Insulin Secretory Granules Research Articles

ATP enhances exocytosis of insulin secretory granules in pancreatic islets under Ca 2+-depleted condition

Glucose and other nutrients have been shown to stimulate insulin release from pancreatic islets under Ca 2+-depleted condition when protein kinase A (PKA) and protein kinase C (PKC) are activated simultaneously. We investigated the role of metabolic nucleotide signals including ATP, ADP, and GTP in exocytosis of insulin secretory granules under Ca 2+-depleted condition using electrically permeabilized rat islets. ATP under PKC activation augmented insulin release concentration-dependently by 100 nM 12- O-tetradecanoyl-phorbol-13-acetate (TPA) in Ca 2+-depleted condition, while ADP could not suppress ATP-dependent insulin release in this condition. Neither GTP nor activated PKA in the absence of PKC activation increased insulin release under Ca 2+-depleted condition in the presence of ATP, but both enhanced insulin secretion in the presence of ATP when PKC was activated. In conclusion, activated PKC and the presence of ATP both are required in the insulin secretory process under Ca 2+-depleted condition. While PKA activation and GTP cannot substitute for PKC activation and ATP, respectively, under Ca 2+-depleted condition, they enhance ATP-dependent insulin secretion when PKC is activated.

Ectopic Expression of Syncollin in INS-1 β-Cells Sorts It into Granules and Impairs Regulated Secretion

Syncollin was first demonstrated to be a protein capable of affecting granule fusion in a cell-free system, but later studies revealed its luminal localization in zymogen granules. To determine its possible role in exocytosis in the intact cell, syncollin and a truncated form of the protein (lacking the N-terminal hydrophobic domain) were stably transfected in insulin-secreting INS-1 cells since these well-studied exocytotic cells appear not to express the protein per se. Studies by subcellular fractionation analysis, double immunofluorescence staining, and electron microscopy examination revealed that transfection of syncollin produced strong signals in the insulin secretory granules, whereas the product from transfecting the truncated syncollin was predominantly associated with the Golgi apparatus and to a lesser degree with the endoplasmic reticulum. The expressed products were associated with membranes and not the soluble fractions in either cytoplasm or the lumens of organelles. Importantly, insulin release stimulated by various secretagogues was severely impaired in cells expressing syncollin, but not affected by expressing truncated syncollin. Transfection of syncollin appeared not to impede insulin biosynthesis and processing, since cellular contents of proinsulin and insulin and the number of secretory granules were not altered. In addition, the early signals (membrane depolarization and Ca(2+) responses) for regulated insulin secretion were unaffected. These findings indicate that syncollin may be targeted to insulin secretory granules specifically and impair regulated secretion at a distal stage.

Docking and fusion of insulin secretory granules in SUR1 knock out mouse β-cells observed by total internal reflection fluorescence microscopy

To explore how the sulfonylurea receptor (SUR1) is involved in docking and fusion of insulin granules, dynamic motion of single insulin secretory granules near the plasma membrane was examined in SUR1 knock-out (Sur1KO) β-cells by total internal reflection fluorescence microscopy. Sur1KO β-cells exhibited a marked reduction in the number of fusion events from previously docked granules. However, the number of docked granules declined during stimulation as a consequence of the release of docked granules into the cytoplasm vs. fusion with the plasma membrane. Thus, the impaired docking and fusion results in decreased insulin exocytosis from Sur1KO β-cells.

