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High extracellular glucose inhibits exocytosis through disruption of syntaxin 1A-containing lipid rafts
Biochemical and Biophysical Research Communications 389 (2009) 241–246
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
High extracellular glucose inhibits exocytosis through disruption of syntaxin 1A-containing lipid rafts Sangeeta Somanath a, Sebastian Barg b, Catriona Marshall a, Christopher J. Silwood a, Mark D. Turner a,* a
Centre for Diabetes, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Whitechapel, London E1 2AT, UK b Department of Medical Cell Biology, Biomedical Center, Uppsala University, 751 23 Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 20 August 2009 Available online 28 August 2009 Keywords: Hyperglycemia Cholesterol Insulin secretion SNAREs
a b s t r a c t Diabetes is characterized by high blood glucose which eventually impairs the secretion of insulin. Glucose directly affects cholesterol biosynthesis and may in turn affect cellular structures that depend on the sterol, including lipid rafts that help organize the secretory apparatus. Here, we investigated the long-term effects of glucose upon lipid rafts and secretory granule dynamics in pancreatic b-cells. Raft fractions, identified by the presence of GM1 and flotillin, contained characteristically high levels of cholesterol and syntaxin 1A, the t-SNARE which tethers granules to the plasma membrane. Seventy-two hours exposure to 28 mM glucose resulted in 30% reduction in membrane cholesterol, with consequent redistribution of raft markers and syntaxin 1A throughout the plasma membrane. Live cell imaging indicated loss of syntaxin 1A from granule docking sites, and fewer docked granules. In conclusion, glucose-mediated inhibition of cholesterol biosynthesis perturbs lipid raft stability, resulting in a loss of syntaxin 1A from granule docking sites and inhibition of insulin secretion. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Type 2 diabetes (T2D) is characterized by failure to control glucose homeostasis, and numerous diabetic complications are attributable to exposure of tissues to high glucose. Glucotoxicity leads to a gradual decline in both b-cell function and b-cell mass [1]. Pancreatic b-cells also undergo multiple changes in gene expression in response to either acute or chronic high glucose. The functions of these genes are widespread and affect metabolism, signaling, transcription, oligonucleotide splicing, cell cycle, apoptosis, and the secretory pathway [2,3]. However, the precise mechanisms by which high glucose inhibits insulin secretion still remain poorly understood. Materials and methods Materials. Anti-3-hydroxy-methylglutaryl coenzyme A reductase antibody was obtained from Millipore (Consett, UK), anti-flotillin antibody from BD Biosciences (Franklin Lakes, NJ, USA), anti-mouse secondary antibody from Dako Cytomation Ltd. (Ely, UK), and antirabbit secondary antibody from Bio-Rad Laboratories (Hemel * Corresponding author. Address: Centre for Diabetes, Blizard Institute for Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, 4 Newark Street, Whitechapel, London E1 2AT, UK. Fax: +44 (0) 20 7882 2186. E-mail address: [email protected] (M.D. Turner). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.08.126
Hempstead, UK). Unless otherwise stated, all other reagents were obtained from Sigma–Aldrich Company Ltd. (Poole, UK). Cell culture and insulin secretion. INS-1 b-cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum, and either 11 mM or 28 mM glucose, for 72 h. Media was also supplemented for 1 h with soluble cholesterol, where indicated. Cells were then washed with Krebs–Ringer (KRH) solution (125 mM NaCl, 1.2 mM KH2PO4, 2 mM MgSO4, 1 mM CaCl2, 1.67 mM glucose, 0.1% BSA, 25 mM HEPES; pH 7.4) and incubated for 30 min in KRH ±60 mM KCl. Supernatant was collected and insulin concentration determined following ELISA kit protocols (Mercodia; Uppsala, Sweden). Cellular insulin content. INS-1 cells were grown in RPMI-1640 media supplemented with glucose, in parallel to those used for secretion experiments. Media was removed and 500 ll of extraction solution containing 1.5% hydrochloric