Insulin secretion from beta cells in intact mouse islets is targeted towards the vasculature

Abstract The role of depolarization in the inverse glucose-dependence of glucagon secretion was investigated by comparing the effects of KATP channel block and of high potassium. The secretion of glucagon and insulin by perifused mouse islets was simultaneously measured. Lowering glucose raised glucagon secretion before it decreased insulin secretion, suggesting an alpha cell–intrinsic signal recognition. Raising glucose affected glucagon and insulin secretion at the same time. However, depolarization by tolbutamide, gliclazide, or 15 mM KCl increased insulin secretion before the glucagon secretion receded. In contrast to the robust depolarizing effect of arginine and KCl (15 and 40 mM) on single alpha cells, tolbutamide was of variable efficacy. Only when applied before other depolarizing agents had tolbutamide a consistent depolarizing effect and regularly increased the cytosolic Ca2+ concentration. When tested on inside-out patches tolbutamide was as effective on alpha cells as on beta cells. In the presence of 1 µM clonidine, to separate insulinotropic from glucagonotropic effects, both 500 µM tolbutamide and 30 µM gliclazide increased glucagon secretion significantly, but transiently. The additional presence of 15 or 40 mM KCl in contrast led to a marked and lasting increase of the glucagon secretion. The glucagon secretion by SUR1 knockout islets was not increased by tolbutamide, whereas 40 mM KCl was of unchanged efficiency. In conclusion a strong and sustained depolarization is compatible with a marked and lasting glucagon secretion. KATP channel closure in alpha cells is less readily achieved than in beta cells, which may explain the moderate and transient glucagonotropic effect.

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Dose- and Glucose-Dependent Effects of Amino Acids on Insulin Secretion from Isolated Mouse Islets and Clonal INS-1E Beta-Cells

The Review of Diabetic Studies ◽

10.1900/rds.2008.5.232 ◽

2008 ◽

Vol 5 (4) ◽

pp. 232-244 ◽

Cited By ~ 39

Author(s):

Zhenping Liu ◽

Per B. Jeppesen ◽

Søren Gregersen ◽

Xiaoping Chen ◽

Kjeld Hermansen

Keyword(s):

Amino Acids ◽

Insulin Secretion ◽

Beta Cells ◽

Mouse Islets

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Inhibitors of phosphatidylinositol 3-kinase amplify insulin release from islets of lean but not obese mice

Journal of Endocrinology ◽

10.1677/joe.0.1740247 ◽

2002 ◽

Vol 174 (2) ◽

pp. 247-258 ◽

Cited By ~ 39

Author(s):

WS Zawalich ◽

GJ Tesz ◽

KC Zawalich

Keyword(s):

Insulin Secretion ◽

Western Blot ◽

Insulin Receptor ◽

Beta Cells ◽

Phosphatidylinositol 3 Kinase ◽

Peripheral Tissues ◽

Pi3k Activation ◽

Insulin Receptor Signaling ◽

Mouse Islets ◽

Phosphatidylinositol 3

We examined the effects of phosphatidylinositol 3-kinase (PI3K) inhibition by wortmannin or LY294002 on glucose-induced secretion from mouse islets. Islets were collagenase isolated and perifused or subjected to Western blot analyses and probed for insulin receptor-signaling components. In agreement with previous studies, mouse islets, when compared with rat islets, were minimally responsive to 10 mM glucose stimulation. The inclusion of 50 nM wortmannin or 10 microM LY294002 significantly amplified 10 mM glucose-induced release from mouse islets. The effect of wortmannin was abolished by the calcium channel antagonist nitrendipine or by lowering the glucose level to 3 mM. Wortmannin had no effect on 10 mM alpha-ketoisocaproate-induced secretion. In contrast to its potentiating effect on islets from CD-1 mice, wortmannin had no effect on 10 mM glucose-induced release from ob/ob mouse islets. Western blot analyses revealed the presence of the insulin receptor, insulin receptor substrate proteins 1 and 2 and PI3K in CD-1 islets. These results support the concept that a PI3K-dependent signaling pathway exists in beta-cells and that it may function to restrain glucose-induced insulin secretion from beta-cells. They also suggest that, as insulin resistance develops in peripheral tissues, a potential result of impaired PI3K activation, the same biochemical anomaly in beta-cells promotes a linked increase in insulin secretion to maintain glucose homeostasis.

