Rui Cheng-Xue

The GluCre-ROSA26EYFP mouse: A new model for easy identification of living pancreatic α-cells

The control of glucagon secretion by pancreatic α‐cells is poorly understood, largely because of the difficulty to recognize living α‐cells. We describe a new mouse model, referred to as GluCre‐ROSA26EYFP (or GYY), allowing easy α‐cell identification because of specific expression of EYFP. GYY mice displayed normal glycemic control during a fasting/refeeding test or intraperitoneal insulin injection. Glucagon secretion by isolated islets was normally inhibited by glucose and stimulated by adrenaline. [Ca2+]c responses to arginine, adrenaline, diazoxide and tolbutamide, were similar in GYY and control mice. Hence, this new mouse model is a reliable and powerful tool to specifically study α‐cells.

Glucose and Pharmacological Modulators of ATP-Sensitive K+ Channels Control [Ca2+]c by Different Mechanisms in Isolated Mouse α-Cells

OBJECTIVE—We studied how glucose and ATP-sensitive K+ (KATP) channel modulators affect α-cell [Ca2+]c. RESEARCH DESIGN AND METHODS—GYY mice (expressing enhanced yellow fluorescent protein in α-cells) and NMRI mice were used. [Ca2+]c, the KATP current (IKATP, perforated mode) and cell metabolism [NAD(P)H fluorescence] were monitored in single α-cells and, for comparison, in single β-cells. RESULTS—In 0.5 mmol/l glucose, [Ca2+]c oscillated in some α-cells and was basal in the others. Increasing glucose to 15 mmol/l decreased [Ca2+]c by ∼30% in oscillating cells and was ineffective in the others. α-Cell IKATP was inhibited by tolbutamide and activated by diazoxide or the mitochondrial poison azide, as in β-cells. Tolbutamide increased α-cell [Ca2+]c, whereas diazoxide and azide abolished [Ca2+]c oscillations. Increasing glucose from 0.5 to 15 mmol/l did not change IKATP and NAD(P)H fluorescence in α-cells in contrast to β-cells. The use of nimodipine showed that L-type Ca2+ channels are the main conduits for Ca2+ influx in α-cells. γ-Aminobutyric acid and zinc did not decrease α-cell [Ca2+]c, and insulin, although lowering [Ca2+]c very modestly, did not affect glucagon secretion. CONCLUSIONS—α-Cells display similarities with β-cells: KATP channels control Ca2+ influx mainly through L-type Ca2+ channels. However, α-cells have distinct features from β-cells: Most KATP channels are already closed at low glucose, glucose does not affect cell metabolism and IKATP, and it slightly decreases [Ca2+]c. Hence, glucose and KATP channel modulators exert distinct effects on α-cell [Ca2+]c. The direct small glucose-induced drop in α-cell [Ca2+]c contributes likely only partly to the strong glucose-induced inhibition of glucagon secretion in islets.

Tolbutamide Controls Glucagon Release From Mouse Islets Differently Than Glucose: Involvement of KATP Channels From Both α-Cells and δ-Cells

We evaluated the role of ATP-sensitive K+ (KATP) channels, somatostatin, and Zn2+ in the control of glucagon secretion from mouse islets. Switching from 1 to 7 mmol/L glucose inhibited glucagon release. Diazoxide did not reverse the glucagonostatic effect of glucose. Tolbutamide decreased glucagon secretion at 1 mmol/L glucose (G1) but stimulated it at 7 mmol/L glucose (G7). The reduced glucagon secretion produced by high concentrations of tolbutamide or diazoxide, or disruption of KATP channels (Sur1−/− mice) at G1 could be inhibited further by G7. Removal of the somatostatin paracrine influence (Sst−/− mice or pretreatement with pertussis toxin) strongly increased glucagon release, did not prevent the glucagonostatic effect of G7, and unmasked a marked glucagonotropic effect of tolbutamide. Glucose inhibited glucagon release in the absence of functional KATP channels and somatostatin signaling. Knockout of the Zn2+ transporter ZnT8 (ZnT8−/− mice) did not prevent the glucagonostatic effect of glucose. In conclusion, glucose can inhibit glucagon release independently of Zn2+, KATP channels, and somatostatin. Closure of KATP channels controls glucagon secretion by two mechanisms, a direct stimulation of α-cells and an indirect inhibition via somatostatin released from δ-cells. The net effect on glucagon release results from a balance between both effects.