acid, 18.5% distilled water, 80% ethanol added. After 24 h at 4 °C, 500 ll 0.1 M sodium hydroxide was added to neutralize the samples, then insulin content assayed using standard ELISA protocols (Mercodia; Uppsala, Sweden). Cholesterol quantitation. Islets were isolated from Wistar rats following previously published protocols [4]. Islets were lysed with 200 ll of RIPA buffer. INS-1 membrane and cytosol fractions were isolated from cell lysates as detailed previously [5]. Two hundred microliters of chloroform–1% Triton X-100 was added to each sample, spun for 15 min at 14,000 rpm, and the lower organic phase collected. Samples were air dried at 50 °C for 30 min, then vacuum
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dried for a further 45 min. The resulting pellet was dissolved in 150 ll of cholesterol reaction buffer. Fifty microliters of reaction mix (46 ll reaction buffer, 2 ll cholesterol probe, 2 ll enzyme) was added to 50 ll sample and incubated at 37 °C for 60 min. OD was measured at 570 nm. Discontinuous density fractionation. INS-1 cells were grown in RPMI-1640, plus 11.7 mM or 28 mM glucose, for 72 h, then resuspended in lysis buffer (1% Triton X-100, 25 mM Tris–HCl, 1 mM EDTA, 150 mM NaCl, 1 mM NaVO4, 1 mM NaF) plus Complete protease inhibitor (Roche Diagnostics, Basel, Switzerland). Cells were homogenized by passing 8 through a 25 gauge syringe needle, then samples centrifuged at 500g for 10 min at 4 °C to remove nuclei and cell debris. A three-step discontinuous OptiPrep gradient was prepared utilizing 40%, 30%, and 0% OptiPrep. Gradients were centrifuged at 17,000g for 2 h at 4 °C in a Beckman XL-70 ultracentrifuge, using a swinging 55-Ti rotor. Eight fractions were generated per gradient. An aliquot was kept for Western blotting while the rest were freeze dried and cholesterol assay performed as per protocol. Western and dot blotting. INS-1 cell homogenates (after normalizing samples for cellular protein content), or gradient fractions, were boiled in Laemmli sample buffer and separated on 10% SDS–PAGE gels. Protein was transferred onto PVDF membrane using a Hoefer TE 70 semi-dry transfer unit (Amersham Pharmacia, Little Chalfont, UK). In the case of GM1, fractions were dotted directly onto PVDF membrane and allowed to dry. Protein was detected using anti-3-hydroxy-methylglutaryl coenzyme A reductase, anti-flotillin and anti-syntaxin 1 primary antibodies, and either anti-mouse or anti-rabbit HRP-conjugated secondary
A
antibody, or cholera toxin B subunit peroxidase conjugate. Antibody binding was detected with ECL Plus (Amersham Pharmacia; Little Chalfont, UK) and visualized with Hyperfilm ECL (Amersham Pharmacia; Little Chalfont, UK). TIRF. INS-1 cells were cultured in 11 or 28 mM glucose for 72 h. On day two, cells were plated onto polylysine coated coverslips and transfected with 1 lg syntaxin 1A-EGFP and 0.3 lg NPY-cherry using Lipofectamine 2000 (Invitrogen; Paisley, UK). On day 3, cells were imaged in a custom-built lens-type total internal reflection (TIRF) microscope with a 100/1.45 objective (Carl Zeiss MicroImaging; Thornwood, NY, USA). Excitation was at 473 and 555 nm (CrystaLaser; Reno, NV, USA). The emission light was separated onto two halves of an EMCCD camera (Roper Cascade 512B) using an image splitter (Photometrics; Tucson, AZ, USA) and emission filters (ET520/40 and ET630/75; both from Chroma Technology; Rockingham, VT, USA). Images shown are averages of 1 s movies acquired at 50 Hz and 20 ms exposure at 100 nm/pixel. Average fluorescence was read automatically by a macro written as MetaMorph journal in (1) a circle of 0.7 lm diameter and (2) in a surrounding annulus with an outer diameter of 1.1 lm. The circle represents fluorescence at the site of the granule, while the annulus represents local background. The annulus value was subtracted from that of the circle to yield the excess syx-EGFP fluorescence beneath a granule. Granules were counted automatically with an in-house algorithm written as MetaMorph journal. Briefly, the image was subjected to morphological tophat, H-Dome, and dilate operations, before internal thresholding and counting of objects. A threshold was set by the operator once for the entire data set.