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Differences in insulin secretion between the rat and mouse: role of cAMP

Acta Endocrinologica ◽

10.1530/eje.0.1320370 ◽

1995 ◽

Vol 132 (3) ◽

pp. 370-376 ◽

Cited By ~ 32

Author(s):

Yan Hui Ma ◽

Jian Wang ◽

Gail G Rodd ◽

Janice L Bolaffi ◽

Gerold M Grodsky

Keyword(s):

Body Weight ◽

Insulin Secretion ◽

Beta Cells ◽

Insulin Release ◽

Second Phase ◽

Perfused Pancreas ◽

Rat Pancreas ◽

Mouse Islets ◽

Camp Levels

Ma YH, Wang J, Rodd GG, Bolaffi JL, Grodsky GM. Differences in insulin secretion between the rat and mouse: role of cAMP. Eur J Endocrinol 1995;132:370–6. ISSN 0804–4643 Although information regarding insulin secretion usually is considered equivalent when generated in the mouse or the rat, it is established that the kinetics of insulin secretion from mouse and rat pancreatic beta cells differ. The mechanisms underlining these differences are not understood. The in vitro perfused pancreas and isolated islets of the mouse or rat were employed in this study to investigate the role of cyclic adenosine monophosphate (cAMP), a major positive modulator of betacell function, as one differentiating signal for the uniquely different insulin release from the beta cells of these commonly used rodents. Glucose-stimulated first-phase insulin release from the perfused pancreas of the rat was higher than the mouse when calculated per gram of pancreas or as fractional secretion, but this phase was identical in the two species when results were adjusted for total body weight. Whether related to insulin content, pancreatic weight or body weight, the rat pancreas responded to glucose with a progressively increasing second-phase insulin release compared to the mouse pancreas, which secreted a flat second-phase of lesser magnitude. Isolated islets from rat and mouse were comparable in insulin content whereas the basal cAMP level of mouse islets was less than half that of the rat. At submaximal stimulation with glucose or glucose + IBMX or forskolin, mouse islets exhibited lower cAMP levels to a given stimulus than the rat. In rat islets cAMP levels increased to approximately 1000 fmol per islet, although insulin secretion maximized by 100–150 fmol. Insulin release at the same 100–150fmol cAMP per mouse islet was one-third that of the rat and secretion had not maximized in mouse islets at 800 fmol. Despite their similar insulin contents, mouse islets consistently secreted less insulin for a given level of cAMP per islet than the rat. The lower capacity of mouse islets to achieve comparable cAMP levels was not the result of increased catabolic rate because the "half-time" disappearance of islet cAMP after a stimulus was similar (~1 min) for both species. It is concluded that, compared to the mouse, beta cells of the rat pancreas elicit a more pronounced secondphase insulin secretion that is due, at least in part, to a greater production of, and sensitivity to, cAMP. Gerold M Grodsky, Metabolic Research Unit, University of California, H5W 1157, Box 0540, 3rd and Parnassus Avenue, San Francisco, CA 94143, USA

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Inhibition of insulin secretion via distinct signaling pathways in alpha2-adrenoceptor knockout mice

Acta Endocrinologica ◽

10.1530/eje.0.1490343 ◽

2003 ◽

pp. 343-350 ◽

Cited By ~ 57

Author(s):

M Peterhoff ◽

A Sieg ◽

M Brede ◽

CM Chao ◽

L Hein ◽

...

Keyword(s):

