Search

Background and aims: The rapid reversibility of recent type 2 diabetes (T2D) by very low-calorie diet in some patients correlates with a marked improvement of glucose-stimulated insulin secretion (GSIS), emphasizing the role of β-cell dysfunction in the early stages of the disease. In search of human-specific mechanisms of β-cell dysfunction, we extensively characterized the glucotoxic alterations of insulin secretion and upstream coupling events in human pancreatic islets cultured for 1 to 3 weeks at ~5, 8, 10 or 20 mmol/l glucose and acutely stimulated by a stepwise increase in glucose concentration. Materials and methods: Islets from 46 non-diabetic (ND) and 6 type 2 diabetic (T2D) donors were obtained from 5 isolation centers over the last 10 years. The islets were precultured 3-7 days in RPMI containing 5 mmol/l glucose and 10% FBS. They were then cultured for 1-3 weeks in the same medium containing 5.5, 8.5, 10.5 or 20.5 mmol/l glucose before measurements of insulin secretion during culture, islet insulin/DNA content ratio, β-cell apoptosis, cytosolic and mitochondrial thiol redox state, and assessment of dynamic insulin secretion and upstream coupling events during stepwise stimulation with glucose (NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)). Results: Culture of ND-islets for 1 to 3 weeks at ~8, 10 or 20 vs 5 mmol/l glucose did not increase β-cell apoptosis or oxidative stress but concentration-dependently decreased insulin content and increased the β-cell sensitivity to subsequent stimulation with glucose. The islet glucose responsiveness (max amplitude of GSIS per islet) was larger after culture at 8 or 10 vs 5 mmol/l glucose but was markedly reduced after culture at 20 vs 5 mmol/l glucose. In the latter islets, [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were bell-shaped, with a maximal stimulation at 5 to 10 mmol/l glucose followed by a rapid sustained inhibition above that concentration. This glucotoxic alteration was a robust characteristic of human islets. It resulted from long-term stimulation of glucose metabolism and was fully reversible after culture at 5 mmol/l glucose. Finally, acute activation/inhibition of glucokinase during perifusion of islets cultured at 20 mmol/l glucose indicated that the acute reduction of [Ca2+]c and insulin secretion above 10 mmol/l glucose was due to overstimulation rather than inhibition of glucose metabolism. Similar results were obtained in islets from T2D-donors. Conclusion: Long-term exposure of human islets to mildly elevated glucose concentrations markedly increases their glucose sensitivity and reveals a bell-shaped glucose response curve for changes in [Ca2+]c and insulin secretion. This human-specific glucotoxic alteration may contribute to β-cell dysfunction in T2D independently from a detectable increase in β-cell apoptosis and oxidative stress.

Endoplasmic reticulum (ER)–plasma membrane (PM) contacts are sites of lipid exchange and Ca2+ transport, and both lipid transport proteins and Ca2+ channels specifically accumulate at these locations. In pancreatic β-cells, both lipid and Ca2+ signaling are essential for insulin secretion. The recently characterized lipid transfer protein TMEM24 (also known as C2CD2L) dynamically localizes to ER–PM contact sites and provides phosphatidylinositol, a precursor of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], to the PM. β-cells lacking TMEM24 exhibit markedly suppressed glucose-induced Ca2+ oscillations and insulin secretion, but the underlying mechanism is not known. We now show that TMEM24 only weakly interacts with the PM, and dissociates in response to both diacylglycerol and nanomolar elevations of cytosolic Ca2+. Loss of TMEM24 results in hyper-accumulation of Ca2+ in the ER and in excess Ca2+ entry into mitochondria, with resulting impairment in glucose-stimulated ATP production., Summary: TMEM24 reversibly localizes to ER–PM contact sites and participates in the regulation of both ER and mitochondrial Ca2+ homeostasis and in glucose-dependent ATP production in insulin-secreting cells.