Mechanisms of control of the free Ca2+ concentration in the endoplasmic reticulum of mouse pancreatic β-cells: interplay with cell metabolism and [Ca2+]c and role of SERCA2b and SERCA3.

OBJECTIVE Sarco-endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and SERCA3 pump Ca2+ in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER Ca2+ concentration ([Ca2+]ER) and the role of SERCA3 in the control of insulin secretion and ER stress. RESEARCH DESIGN AND METHODS β-Cell [Ca2+]ER of SERCA3+/+ and SERCA3−/− mice was monitored with an adenovirus encoding the low Ca2+-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca2+ concentration ([Ca2+]c) and [Ca2+]ER were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied. RESULTS Glucose elicited synchronized [Ca2+]ER and [Ca2+]c oscillations. [Ca2+]ER oscillations were smaller in SERCA3−/− than in SERCA3+/+ β-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dose-dependent [Ca2+]ER rise in SERCA3+/+ and SERCA3−/− β-cells. In a Ca2+-free medium, glucose moderately raised [Ca2+]ER from a highly buffered cytosolic Ca2+ pool. Increasing [Ca2+]c with high [K] elicited a [Ca2+]ER rise that was larger but more transient in SERCA3+/+ than SERCA3−/− β-cells because of the activation of a Ca2+ release from the ER in SERCA3+/+ β-cells. Glucose-induced insulin release was larger in SERCA3−/− than SERCA3+/+ islets. SERCA3 ablation did not induce ER stress. CONCLUSIONS [Ca2+]c and [Ca2+]ER oscillate in phase in response to glucose. Upon [Ca2+]c increase, Ca2+ is taken up by SERCA2b and SERCA3. Strong Ca2+ influx triggers a Ca2+ release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs Ca2+ uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress.

O55 Amplification de la sécrétion d’insuline par le glucose : augmentation du Ca2+ sous-membranaire dans les cellules β ou augmentation de l’action Ca2+

Introduction : Le glucose stimule la secretion d’insuline (SI) en elevant la [Ca2+] dans le cytosol des cellules β (voie declenchante) et en augmentant l’efficacite du Ca2+ sur l’exocytose (voie amplificatrice). Le concept d’amplification repose sur l’observation qu’a [Ca2+]c elevee et stable, le glucose augmente la SI sans augmenter davantage la [Ca2+]c. Toutefois, ces mesures globales de [Ca2+]c n’excluent pas la possibilite qu’une elevation de la [Ca2+] sous la membrane plasmique seulement ([Ca2+]SM) explique l’effet amplificateur du glucose. C’est l’hypothese que nous avons testee. Materiels et Methodes : La [Ca2+]SM a ete mesuree par « Total Internal Reflection fluorescence Microscopy » (TIRF) apres expression adenovirale du rapporteur D3cpv (cytosolique) dans des amas de cellules β de souris ou du LynD3cpv (membranaire) dans les cellules β d’ilots intacts (strategie Rip-Cre-Lox). Resultats : L’expression du D3cpv n’a pas altere les changements de [Ca2+] et de SI dans les amas de cellules β. L’effet amplificateur a ete etudie en presence de diazoxide (pour ouvrir les canaux KATP) et 30 mM KCl (pour stimuler un influx de Ca2+). Dans ces conditions, une augmentation de glucose a abaisse la [Ca2+]SM mais double la SI. Une inhibition de la capture du Ca2+ par le reticulum endoplasmique a attenue la baisse de [Ca2+]SM produite par 15 mM glucose mais n’a pas demasque d’augmentation, meme localisee, de la [Ca2+]SM. En revanche, des augmentations significatives de la [Ca2+]SM ont ete detectees lors de changements repetitifs de la concentration de KCl entre 30 et 32-35 mM, attestant de la sensibilite adequate de notre systeme de detection. Des resultats similaires ont ete obtenus dans les cellules β d’ilots intacts. Conclusion : L’amplification de la SI par le glucose n’est pas due a une augmentation sous-membranaire de la [Ca2+] dans la cellule β et peut donc etre attribuee a une augmentation de l’action du [Ca2+] sur l’exocytose.