Insulin Secretion ◽

Adenylyl Cyclase ◽

Signaling Pathways ◽

Beta Cells ◽

Intracellular Signaling ◽

Islet Cells ◽

Inhibitory Effect ◽

Receptor Subtypes ◽

Mouse Islets ◽

Alpha2 Adrenoceptor

OBJECTIVE: Adrenaline inhibits insulin secretion through activation of alpha(2)-adrenoceptors (ARs). These receptors are linked to pertussis toxin-sensitive G proteins. Agonist binding leads to inhibition of adenylyl cyclase, inhibition of Ca(2+) channels and activation of K(+) channels. Recently, three distinct subtypes of alpha(2)-AR were described, alpha(2A)-AR, alpha(2B)-AR and alpha(2C)-AR. At present, it is unknown which of these alpha(2)-AR subtype(s) may regulate insulin secretion. We used mice deficient in alpha(2)-ARs to analyze the coupling and role of individual alpha(2)-AR subtypes in insulin-secreting beta cells. METHODS: The inhibitory effect of adrenaline on insulin secretion was measured in freshly isolated and cultured wild type (wt) and alpha(2)-AR knockout (KO) mouse islets in order to examine the receptor subtypes which mediate adrenaline-induced inhibition of insulin secretion. Adenylyl cyclase activity was measured in isolated cultured islets. Membrane potential was measured using the amphotericin B permeabilized patch clamp method in isolated and cultured single islet cells. RESULTS: In wt, alpha(2A)- and alpha(2C)-AR KO mouse islets, adrenaline, 1 microM/L, inhibited secretion by 83, 80 and 100% respectively. In contrast, in alpha(2A/2C)-AR double KO mouse islets, adrenaline had no effect on stimulated secretion indicating that both alpha(2A)-AR and alpha(2C)-AR, but not alpha(2B)-AR, are functionally expressed in mouse islets. Surprisingly, glucose (16.7 mM/L)-induced secretion in the presence of 1 microM/L forskolin was greatly impaired in alpha(2A)-AR KO islets. However, when cAMP levels were increased further by the combination of forskolin (5 microM/L) and 3-isobutyl-1-methylxanthine (100 microM/L), secretion was stimulated 2.7-fold (8.5-fold in wt islets). Adrenaline lowered the concentration of cAMP in wt and alpha(2C)-AR KO mouse islets by 74%. Adrenaline also hyperpolarized wt and alpha(2C)-AR KO beta cells. In contrast, adrenaline did not inhibit adenylyl cyclase in islets of alpha(2A)-AR KO mice, nor did it hyperpolarize alpha(2A)-AR KO beta cells. CONCLUSION: Adrenaline inhibits insulin release through alpha(2A)- and alpha(2C)-ARs via distinct intracellular signaling pathways.

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Glucose but not KCl diminishes submembrane granule turnover in mouse beta-cells

Journal of Molecular Endocrinology ◽

10.1530/jme-17-0063 ◽

2017 ◽

Vol 59 (3) ◽

pp. 311-324 ◽

Cited By ~ 8

Author(s):

Dennis Brüning ◽

Kirstin Reckers ◽

Peter Drain ◽

Ingo Rustenbeck

Keyword(s):

Insulin Secretion ◽

Beta Cells ◽

Initial Response ◽

Secretory Response ◽

Insulin Granules ◽

Primary Mouse ◽

Mouse Islets ◽

Glucose Stimulated Insulin Secretion ◽

Secretion Rates ◽

Reversed Order

KCl depolarization is widely used to mimic the depolarization during glucose-stimulated insulin secretion. Consequently, the insulin secretion elicited by KCl is often regarded as the equivalent of the first phase of glucose-induced insulin secretion. Here, the effects of both stimuli were compared by measuring the secretion of perifused mouse islets, the cytosolic Ca2+ concentration of single beta-cells and the mobility of submembrane insulin granules by TIRF microscopy of primary mouse beta-cells. Two cargo-directed granule labels were used namely insulin-EGFP and C-peptide-emGFP. The granule behaviour common to both was used to compare the effect of sequential stimulation with 40 mM KCl and 30 mM glucose and sequential stimulation with the same stimuli in reversed order. At the level of the cell secretory response, the sequential pulse protocol showed marked differences depending on the order of the two stimuli. KCl produced higher maximal secretion rates and diminished the response to the subsequent glucose stimulus, whereas glucose enhanced the response to the subsequent KCl stimulus. At the level of granule behaviour, a difference developed during the first stimulation phase in that the total number of granules, the short-term resident granules and the arriving granules, which are all parameters of granule turnover, were significantly smaller for glucose than for KCl. These differences at both the level of the cell secretory response and granule behaviour in the submembrane space are incompatible with identical initial response mechanisms to KCl and glucose stimulation.

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Inosine-stimulated insulin release and metabolism of inosine in isolated mouse pancreatic islets

Biochemical Journal ◽

10.1042/bj1580335 ◽

1976 ◽

Vol 158 (2) ◽

pp. 335-340 ◽

Cited By ~ 17

Author(s):

K Capito ◽

C J Hedeskov

Keyword(s):

Insulin Secretion ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Pancreatic Islets ◽