Fatty Acid Translocase (FAT/CD36) Is Localized on Insulin-Containing Granules in Human Pancreatic β-Cells and Mediates Fatty Acid Effects on Insulin Secretion

The membrane receptor FAT/CD36 facilitates the major fraction of long-chain fatty acid (FA) uptake by muscle and adipose tissues. In line with the well-known effects of FA metabolism on carbohydrate utilization and insulin responsiveness, altered expression of CD36 has been linked to phenotypic features of the metabolic syndrome including insulin resistance and dyslipidemia. FA metabolism is also known to significantly affect insulin secretion. However, the role of CD36 in this process remains unknown, since its expression levels and function in the pancreas have not been explored. In the present study, freshly isolated human islets and a mouse-derived beta-cell line (MIN6) were shown positive for CD36 expression by RT-PCR, Western blot, and immunofluorescence. The identity of the PCR product was confirmed by microsequencing. The identified transcript was translated and the protein was expressed and subjected to the known posttranslational glycosylation. Fluorescence resonance energy transfer analysis and subcellular protein fractionation indicated that insulin and CD36 are colocalized in the secretory granules of beta-cells. Islet CD36 functioned in FA uptake because this process was blocked by the irreversible CD36 inhibitor sulfosuccinimidyl-oleate. More importantly, sulfosuccinimidyl-oleate reversed enhancing and inhibiting effects, respectively, of acute and long-term palmitate incubations on glucose-dependent insulin secretion. In conclusion, our study demonstrates that human islets express CD36 in the plasma membrane as well as in the insulin secretory granules. CD36 activity appears important for uptake of FA into beta-cells as well as for mediating their modulatory effects on insulin secretion.

Molecular mechanism of docking and fusion of insulin secretory granules in pancreatic β cell

Molecular mechanism of docking and fusion of insulin secretory granules in pancreatic β cell

Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in β-cells

Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic β-cells. Here, we show that exocytosis of SGs and insertion of ICA512 in the plasma membrane promotes the Ca2+-dependent cleavage of ICA512 cytoplasmic domain by μ-calpain. This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression. Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in β-cells, whose impaired insulin production and secretion causes diabetes.

A molecular basis underlying differences in the toxicity of botulinum serotypes A and E.

Botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of the proteins responsible for vesicle exocytosis. Paradoxically, two serotypes of BoNTs, A and E, cleave the same molecule, synaptosome-associated protein with relative molecular mass 25K (SNAP-25), and yet they cause synaptic blockade with very different properties. Here we compared the action of BoNTs A and E on the plasma membrane fusion machinery composed of syntaxin and SNAP-25. We now show that the BoNT/A-cleaved SNAP-25 maintains its association with two syntaxin isoforms in vitro, which is mirrored by retention of SNAP-25 on the plasma membrane in vivo. In contrast, BoNT/E severely compromises the ability of SNAP-25 to bind the plasma membrane syntaxin isoforms, leading to dissociation of SNAP-25. The distinct properties of botulinum intoxication, therefore, can result from the ability of shortened SNAP-25 to maintain its association with syntaxins-in the case of BoNT/A poisoning resulting in unproductive syntaxin/SNAP-25 complexes that impede vesicle exocytosis.

PPAR-γ overexpression selectively suppresses insulin secretory capacity in isolated pancreatic islets through induction of UCP-2 protein

Peroxisome proliferator-activated receptor-γ (PPAR-γ) regulates several cellular functions, but its physiological role in pancreatic islet cells remains to be investigated. In this study, we confirmed the presence of PPAR-γ in rat isolated islets and examined its role on insulin and glucagon secretion by using PPAR-γ-overexpressed islets. PPAR-γ overexpression significantly suppressed insulin secretion induced by stimulatory concentration of glucose (p<0.05). In addition, insulin secretion evoked by high potassium depolarization also was significantly decreased from PPAR-γ-overexpressed islets (p<0.05). On the other hand, no significant change in glucagon release was observed after high potassium depolarization between PPAR-γ-overexpressed and control islets. Insulin and glucagon content in islets was not statistically different between the two groups. In addition, the expression of uncoupling protein-2 (UCP-2) was found to be induced in PPAR-γ-overexpressed islets. This result clearly indicates that the deteriorative effect of PPAR-γ overexpression on the secretory machinery is selective for pancreatic β-cells. And it is possible that its site of action can be located in the energy-consuming exocytotic process of insulin secretory granules, and that the reduction of ATP production through increased UCP-2 reduces insulin exocytosis.

