GLP-1 and GIP receptors signal through distinct ß-arrestin 2-dependent pathways to regulate pancreatic ß cell function.

Glucagon-like peptide 1 (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIPR) receptors are G-protein-coupled receptors involved in glucose homeostasis. Diabetogenic conditions decrease ß-arrestin 2 (ARRB2) levels in human islets. In mouse ß cells, ARRB2 dampens insulin secretion by partially uncoupling cyclic AMP (cAMP)/protein kinase A (PKA) signaling at physiological doses of GLP-1, whereas at pharmacological doses, the activation of extracellular signal-related kinase (ERK)/cAMP-responsive element-binding protein (CREB) requires ARRB2. In contrast, GIP-potentiated insulin secretion needs ARRB2 in mouse and human islets. The GIPR-ARRB2 axis is not involved in cAMP/PKA or ERK signaling but does mediate GIP-induced F-actin depolymerization. Finally, the dual GLP-1/GIP agonist tirzepatide does not require ARRB2 for the potentiation of insulin secretion. Thus, ARRB2 plays distinct roles in regulating GLP-1R and GIPR signaling, and we highlight (1) its role in the physiological context and the possible functional consequences of its decreased expression in pathological situations such as diabetes and (2) the importance of assessing the signaling pathways engaged by the agonists (biased/dual) for therapeutic purposes.

Metabolic Stress Impairs Pericyte Response to Optogenetic Stimulation in Pancreatic Islets.

Pancreatic islets are highly vascularized micro-organs ensuring whole body glucose homeostasis. Islet vascular cells play an integral part in sustaining adequate insulin release by beta cells. In particular, recent studies have demonstrated that islet pericytes regulate local blood flow velocity and are required for maintenance of beta cell maturity and function. In addition, increased metabolic demand accompanying obesity alters islet pericyte morphology. Here, we sought to explore the effects of metabolic stress on islet pericyte functional response to stimulation in a mouse model of type 2 diabetes, directly in the pancreas in vivo . We found that high fat diet induced islet pericyte hypertrophy without alterations in basal local blood flow. However, optogenetic stimulation of pericyte activity revealed impaired islet vascular responses, despite increased expression of genes encoding proteins directly or indirectly involved in cell contraction. These findings suggest that metabolic stress impinges upon islet pericyte function, which may contribute to beta cell failure during T2D.

The nuclear receptor REV-ERBα is implicated in the alteration of ß-cell autophagy and survival under diabetogenic conditions.

Pancreatic ß-cell failure in type 2 diabetes mellitus (T2DM) is associated with impaired regulation of autophagy which controls ß-cell development, function, and survival through clearance of misfolded proteins and damaged organelles. However, the mechanisms responsible for defective autophagy in T2DM ß-cells remain unknown. Since recent studies identified circadian clock transcriptional repressor REV-ERBα as a novel regulator of autophagy in cancer, in this study we set out to test whether REV-ERBα-mediated inhibition of autophagy contributes to the ß-cell failure in T2DM. Our study provides evidence that common diabetogenic stressors (e.g., glucotoxicity and cytokine-mediated inflammation) augment ß-cell REV-ERBα expression and impair ß-cell autophagy and survival. Notably, pharmacological activation of REV-ERBα was shown to phenocopy effects of diabetogenic stressors on the ß-cell through inhibition of autophagic flux, survival, and insulin secretion. In contrast, negative modulation of REV-ERBα was shown to provide partial protection from inflammation and glucotoxicity-induced ß-cell failure. Finally, using bioinformatic approaches, we provide further supporting evidence for augmented REV-ERBα activity in T2DM human islets associated with impaired transcriptional regulation of autophagy and protein degradation pathways. In conclusion, our study reveals a previously unexplored causative relationship between REV-ERBα expression, inhibition of autophagy, and ß-cell failure in T2DM.

Mechanisms of Beta-Cell Apoptosis in Type 2 Diabetes-Prone Situations and Potential Protection by GLP-1-Based Therapies.