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Fig. 1. Effect of high glucose on b-cell cholesterol content. (A) INS-1 cells were cultured for 72 h in RPMI-1640 media containing either 11 mM or 28 mM glucose. Cell homogenates were boiled in SDS dissociation buffer, then separated on 10% SDS gel, immunoblotted with anti-3-hydroxy-methylglutaryl coenzyme A reductase antibody and detected using ECL chemiluminescence. (B) Rat islets were isolated by collagenase digestion and incubated for 72 h in either 11 mM or 28 mM glucose. Cholesterol concentration was determined following extraction with chloroform/Triton X-100. OD was measured at 570 nm to quantify cholesterol concentration. (C) INS-1 membrane and cytosol fractions were isolated by differential centrifugation. Cholesterol concentration was determined following extraction with chloroform/Triton X-100. OD was measured at 570 nm to quantify cholesterol concentration.
S. Somanath et al. / Biochemical and Biophysical Research Communications 389 (2009) 241–246
Statistical analysis. Bar graphs represent mean ± SEM (n = 5 or more). Comparisons of parameters between the different media conditions were by one-way ANOVA, followed by Dunnett t posthoc test (2-way).
of INS-1 b-cells in 28 mM glucose, we observed a significant reduction in 3-hydroxy-methylglutaryl coenzyme A reductase (Fig. 1A). Densitometry analysis of films from 6 independent experiments showed that there was 69.0 ± 8.2% reduction (p < 0.01) in 3-hydroxy-methylglutaryl coenzyme A reductase protein. In order to determine the effect of high glucose upon islet cholesterol content, we isolated rat islets and exposed them to either 11 or 28 mM glucose for 72 h. Exposure to 28 mM glucose reduced total islet cholesterol content by 23.2 ± 3.8% (Fig. 1B). However islets consist of multiple cell types, and it is also not clear to what extent glucose would affect metabolic cholesterol in the cytosol, or membraneassociated cholesterol. This is particularly important, given that high serum cholesterol can inhibit insulin secretion through downregulation of b-cell metabolism [19]. Following 72 h exposure of INS-1 b-cells to 28 mM glucose we observed a 30.5 ± 5.9% reduction in membrane cholesterol, and a 25.7 ± 6.3% inhibition in cytosolic cholesterol (Fig. 1C). In order to investigate the effect of glucose upon b-cell exocytosis, without the consequences of altered glucose metabolism, we cultured INS-1 cells in either 11 mM or 28 mM glucose for 72 h, then depolarized cells with 60 mM KCl in the presence of 1.67 mM glucose. We observed 72.7 ± 6.7% inhibition of KCl-stimulated insulin secretion from cells which had been pre-incubated in 28 mM glucose (Fig. 2A). Interestingly, when cells were incubated in media containing 28 mM glucose which was also supplemented with soluble cholesterol for 1 h immediately prior to stimulation (Fig. 2A), we observed a concentration-dependent reduction in glucose-mediated inhibition of stimulated insulin secretion. In particular, we observed a reduction to only 27.9 ± 2.5% inhibition from cells incubated with 5 mM cholesterol. Crucially, cells are extremely sensitive to the extracellular choles-
Results and discussion Lipid rafts are specialized membrane nanodomains enriched with glycosphingolipids and cholesterol. The resultant tight packing of these acyl lipid chains is thought to slow lateral protein diffusion rates [6–8]. Interestingly, cholesterol depletion inhibits a number of fundamental intracellular transport steps, including both endocytosis [9,10] and exocytosis [11,12]. Exocytosis is the final step in the secretory pathway, and is mediated by soluble N ethyl maleimide sensitive fusion protein attachment receptor (SNARE) complex proteins. SNAREs form helical structures that together form helical bundles between granules/vesicles (v-SNAREs) and target membranes (t-SNAREs) to facilitate membrane fusion [13,14]. In addition to having a highly enriched t-SNARE content, lipid