Secretory protein trafficking: genetic and biochemical analysis.

Acute insulin secretion from stimulated pancreatic beta-cells is derived from the intracellular pool of insulin secretory granules wherein insulin is packaged in a highly concentrated (and in some species, crystalline) state. Here we review experimental work, principally from our laboratory, on the question of biogenesis of mature secretory granules within the broader context of intracellular protein trafficking. Events occurring in the lumen of organelles at various stages of intracellular transport within the secretory pathway and events at the limiting membrane of newly forming secretory granules each contribute to formation of the insulin storage compartment comprising the readily releasable pool.

The Cytokine Interleukin-1β Reduces the Docking and Fusion of Insulin Granules in Pancreatic β-Cells, Preferentially Decreasing the First Phase of Exocytosis

The prediabetic period in type I diabetes mellitus is characterized by the loss of first phase insulin release. This might be due to islet infiltration mediated by mononuclear cells and local release of cytokines, but the mechanisms involved are unknown. To determine the role of cytokines in insulin exocytosis, we have presently utilized total internal reflection fluorescence microscopy (TIRFM) to image and analyze the dynamic motion of single insulin secretory granules near the plasma membrane in live beta-cells exposed for 24 h to interleukin (IL)-1beta or interferon (IFN)-gamma. Immunohistochemistry observed via TIRFM showed that the number of docked insulin granules was decreased by 60% in beta-cells treated with IL-1beta, while it was not affected by exposure to IFN-gamma. This effect of IL-1beta was paralleled by a 50% reduction in the mRNA and the number of clusters of SNAP-25 in the plasma membrane. TIRF images of single insulin granule motion during a 15-min stimulation by 22 mm glucose in IL-1beta-treated beta-cells showed a marked reduction in the fusion events from previously docked granules during the first phase insulin release. Fusion from newcomers, however, was well preserved during the second phase of insulin release of IL-1beta-treated beta-cells. The present observations indicate that IL-1beta, but not IFN-gamma, has a preferential inhibitory effect on the first phase of glucose-induced insulin release, mostly via an action on previously docked granules. This suggests that beta-cell exposure to immune mediators during the course of insulitis might be responsible for the loss of first phase insulin release.

Impaired glucose homeostasis and mitochondrial abnormalities in offspring of rats fed a fat-rich diet in pregnancy.

We previously reported that prenatal and suckling exposure to a maternal diet rich in animal fat leads to cardiovascular dysfunction in young adult rat offspring with subsequent development of dyslipidemia and hyperglycemia. We have further investigated glucose homeostasis in adult female offspring by euglycemic-hyperinsulinemic clamp and by dynamic assessment of glucose-stimulated insulin secretion in isolated, perifused pancreatic islet cells. Additionally, given the link between reduced mitochondrial DNA (mtDNA) content and the development of type 2 diabetes mellitus, we have measured mtDNA in organs from young adult animals. Sprague-Dawley rats were fed a diet rich in animal fat or normal chow throughout pregnancy and weaning. Infusion of insulin (5 mU.kg(-1).min(-1)) resulted in a higher steady-state plasma insulin concentration in 1-year-old offspring of fat-fed dams (OHF, n = 4) vs. offspring of control dams (OC, n = 4, P < 0.01). Glucose-stimulated insulin secretion in isolated islets from 9-mo-old OHF was significantly reduced compared with OC (n = 4, P < 0.05). Transmission electron micrography showed altered insulin secretory granule morphology in OHF pancreatic beta-cells. Kidney mtDNA was reduced in 3-mo-old OHF [16S-to-18S gene ratio: OC (n = 10) 1.05 +/- 0.19 vs. OHF (n = 10) 0.66 +/- 0.06, P < 0.05]. At 6 mo, gene chip microarray of OHF aorta showed reduced expression of the mitochondrial genome. Prenatal and suckling exposure to a diet rich in animal fat leads to whole body insulin resistance and pancreatic beta-cell dysfunction in adulthood, which is preceded by reduced tissue mtDNA content and altered mitochondrial gene expression.