International audience; no abstract

International audience; Background and aims : The scaffold protein beta-arrestin2 (ARRB2) is known to uncouple G protein coupled receptors (GPCRs) from the G protein and to recruit new signaling pathways (such as the ERK1/2, PI3K, FAK…). In non beta cells, ARRB2 interacts with a wide range of GPCRs, but its interaction with the GIP receptor (GIPR) is still unclear. Our aim is to determine if ARRB2 is involved in the signaling of the GIPR in pancreatic beta cells.Materials and methods : The experiments were carried out in beta cells from five-month-old Arrb2+/+ and Arrb2-/- male mice. cAMP production (CAMPS-EPAC), endogenous PKA (AKAR3) and ERK1/2 (EKAR) activations, [Ca2+] in the cytosol ([Ca2+]c ; Fura2-LR) and in the endoplasmic reticulum ([Ca2+]ER ; D4ER) were assessed by live cell imaging in mouse pancreatic beta cells. EPAC2 (EPAC2-GFP) recruitment beneath the plasma membrane was monitored by total internal reflection fluorescence microscopy. Actin-F depolymerisation was evaluated by phalloidin staining (Alexa Fluor 488-conjugated phalloidin) and the phosphorylation of Focal Adhesion Kinase (FAK) by immunofluorescence.Results: Insulin secretion from Arrb2-/- islets was reduced by 50% compared to Arrb2+/+ islets in response to GIP (100pM-10nM, p

Hypersecretion of glucagon from pancreatic α-cells strongly contributes to diabetic hyperglycemia. Moreover, failure of α-cells to increase glucagon secretion in response to falling blood glucose concentrations compromises the defense against hypoglycemia, a common complication in diabetes therapy. However, the mechanisms underlying glucose regulation of glucagon secretion are poorly understood and likely involve both α-cell–intrinsic and intraislet paracrine signaling. Among paracrine factors, glucose-stimulated release of the GABA metabolite γ-hydroxybutyric acid (GHB) from pancreatic β-cells might mediate glucose suppression of glucagon release via GHB receptors on α-cells. However, the direct effects of GHB on α-cell signaling and glucagon release have not been investigated. Here, we found that GHB (4–10 μm) lacked effects on the cytoplasmic concentrations of the secretion-regulating messengers Ca2+ and cAMP in mouse α-cells. Glucagon secretion from perifused mouse islets was also unaffected by GHB at both 1 and 7 mm glucose. The GHB receptor agonist 3-chloropropanoic acid and the antagonist NCS-382 had no effects on glucagon secretion and did not affect stimulation of secretion induced by a drop in glucose from 7 to 1 mm. Inhibition of endogenous GHB formation with the GABA transaminase inhibitor vigabatrin also failed to influence glucagon secretion at 1 mm glucose and did not prevent the suppressive effect of 7 mm glucose. In human islets, GHB tended to stimulate glucagon secretion at 1 mm glucose, an effect mimicked by 3-chloropropanoic acid. We conclude that GHB does not mediate the inhibitory effect of glucose on glucagon secretion.

BACKGROUND AND AIMS: SGLT2 inhibitors (SGLT2i), such as dapagliflozin and empagliflozin, are effective glucose-lowering medications with a unique, insulin-independent mechanism that is to promote glycosuria. SGTL2i-induced glycosuria is associated with metabolic responses including increases in fatty acid mobilization, hepatic glucose and ketone body production, and circulating glucagon levels. These metabolic adaptations are considered causative for rare cases of euglycemic ketoacidosis reported for SGLT2i. One potential cause of euglycemic ketoacidosis is the SGLT2i-mediated increase in glucagonemia and drop in insulinemia. It has been suggested that direct inhibition of α-cell SGLT2 stimulates glucagon release. Here, we investigated the potential role of SGLT1 and SGLT2 in the control of glucagon and insulin secretion from rat, mouse and human islets [...]

Background and aims: Pancreatic β-cell failure is central in the pathogenesis of diabetes. In vitro differentiation of human induced pluripotent stem cells (iPSCs) into β-cells represents a novel cell source for diabetes research. Here we differentiated iPSC-β-cells and tested their function in vitro and in vivo in humanized mice [...]