Beta Cells ◽

Insulin Release ◽

Lactate Production ◽

Islet Metabolism ◽

Mouse Pancreatic Islets ◽

Mouse Islets

Inosine is a potent primary stimulus of insulin secretion from isolated mouse islets. The inosine-induced insulin secretion was totally depressed during starvation, but was completely restored by the addition of 5 mM-caffeine to the medium and partially restored by the addition of 5 mM-glucose. Mannoheptulose (3 mg/ml) potentiated the effect of 10 mM-inosine in islets from fed mice. The mechanism of the stimulatory effect of inosine was further investigated, and it was demonstrated that pancreatic islets contain a nucleoside phosphorylase capable of converting inosine into hypoxanthine and ribose 1-phosphate. Inosine at 10 mM concentration increased the lactate production and the content of ATP, glucose 6-phosphate (fructose 1,6-diphosphate + triose phosphates) and cyclic AMP in islets from fed mice. In islets from starved mice inosine-induced lactate production was decreased and no change in the concentration of cyclic AMP could be demonstrated, whereas the concentration of ATP and glucose 6-phosphate rose. Inosine (10 mM) induced a higher concentration of (fructose 1,6-diphosphate + triose phosphates) in islets from starved mice than in islets from fed mice suggesting that in starvation the activities of glyceraldehyde 3-phosphate dehydrogenase or other enzymes below this step in glycolysis are decreased. Formation of glucose from inosine was negligible. Inosine had no direct effect on adenylate cyclase activity in islet homogenates. The observed changes in insulin secretion and islet metabolism mimic what is seen when glucose and glyceraldehyde stimulate insulin secretion, and as neither ribose nor hypoxanthine-stimulated insulin release, the results are interpreted as supporting the substrate-site hypothesis for glucose-induced insulin secretion according to which glucose has to be metabolized in the beta-cells before secretion is initiated.

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Pancreatic beta cell selective deletion of mitofusins 1 and 2 (Mfn1 and Mfn2) disrupts mitochondrial architecture and abrogates glucose-stimulated insulin secretion in vivo

10.1101/2020.04.22.055384 ◽

2020 ◽

Cited By ~ 1

Author(s):

Eleni Georgiadou ◽

Charanya Muralidharan ◽

Michelle Martinez ◽

Pauline Chabosseau ◽

Alejandra Tomas ◽

...

Keyword(s):

Insulin Secretion ◽

Membrane Potential ◽

Beta Cell ◽

Beta Cells ◽

Glucose Sensing ◽

Mitochondrial Fusion ◽

Intravital Imaging ◽

Mouse Islets

AbstractBackground and aimsMitochondria are highly dynamic organelles, fundamental to cellular energy homeostasis. Mitochondrial metabolism of glucose is essential for the initiation of insulin release from pancreatic beta cells. Whether mitochondrial ultra-structure, and the proteins controlling fission and fusion, are important for glucose recognition are unclear. Mitochondrial fusion is supported by proteins including mitofusin 1 (MFN1), mitofusin 2 (MFN2) and optic atrophy (OPA1), and fission by dynamin-related protein 1 (DRP1). Here, we generated mice with beta cell-selective, adult-restricted deletion of Mfn1 and Mfn2 (βMfn1/2-KO), and explored the impact on insulin secretion and glucose homeostasis in vivo and in vitro.Materials and methodsC57BL/6J mice bearing Mfn1 and Mfn2 alleles with loxP sites, were crossed to animals carrying an inducible Cre recombinase at the Pdx1 locus (PdxCreERT). Isolated islets were used for live beta cell fluorescence imaging of cytosolic (Cal-520) or mitochondrial (Pericam) free Ca2+ concentration and membrane potential (TMRE). Mitochondrial network characteristics were quantified using super resolution fluorescence and transmission electron microscopy. Beta cell-beta cell connectivity was assessed using the Pearson (R) analysis and Monte Carlo simulation in intact mouse islets. Intravital imaging was performed in mice injected with an adeno-associated virus to express the cytosolic Ca2+ sensor GCaMP6s selectively in beta cells and TMRM to visualise mitochondria using multiphoton microscopy.ResultsβMfn1/2-KO mice displayed higher fasting glycaemia than control littermates at 14 weeks (8.6 vs 6.4 mmol/L, p>0.05) and a >five-fold decrease in plasma insulin post-intraperitoneal glucose injection (5-15 min, p<0.0001). Mitochondrial length, and glucose-induced Ca2+ accumulation, mitochondrial hyperpolarisation and beta cell connectivity were all significantly reduced in βMfn1/2-KO mouse islets. Examined by intravital imaging of the exteriorised pancreas, antiparallel changes in cytosolic Ca2+ and mitochondrial membrane potential, observed in control animals in vivo, were suppressed after Mfn1/2 deletion.ConclusionMitochondrial fusion and fission cycles are essential in the beta cell to maintain normal mitochondrial bioenergetics and glucose sensing both in vitro and in the living mouse. Such cycles may be disrupted in some forms of diabetes to impair mitochondrial function and, consequently, insulin secretion.

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