Type 2 diabetes (T2D) is characterized by chronic hyperglycemia secondary to the decline of functional beta-cells and is usually accompanied by a reduced sensitivity to insulin. Whereas altered beta-cell function plays a key role in T2D onset, a decreased beta-cell mass was also reported to contribute to the pathophysiology of this metabolic disease. The decreased beta-cell mass in T2D is, at least in part, attributed to beta-cell apoptosis that is triggered by diabetogenic situations such as amyloid deposits, lipotoxicity and glucotoxicity. In this review, we discussed the molecular mechanisms involved in pancreatic beta-cell apoptosis under such diabetes-prone situations. Finally, we considered the molecular signaling pathways recruited by glucagon-like peptide-1-based therapies to potentially protect beta-cells from death under diabetogenic situations.

Methods to Study Roles of ß-Arrestins in the Regulation of Pancreatic ß-Cell Function.

Novel findings reveal important functional roles for ß-arrestin 1 and ß-arrestin 2 in the regulation of insulin secretion, ß-cell survival, and ß-cell mass plasticity not only by glucose but also by G-protein-coupled receptors, such as the glucagon-like peptide-1 (GLP-1) and the pituitary adenylate cyclase-activating polypeptide (PACAP) receptors or GPR40, or tyrosine kinase receptors, such as the insulin receptor. Here, we describe experimental protocols to knock down ß-arrestins by small interference RNA, to follow subcellular localization of ß-arrestins in the cytosol and nucleus of the insulinoma INS-1E rat pancreatic ß-cell line, and to analyze ß-arrestin protein expression by Western blot using INS-1E cells and isolated mouse or human pancreatic islets. We also provide details on how to genotype ß-arrestin 2 knockout (Arrb2-/-) mice and to evaluate ß-arrestin-mediated roles in ß-cell mass plasticity and ß-cell signaling using immunocytochemistry on pancreatic sections or on primary dispersed ß-cells from wild-type mice and Arrb2-/- mice.

Proteasomal degradation of the histone acetyl transferase p300 contributes to beta-cell injury in a diabetes environment.

In type 2 diabetes, amyloid oligomers, chronic hyperglycemia, lipotoxicity, and pro-inflammatory cytokines are detrimental to beta-cells, causing apoptosis and impaired insulin secretion. The histone acetyl transferase p300, involved in remodeling of chromatin structure by epigenetic mechanisms, is a key ubiquitous activator of the transcriptional machinery. In this study, we report that loss of p300 acetyl transferase activity and expression leads to beta-cell apoptosis, and most importantly, that stress situations known to be associated with diabetes alter p300 levels and functional integrity. We found that proteasomal degradation is the mechanism subserving p300 loss in beta-cells exposed to hyperglycemia or pro-inflammatory cytokines. We also report that melatonin, a hormone produced in the pineal gland and known to play key roles in beta-cell health, preserves p300 levels altered by these toxic conditions. Collectively, these data imply an important role for p300 in the pathophysiology of diabetes.

ERK1 is dispensable for mouse pancreatic beta cell function but is necessary for glucose-induced full activation of MSK1 and CREB.

AIMS/HYPOTHESIS: Insufficient insulin secretion from pancreatic beta cells, which is associated with a decrease in beta cell mass, is a characteristic of type 2 diabetes. Extracellular signal-related kinase 1 and 2 (ERK1/2) inhibition in beta cells has been reported to affect insulin secretion, gene transcription and survival, although whether ERK1 and ERK2 play distinct roles is unknown. The aim of this study was to assess the individual roles of ERK1 and ERK2 in beta cells using ERK1 (also known as Mapk3)-knockout mice (Erk1 -/- mice) and pharmacological approaches. METHODS: NAD(P)H, free cytosolic Ca2+ concentration and insulin secretion were determined in islets. ERK1 and ERK2 subplasmalemmal translocation and activity was monitored using total internal reflection fluorescence microscopy. ERK1/2, mitogen and stress-activated kinase1 (MSK1) and cAMP-responsive element-binding protein (CREB) activation were evaluated by western blot and/or immunocytochemistry. The islet mass was determined from pancreatic sections. RESULTS: Glucose induced rapid subplasmalemmal recruitment of ERK1 and ERK2. When both ERK1 and ERK2 were inhibited simultaneously, the rapid transient peak of the first phase of glucose-induced insulin secretion was reduced by 40% (p < 0.01), although ERK1 did not appear to be involved in this process. By contrast, ERK1 was required for glucose-induced full activation of several targets involved in beta cell survival; MSK1 and CREB were less active in Erk1 -/- mouse beta cells (p < 0.01) compared with Erk1 +/+ mouse beta cells, and their phosphorylation could only be restored when ERK1 was re-expressed and not when ERK2 was overexpressed. Finally, the islet mass of Erk1 -/- mice was slightly increased in young animals (4-month-old mice) vs Erk1 +/+ mice (section occupied by islets [mean ± SEM]: 0.74% ± 0.03% vs 0.62% ± 0.04%; p < 0.05), while older mice (10 months old) were less prone to age-associated pancreatic peri-insulitis (infiltrated islets [mean ± SEM]: 7.51% ± 1.34% vs 2.03% ± 0.51%; p < 0.001). CONCLUSIONS/INTERPRETATION: ERK1 and ERK2 play specific roles in beta cells. ERK2 cannot always compensate for the lack of ERK1 but the absence of a clear-cut phenotype in Erk1 -/- mice shows that ERK1 is dispensable in normal conditions.

Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1.

The metabolic syndrome covers metabolic abnormalities including obesity and type 2 diabetes (T2D). T2D is characterized by insulin resistance resulting from both environmental and genetic factors. A genome-wide association study (GWAS) published in 2010 identified TP53INP1 as a new T2D susceptibility locus, but a pathological mechanism was not identified. In this work, we show that mice lacking TP53INP1 are prone to redox-driven obesity and insulin resistance. Furthermore, we demonstrate that the reactive oxygen species increase in TP53INP1-deficient cells results from accumulation of defective mitochondria associated with impaired PINK/PARKIN mitophagy. This chronic oxidative stress also favors accumulation of lipid droplets. Taken together, our data provide evidence that the GWAS-identified TP53INP1 gene prevents metabolic syndrome, through a mechanism involving prevention of oxidative stress by mitochondrial homeostasis regulation. In conclusion, this study highlights TP53INP1 as a molecular regulator of redox-driven metabolic syndrome and provides a new preclinical mouse model for metabolic syndrome clinical research.

Calcium signaling in pancreatic ß-cells in health and in Type 2 diabetes.

Changes in cytosolic free Ca(2+) concentration ([Ca(2+)]c) play a crucial role in the control of insulin secretion from the electrically excitable pancreatic ß-cell. Secretion is controlled by the finely tuned balance between Ca(2+) influx (mainly through voltage-dependent Ca(2+) channels, but also through voltage-independent Ca(2+) channels like store-operated channels) and efflux pathways. Changes in [Ca(2+)]c directly affect [Ca(2+)] in various organelles including the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus, secretory granules and lysosomes, as imaged using recombinant targeted probes. Because most of these organelles have specific Ca(2+) influx and efflux pathways, they mutually influence free [Ca(2+)] in the others. In this article, we review the mechanisms of control of [Ca(2+)] in various compartments and particularly the cytosol, the endoplasmic reticulum ([Ca(2+)]ER), acidic stores and mitochondrial matrix ([Ca(2+)]mito), focusing chiefly on the most important physiological stimulus of ß-cells, glucose. We also briefly review some alterations of ß-cell Ca(2+) homeostasis in Type 2 diabetes.

ß-Arrestin2 plays a key role in the modulation of the pancreatic beta cell mass in mice.