Glucokinase is an integral component of the insulin granules in glucose-responsive insulin secretory cells and does not translocate during glucose stimulation.

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.

Ryanodine receptors in human pancreatic beta cells: localization and effects on insulin secretion.

It is clear that pancreatic beta-cell dysfunction, including basal hyperinsulinemia and reduced insulin release in response to glucose, is a key determinant of disease progression in type 2 diabetes, but the underlying molecular defects are not known. In diabetes, the expression and function of ryanodine receptor (RyR) Ca2+ release channels are reduced. The present studies were undertaken to define the subcellular location and role of RyR in the control of stimulated and basal insulin release from human pancreatic beta cells. Using confocal microscopy, we observed RyR immunoreactivity in a vesicular pattern. RyRs did not colocalize with insulin secretory granules but partially colocalized with endosomes. Direct activation with nanomolar concentrations of ryanodine evoked increases in cytosolic Ca2+ that were coupled to transient insulin release. Insulin release stimulated by 1 nM ryanodine was sensitive to BAPTA-AM preincubation but independent of thapsigargin-sensitive endoplasmic reticulum (ER) Ca2+ pools. Blocking RyRs with micromolar concentrations of ryanodine led to BAPTA-resistant insulin release that was not associated with an increase in cytosolic Ca2+, which implicated alterations in luminal Ca2+. However, neither Ca2+ signals nor insulin release stimulated by glucose was blocked by 10-50 microM ryanodine, which suggests that the CD38/cyclic ADP-ribose/RyR pathway is not a primary mechanism of glucose action in nontransformed beta cells. We provide the first evidence that RyRs directly control insulin secretion in primary beta cells. Unexpectedly, stimulation of insulin secretion by ryanodine occurs independently of glucose and by two mechanisms, including a novel cytosolic Ca2+-independent mechanism likely involving changes in Ca2+ within the lumens of non-ER organelles, such as endosomes.

Misfolded proinsulin accumulates in expanded pre‐Golgi intermediates and endoplasmic reticulum subdomains in pancreatic beta cells of Akita mice

A missense mutation of the insulin 2 gene (Cys96Tyr) in Akita mice disrupting one of the two interchain disulfide bonds results in intracellular accumulation of misfolded proinsulin. We analyzed the secretory pathway of pancreatic beta cells by electron microscopy and morphometry and identified sites of proinsulin accumulation by quantitative immunogold electron microscopy in this protein-folding disease. In Akita mice beta cells, the volume density of dilated endoplasmic reticulum subdomains was increased by 2.9-fold, resulting in a 1.7-fold increased volume density of the entire rough endoplasmic reticulum. The volume density of pre-Golgi intermediates was increased by 4.9-fold, and that of the Golgi apparatus was increase by 3.4-fold. The relative labeling intensity for proinsulin was 2.1-fold higher in dilated endoplasmic reticulum subdomains and 2.9-fold higher in pre-Golgi intermediates as compared with narrow endoplasmic reticulum, resulting in a significantly different distribution pattern between Akita and control mice beta cells (Chi2= 29.97, P<0.001). The numerical density of insulin secretory granules was equal in Akita and control mice beta cells. However, their volume density and average volume were reduced to 20% and their average diameter to 58% in Akita mice. Together, these data demonstrate that misfolded proinsulin accumulates mainly in pre-Golgi intermediates and to a lesser extent in dilated endoplasmic reticulum subdomains, providing evidence for the importance of pre-Golgi intermediates in a protein folding disease.

