induced insulin release by kisspeptin, but intracellular signalling pathways involving phospholipase C, p42/44 MAPK and increased [Ca 2+ ] i are required for the stimulatory effects on insulin secretion. The observation that kisspeptin is also capable of stimulating insulin release in vivo supports the conclusion that kisspeptin is a regulator of beta cell function.

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“…ethanol) then sonicated. Tissue insulin, glucagon and somatostatin contents were measured using in-house RIA, as previously described [24,25] and a commercially available somatostatin RIA kit (Euro-Diagnostica, Malmö, Sweden). Serum samples were obtained from non-fasted mice and insulin was measured using an ELISA (Mercodia, Uppsala, Sweden).…”

Section: Graft Functionmentioning

confidence: 99%

Co-transplantation of mesenchymal stem cells maintains islet organisation and morphology in mice

et al. 2011

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Aims/hypothesis Recent studies have shown that mesenchymal stem cells (MSCs) secrete several factors that improve survival and function of transplanted islets. Implantation of islets beneath the kidney capsule results in morphological changes, due to interactions of the graft with the host, thus impairing islet function. We co-transplanted MSCs with islets to determine their effects on the remodelling process and studied graft function in a mouse model of minimal islet mass. Methods Islets were syngeneically transplanted, either alone or with kidney-derived MSCs, underneath the kidney capsule of streptozotocin-induced diabetic C57Bl/6 mice. Blood glucose levels were monitored and intraperitoneal glucose tolerance tests carried out. Hormone contents of grafts and pancreas were assessed by radioimmunoassay. Graft morphology and vascularisation were evaluated by immunohistochemistry. Results MSCs improved the capacity of islet grafts to reverse hyperglycaemia, with 92% of mice co-transplanted with MSCs reverting to normoglycaemia, compared with 42% of those transplanted with islets alone. Average blood glucose concentrations were lower throughout the 1 month monitoring period in MSC co-transplanted mice. MSCs did not alter graft hormone content. Islets co-transplanted with MSCs maintained a morphology that more closely resembled that of islets in the endogenous pancreas, both in terms of size, and of endocrine and endothelial cell distribution. Vascular engraftment was superior in MSC co-transplanted mice, as shown by increased endothelial cell numbers within the endocrine tissue. Conclusions/interpretation Co-transplantation of islets with MSCs had a profound impact on the remodelling process, maintaining islet organisation and improving islet revascularisation. MSCs also improved the capacity of islets to reverse hyperglycaemia.

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Protein phosphorylation in electrically permeabilized islets of Langerhans. Effects of Ca2+, cyclic AMP, a phorbol ester and noradrenaline

Cited by 161 publications

References 51 publications

The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis

The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis

Kisspeptin stimulation of insulin secretion: mechanisms of action in mouse islets and rats

Co-transplantation of mesenchymal stem cells maintains islet organisation and morphology in mice

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