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.

OBJECTIVESarco-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 METHODSβ-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.RESULTSGlucose 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.

OBJECTIVE— We studied how glucose and ATP-sensitive K + (K ATP ) channel modulators affect α-cell [Ca 2+ ] c . RESEARCH DESIGN AND METHODS— GYY mice (expressing enhanced yellow fluorescent protein in α-cells) and NMRI mice were used. [Ca 2+ ] c , the K ATP current (I KATP , 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, [Ca 2+ ] c oscillated in some α-cells and was basal in the others. Increasing glucose to 15 mmol/l decreased [Ca 2+ ] c by ∼30% in oscillating cells and was ineffective in the others. α-Cell I KATP was inhibited by tolbutamide and activated by diazoxide or the mitochondrial poison azide, as in β-cells. Tolbutamide increased α-cell [Ca 2+ ] c , whereas diazoxide and azide abolished [Ca 2+ ] c oscillations. Increasing glucose from 0.5 to 15 mmol/l did not change I KATP and NAD(P)H fluorescence in α-cells in contrast to β-cells. The use of nimodipine showed that L-type Ca 2+ channels are the main conduits for Ca 2+ influx in α-cells. γ-Aminobutyric acid and zinc did not decrease α-cell [Ca 2+ ] c , and insulin, although lowering [Ca 2+ ] c very modestly, did not affect glucagon secretion. CONCLUSIONS— α-Cells display similarities with β-cells: K ATP channels control Ca 2+ influx mainly through L-type Ca 2+ channels. However, α-cells have distinct features from β-cells: Most K ATP channels are already closed at low glucose, glucose does not affect cell metabolism and I KATP , and it slightly decreases [Ca 2+ ] c . Hence, glucose and K ATP channel modulators exert distinct effects on α-cell [Ca 2+ ] c . The direct small glucose-induced drop in α-cell [Ca 2+ ] c contributes likely only partly to the strong glucose-induced inhibition of glucagon secretion in islets.

The control of glucagon secretion by pancreatic acells is poorly understood, largely because of the difficulty to recognize living a-cells. We describe a new mouse model, referred to as GluCre-ROSA26EYFP (or GYY), allowing easy a-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. [Ca 2+ ] 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 a-cells.

Aims/hypothesis Glucose-induced insulin secretion is attributed to a rise of beta cell cytosolic free [Ca2+] ([Ca2+]c) (triggering pathway) and amplification of the action of Ca2+. This concept of amplification rests on observations that glucose can increase Ca2+-induced insulin secretion without further elevating an imposed already high [Ca2+]c. However, it remains possible that this amplification results from an increase in [Ca2+] just under the plasma membrane ([Ca2+]SM), which escaped detection by previous measurements of global [Ca2+]c. This was the hypothesis that we tested here by measuring [Ca2+]SM. Methods The genetically encoded Ca2+ indicators D3-cpv (untargeted) and LynD3-cpv (targeted to plasma membrane) were expressed in clusters of mouse beta cells. LynD3-cpv was also expressed in beta cells within intact islets. [Ca2+]SM changes were monitored using total internal reflection fluorescence microscopy. Insulin secretion was measured in parallel. Results Beta cells expressing D3cpv or LynD3cpv displayed normal [Ca2+] changes and insulin secretion in response to glucose. Distinct [Ca2+]SM fluctuations were detected during repetitive variations of KCl between 30 and 32–35 mmol/l, attesting to the adequate sensitivity of our system. When the amplifying pathway was evaluated (high KCl+diazoxide), increasing glucose from 3 to 15 mmol/l consistently lowered [Ca2+]SM while stimulating insulin secretion approximately two fold. Blocking Ca2+ uptake by the endoplasmic reticulum largely attenuated the [Ca2+]SM decrease produced by high glucose but did not unmask localised [Ca2+]SM increases. Conclusions/interpretation Glucose can increase Ca2+-induced insulin secretion without causing further elevation of beta cell [Ca2+]SM. The phenomenon is therefore a true amplification of the triggering action of Ca2+.