Stabilization of mRNA by PTB promotes the biogenesis of insulin secretory granules

Stabilization of mRNA by PTB promotes the biogenesis of insulin secretory granules

Cleavage and nuclear translocation of ICA512 upon exocytosis of insulin secretory granules

Islet Cell Autoantigen 512 (ICA512), is a receptor tyrosine phosphatase-like protein associated with insulin-containing secretory granules (SGs) of pancreatic ß-cells, and neurosecretory granules in general. We have previously shown that stimulation of exocytosis from insulinoma INS-1 cells leads to a significant degradation of the ICA512 pool associated with SGs. The cytosolic tail of ICA512 contains a PEST domain. Proteins with PEST domains are often cleaved by Ca2+-dependent proteases of the calpain family. Further evidence suggested that ICA512 is cleaved by µ-calpain. We have now proven this hypothesis by showing that over-expression of µ-calpain increases ICA512 cleavage in stimulated INS-1 cells. Conversely, down-regulation of µ-calpain by RNA interference reduces ICA512 cleavage. Confocal microscopy shows that m-calpain is enriched at the plasma membrane of INS-1 cells. Transfection of INS-1 cells with tetanus toxin, which prevents fusion of SGs with the plasma membrane, inhibits ICA512 cleavage. ICA512 cleavage is also reduced upon over-expression of dynamin I-GFP or dynamin II-GFP, which increase the rate of endocytosis, while it is enhanced by expression of dynamin I (K44A)-GFP or dynamin II (K44A)-GFP, which interfere with clathrin-mediated endocytosis. To follow the fate of the ICA512 cytoplasmic fragment generated by µ-calpain, the C-terminal domain of ICA512 was tagged with GFP. ICA512-GFP is correctly processed in the ectodomain and sorted in insulin-containing SGs. Preliminary studies by confocal microscopy suggest that a fraction of ICA512-GFP is present in the nuclei of stimulated cells, whereas virtually no nuclear GFP labeling is present in resting cells. Taken together, these data demonstrate that ICA512 is cleaved by µ-calpain at the plasma membrane following SG exocytosis, and prior to its internalization by endocytosis of SG membranes. Moreover, they suggest that the resulting ICA512 cytosolic fragment translocates to the nucleus. Thus, ICA512 may act as a feedback signal that couples regulated secretion and gene expression in ß-cells.

Polypyrimidine tract-binding protein promotes insulin secretory granule biogenesis.

Pancreatic beta-cells store insulin in secretory granules that undergo exocytosis upon glucose stimulation. Sustained stimulation depletes beta-cells of their granule pool, which must be quickly restored. However, the factors promoting rapid granule biogenesis are unknown. Here we show that beta-cell stimulation induces the nucleocytoplasmic translocation of polypyrimidine tract-binding protein (PTB). Activated cytosolic PTB binds and stabilizes mRNAs encoding proteins of secretory granules, thus increasing their translation, whereas knockdown of PTB expression by RNA interference (RNAi) results in the depletion of secretory granules. These findings may provide insight for the understanding and treatment of diabetes, in which insulin secretion is typically impaired.

Pancreatic beta-cell granule peptides form heteromolecular complexes which inhibit islet amyloid polypeptide fibril formation.

Islet amyloid polypeptide (IAPP), or 'amylin', is co-stored with insulin in secretory granules of pancreatic islet beta-cells. In Type 2 diabetes, IAPP converts into a beta-sheet conformation and oligomerizes to form amyloid fibrils and islet deposits. Granule components, including insulin, inhibit spontaneous IAPP fibril formation in vitro. To determine the mechanism of this inhibition, molecular interactions of insulin with human IAPP (hIAPP), rat IAPP (rIAPP) and other peptides were examined using surface plasmon resonance (BIAcore), CD and transmission electron microscopy (EM). hIAPP and rIAPP complexed with insulin, and this reaction was concentration-dependent. rIAPP and insulin, but not pro-insulin, bound to hIAPP. Insulin with a truncated B-chain, to prevent dimerization, also bound hIAPP. In the presence of insulin, hIAPP did not spontaneously develop beta-sheet secondary structure or form fibrils. Insulin interacted with pre-formed IAPP fibrils in a regular repeating pattern, as demonstrated by immunoEM, suggesting that the binding sites for insulin remain exposed in hIAPP fibrils. Since rIAPP and hIAPP form complexes with insulin (and each other), this could explain the lack of amyloid fibrils in transgenic mice expressing hIAPP. It is likely that IAPP fibrillogenesis is inhibited in secretory granules (where the hIAPP concentration is in the millimolar range) by heteromolecular complex formation with insulin. Alterations in the proportions of insulin and IAPP in granules could disrupt the stability of the peptide. The increase in the proportion of unprocessed pro-insulin produced in Type 2 diabetes could be a major factor in destabilization of hIAPP and induction of fibril formation.