AIMS/HYPOTHESIS: Beta cell failure due to progressive secretory dysfunction and limited expansion of beta cell mass is a key feature of type 2 diabetes. Beta cell function and mass are controlled by glucose and hormones/neurotransmitters that activate G protein-coupled receptors or receptor tyrosine kinases. We have investigated the role of ß-arrestin (ARRB)2, a scaffold protein known to modulate such receptor signalling, in the modulation of beta cell function and mass, with a specific interest in glucagon-like peptide-1 (GLP-1), muscarinic and insulin receptors. METHODS: ß-arrestin2-knockout mice and their wild-type littermates were fed a normal or a high-fat diet (HFD). Glucose tolerance, insulin sensitivity and insulin secretion were assessed in vivo. Beta cell mass was evaluated in pancreatic sections. Free cytosolic [Ca(2+)] and insulin secretion were determined using perifused islets. The insulin signalling pathway was evaluated by western blotting. RESULTS: Arrb2-knockout mice exhibited impaired glucose tolerance and insulin secretion in vivo, but normal insulin sensitivity compared with wild type. Surprisingly, the absence of ARRB2 did not affect glucose-stimulated insulin secretion or GLP-1- and acetylcholine-mediated amplifications from perifused islets, but it decreased the islet insulin content and beta cell mass. Additionally, there was no compensatory beta cell mass expansion through proliferation in response to the HFD. Furthermore, Arrb2 deletion altered the islet insulin signalling pathway. CONCLUSIONS/INTERPRETATION: ARRB2 is unlikely to be involved in the regulation of insulin secretion, but it is required for beta cell mass plasticity. Additionally, we provide new insights into the mechanisms involved in insulin signalling in beta cells.

Tolbutamide controls glucagon release from mouse islets differently than glucose: involvement of K(ATP) channels from both α-cells and δ-cells.

We evaluated the role of ATP-sensitive K⁺ (K(ATP)) channels, somatostatin, and Zn²âº 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 K(ATP) 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 K(ATP) channels and somatostatin signaling. Knockout of the Zn²âº transporter ZnT8 (ZnT8(-/-) mice) did not prevent the glucagonostatic effect of glucose. In conclusion, glucose can inhibit glucagon release independently of Zn²âº, K(ATP) channels, and somatostatin. Closure of K(ATP) 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.

Frequency-dependent mitochondrial Ca(2+) accumulation regulates ATP synthesis in pancreatic ß cells.

Pancreatic ß cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive K(+) channels and electrical spiking. The latter results in oscillatory Ca(2+) influx through voltage-gated Ca(2+) channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca(2+)]cyt and [Ca(2+)]mit, respectively) during these cycles is poorly understood. Importantly, the activation of Ca(2+)-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca(2+) pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary ß cells, we have deployed cytosolic (Fura red, Indo1) or green fluorescent protein-based recombinant-targeted (Pericam, 2mt8RP for mitochondria; D4ER for the ER) probes for Ca(2+) and cytosolic ATP/ADP (Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca(2+) uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca(2+) oscillation frequency as stable increases in [Ca(2+)]mit and cytosolic ATP/ADP; (3) mitochondrial Ca(2+) uptake rates remained constant between individual spikes, arguing against activity-dependent regulation ("plasticity") and (4) the relationship between [Ca(2+)]cyt and [Ca(2+)]mit is essentially unaffected by changes in endoplasmic reticulum Ca(2+) ([Ca(2+)]ER). Our findings thus highlight new aspects of Ca(2+) signalling in ß cells of relevance to the actions of both glucose and sulphonylureas.

The mitochondrial Ca2+ uniporter MCU is essential for glucose-induced ATP increases in pancreatic ß-cells.

Glucose induces insulin release from pancreatic ß-cells by stimulating ATP synthesis, membrane depolarisation and Ca(2+) influx. As well as activating ATP-consuming processes, cytosolic Ca(2+) increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca(2+) transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca(2+) uptake into these organelles, or to examine the impact on ß-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca(2+) and ATP/ADP ratio in single primary mouse ß-cells, using recombinant targeted (Pericam or Perceval, respectively) as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca(2+) uniporter, MCU, is required for depolarisation-induced mitochondrial Ca(2+) increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na(+)-Ca(2+) exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca(2+) uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca(2+) accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreatic ß-cells, and becomes defective in conditions mimicking the diabetic milieu.

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

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

Emerging roles for ß-arrestin-1 in the control of the pancreatic ß-cell function and mass: new therapeutic strategies and consequences for drug screening.

Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase in ß-cell mass, decreased ß-cell survival and impaired glucose-dependent insulin release. Pancreatic ß-cell proliferation, survival and secretion are thought to be regulated by signalling pathways linked to G-protein coupled receptors (GPCRs), such as the glucagon-like peptide-1 (GLP-1) and the pituitary adenylate cyclase-activating polypeptide (PACAP) receptors. ß-arrestin-1 serves as a multifunctional adaptor protein that mediates receptor desensitization, receptor internalization, and links GPCRs to downstream pathways such as tyrosine kinase Src, ERK1/2 or Akt/PKB. Importantly, recent studies found that ß-arrestin-1 mediates GLP-1 signalling to insulin secretion, GLP-1 antiapoptotic effect by phosphorylating the proapoptotic protein Bad through ERK1/2 activation, and PACAP potentiation of glucose-induced long-lasting ERK1/2 activation controlling IRS-2 expression. Together, these novel findings reveal an important functional role for ß-arrestin-1 in the regulation of insulin secretion and ß-cell survival by GPCRs.

Isolation and culture of mouse pancreatic islets for ex vivo imaging studies with trappable or recombinant fluorescent probes.

The endocrine pancreas contains small clusters of 1,000-2,000 neuroendocrine cells termed islets of Langerhans. By secreting insulin, glucagon, or other hormones as circumstances dictate, islets play a central role in the control of glucose homeostasis in mammals. Islets are dispersed throughout the exocrine tissue and comprise only 1-2% of the volume of the whole organ; human pancreas contains about 10(6) islets whereas rodents have approximately 2 x 10(3) islets. The isolation of islets from the exocrine tissue usually begins with digestion of the pancreas with collagenase. Collagenase-containing medium is either injected into the pancreatic duct, and the organ left to digest in situ, or added after isolation of the pancreas and its dissection into small pieces ex vivo. Islets can then be separated from the exocrine tissue by gradient density or by handpicking. The islets obtained can either be used intact, for example, to measure insulin or glucagon secretion or be dispersed into single cells with a Ca(2+)-free medium or with trypsin/dispase. The latter facilitates the introduction of recombinant or trappable probes and microimaging studies of, for example, changes in cytosolic-free Ca(2+) concentration or the dynamics of individual organelles or proteins.

Glucose controls cytosolic Ca2+ and insulin secretion in mouse islets lacking adenosine triphosphate-sensitive K+ channels owing to a knockout of the pore-forming subunit Kir6.2.

Glucose-induced insulin secretion is classically attributed to the cooperation of an ATP-sensitive potassium (K ATP) channel-dependent Ca2+ influx with a subsequent increase of the cytosolic free Ca2+ concentration ([Ca2+]c) (triggering pathway) and a K ATP channel-independent augmentation of secretion without further increase of [Ca2+]c (amplifying pathway). Here, we characterized the effects of glucose in beta-cells lacking K ATP channels because of a knockout (KO) of the pore-forming subunit Kir6.2. Islets from 1-yr and 2-wk-old Kir6.2KO mice were used freshly after isolation and after 18 h culture to measure glucose effects on [Ca2+]c and insulin secretion. Kir6.2KO islets were insensitive to diazoxide and tolbutamide. In fresh adult Kir6.2KO islets, basal [Ca2+]c and insulin secretion were marginally elevated, and high glucose increased [Ca2+]c only transiently, so that the secretory response was minimal (10% of controls) despite a functioning amplifying pathway (evidenced in 30 mm KCl). Culture in 10 mm glucose increased basal secretion and considerably improved glucose-induced insulin secretion (200% of controls), unexpectedly because of an increase in [Ca2+]c with modulation of [Ca2+]c oscillations. Similar results were obtained in 2-wk-old Kir6.2KO islets. Under selected conditions, high glucose evoked biphasic increases in [Ca2+]c and insulin secretion, by inducing K ATP channel-independent depolarization and Ca2+ influx via voltage-dependent Ca2+ channels. In conclusion, Kir6.2KO beta-cells down-regulate insulin secretion by maintaining low [Ca2+]c, but culture reveals a glucose-responsive phenotype mainly by increasing [Ca2+]c. The results support models implicating a K ATP channel-independent amplifying pathway in glucose-induced insulin secretion, and show that K ATP channels are not the only possible transducers of metabolic effects on the triggering Ca2+ signal.

Imaging a target of Ca2+ signalling: dense core granule exocytosis viewed by total internal reflection fluorescence microscopy.