Site of Docking and Fusion of Insulin Secretory Granules in Live MIN6 β Cells Analyzed by TAT-conjugated Anti-syntaxin 1 Antibody and Total Internal Reflection Fluorescence Microscopy

To determine the site of insulin exocytosis in the pancreatic beta cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled with Cy3 then conjugated with the protein transduction domain of HIV-1 TAT. TAT-conjugated Cy3-labeled anti-syntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 beta cells, which enabled us to observe the spatial organization and distribution of endogenous syntaxin 1. TIRFM imaging revealed that syntaxin 1 is distributed in numerous separate clusters in the intact plasma membrane, where insulin secretory granules were docked preferentially to the sites of syntaxin 1 clusters, colocalizing with synaptosomal-associated protein of 25 kDa (SNAP-25) clusters. TIRFM imaging analysis of the motion of single insulin granules demonstrated that the fusion of insulin secretory granules stimulated by 50 mm KCl occurred exclusively at the sites of the syntaxin 1 clusters. Cholesterol depletion by methyl-beta-cyclodextrin treatment, in which the syntaxin 1 clusters were disintegrated, decreased the number of docked insulin granules, and, eventually the number of fusion events was significantly reduced. Our results indicate that 1) insulin exocytosis occurs at the site of syntaxin 1 clusters; 2) syntaxin 1 clusters are essential for the docking and fusion of insulin granules in MIN6 beta cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.

Fluorescence microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic β-cell ATP-sensitive K + currents

Hypoglycemic sulfonylureas (e.g. tolbutamide, glibenclamide) exert their stimulatory effects on pancreatic β-cells by closure of ATP-sensitive K + (K ATP) channels. Pancreatic K ATP channels are composed of two subunits, a pore-forming inwardly rectifying K + channel (Kir6.2) subunit and a regulatory subunit (the sulfonylurea receptor of subtype 1 (SUR1)) in a (SUR1/Kir6.2) 4 stoichiometry. The aim of the present study was to characterize the interaction of green-fluorescent 3-[3-(4,4 difluoro-5,7-dimethyl-4-bora-3 a,4 a-diaza- S-indacen-3-yl)propanamido] glibenclamide (Bodipy-glibenclamide) with pancreatic β-cell K ATP channels using patch-clamp and fluorescence microscopy techniques. Bodipy-glibenclamide inhibited K ATP currents from the clonal insulinoma cell line RINm5F half-maximally at a concentration of 0.6 nM. Using laser-scanning confocal microscopy Bodipy-glibenclamide was shown to induce a diffuse fluorescence across the RINm5F cell, but only about 17% of total Bodipy-glibenclamide-induced fluorescence intensity in RINm5F cells was due to specific binding to SUR1. Using fluorescence correlation spectroscopy, it could be demonstrated that the fluorescence label contributes to the protein binding and, therefore, possibly also to the non-specific binding of Bodipy-glibenclamide observed in RINm5F cells. Specific binding of Bodipy-glibenclamide to SUR1 in RINm5F cells might be localized to different intracellular structures (nuclear envelope, endoplasmic reticulum, Golgi compartment, insulin secretory granules) as well as to the plasma membrane. In conclusion, Bodipy-glibenclamide is a high-affinity blocker of pancreatic β-cell K ATP currents and can be used for visualizing SUR1 in intact pancreatic β-cells, although non-specific binding must be taken into account in confocal microscopy experiments on intact β-cells.