Ca2+ ions are the most ubiquitous second messenger found in all cells, and play a significant role in controlling regulated secretion from neurons, endocrine, neuroendocrine and exocrine cells. Here, we describe microscopic techniques to image regulated secretion, a target of Ca2+ signalling. The first of these, total internal reflection fluorescence (TIRF), is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation (<150 nm). It is thus particularly useful for studies of regulated hormone secretion. A brief summary of this approach is provided, as well as a description of the physical basis for the technique and the tools to implement TIRF using a standard fluorescence microscope. We also detail the different fluorescent probes which can be used to detect secretion and how to analyze the data obtained. A comparison between TIRF and other imaging modalities including confocal and multiphoton microscopy is also included.

SREBP1 is required for the induction by glucose of pancreatic beta-cell genes involved in glucose sensing.

Previous studies have reported both positive and negative effects of culture of islets at high glucose concentrations on regulated insulin secretion. Here, we have reexamined this question in mouse islets and determined the role of changes in lipid synthesis in the effects of glucose. Glucose-stimulated insulin secretion (GSIS) and gene expression were examined in islets from C57BL/6 mice or littermates deleted for sterol-regulatory element binding protein-1 (SREBP1) after 4 days of culture at high glucose concentrations. Culture of control islets at 30 versus 8 mmol/l glucose led to enhanced secretion at both basal (3 mmol/l) and stimulatory (17 mmol/l) glucose concentrations and to enhanced triacylglycerol accumulation. These changes were associated with increases in the expression of genes involved in glucose sensing (glucose transporter 2, glucokinase, sulfonylurea receptor 1, inwardly rectifying K(+) channel 6.2), differentiation (pancreatic duodenal homeobox 1), and lipogenesis (Srebp1, fatty acid synthase, acetyl-coenzyme A carboxylase 1, stearoyl-coenzyme A desaturase 1). When cultured at either 8 or 30 mmol/l glucose, SREBP1-deficient (SREBP1(-/-)) islets displayed reduced GSIS and triacylglycerol content compared with normal islets. Correspondingly, glucose induction of the above genes in control islets was no longer observed in SREBP1(-/-) mouse islets. We conclude that enhanced lipid synthesis mediated by SREBP1c-dependent genes is required for the adaptive changes in islet gene expression and insulin secretion at high glucose concentrations.

MicroRNA-124a regulates Foxa2 expression and intracellular signaling in pancreatic beta-cell lines.

MicroRNAs (miRNAs) are short non-coding RNAs that have been implicated in fine-tuning gene regulation, although the precise roles of many are still unknown. Pancreatic development is characterized by the complex sequential expression of a gamut of transcription factors. We have performed miRNA expression profiling at two key stages of mouse embryonic pancreas development, e14.5 and e18.5. miR-124a2 expression was strikingly increased at e18.5 compared with e14.5, suggesting a possible role in differentiated beta-cells. Among the potential miR-124a gene targets identified by biocomputation, Foxa2 is known to play a role in beta-cell differentiation. To evaluate the impact of miR-124a2 on gene expression, we overexpressed or down-regulated miR-124a2 in MIN6 beta-cells. As predicted, miR-124a2 regulated Foxa2 gene expression, and that of its downstream target, pancreatic duodenum homeobox-1 (Pdx-1). Foxa2 has been described as a master regulator of pancreatic development and also of genes involved in glucose metabolism and insulin secretion, including the ATP-sensitive K(+) (K(ATP)) channel subunits, Kir6.2 and Sur-1. Correspondingly, miR-124a2 overexpression decreased, and anti-miR-124a2 increased Kir6.2 and Sur-1 mRNA levels. Moreover, miR-124a2 modified basal and glucose- or KCl-stimulated intracellular free Ca(2+) concentrations in single MIN6 and INS-1 (832/13) beta-cells, without affecting the secretion of insulin or co-transfected human growth hormone, consistent with an altered sensitivity of the beta-cell exocytotic machinery to Ca(2+). In conclusion, whereas the precise role of microRNA-124a2 in pancreatic development remains to be deciphered, we identify it as a regulator of a key transcriptional protein network in beta-cells responsible for modulating intracellular signaling.