rafts also serve to compartmentalize the predominant voltage-gated Ca2+ and K+ channels that mediate stimulus-secretion coupling in b-cells [15]. By concentrating these molecules into discreet areas of the plasma membrane where insulin-containing secretory granules are docked [16,17], the exocytotic machinery becomes effectively clustered into multiprotein exitosome complexes [18]. It has previously been shown that 24 h exposure of MIN6 b-cells to high glucose resulted in a 3.5-fold decrease in expression of 3hydroxy-methylglutaryl coenzyme A reductase [2], a key enzyme in cholesterol biosynthesis. Similarly, following 72 h incubation
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Fig. 2. Effect of high glucose on insulin secretion, insulin content and cell viability. (A) INS-1 cells were incubated for 72 h in media supplemented either 11 mM or 28 mM glucose. Media was also supplemented for 1 h with soluble cholesterol where indicated. Cells were then washed three times, and incubated for 30 min in Krebs–Ringer solution ±60 mM KCl. Supernatant was collected and insulin concentration determined using rat insulin ELISA kit (Mercodia, Uppsala, Sweden). Cellular protein content of lysed cells was quantified, and used to normalize secretion data. (B) INS-1 cells were cultured for 72 h in RPMI-1640 media containing either 11 or 28 mM glucose. Media was removed from the wells, and intracellular insulin extracted using hydrochloric acid/ethanol. Insulin content was assayed using standard ELISA protocols. Total protein content was quantified to normalize insulin values obtained. (C) INS-1 cells were incubated in either 11 or 28 mM glucose. Cell viability was determined based on Trypan blue exclusion from nuclei of intact cells.
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terol concentration, as exposure to 12.5 mM cholesterol resulted in 99.0 ± 2.0% inhibition of simulated secretion. This latter observation is also consistent with the recent report of inhibition of insulin secretion from islets exposed to 10 mM extracellular cholesterol [19]. One of the genes sensitive to extracellular glucose concentration is that encoding insulin itself [20,21], and glucotoxicity has previously been shown to partially deplete b-cell insulin content [22]. We also found that INS-1 cells cultured for 72 h in 28 mM glucose had a 35.4 ± 1.2% reduction in cellular insulin content relative to control (Fig. 2B). This was not a result of glucotoxic cell death, as we observed no significant effect on cell viability (Fig. 2C). Although insulin content was partially depleted by high glucose, this alone cannot explain the much larger inhibition in insulin secretion. Moreover, recent work has also shown that high glucose does not deplete b-cells of exocytosis-competent insulin-containing secretory granules [22]. Our observations are therefore indicative of an effect of glucose upon a late exocytotic step. In order to test this hypothesis we investigated whether glucose could affect secretion by altering the distribution of either syntaxin 1A, which is highly enriched within lipid nanodomains [11,15], or the widely utilized lipid raft markers, flotillin and GM1. As lipid raft fractions are resistant to solubilization by the detergent Triton X-100, we exploited this characteristic, along with their buoyancy on sucrose gradients, to purify raft fractions. At 11 mM glucose, cholesterol, flotillin, and GM1 concentrations were highest in fractions 2–3 from discontinuous density fractionation (Fig. 3), indicative of their presence in lipid rafts. Although cells incubated in 28 mM glucose showed a reduction in cholesterol content in all sub-cellular
A Membrane Cholesterol (µg/µg cellular protein)
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fractions (Fig. 3A), there was a marked redistribution of the raft markers, flotillin and GM1 (Fig. 3B and C), and syntaxin 1A (Fig. 3D) to non-raft fractions in cells cultured in 28 mM glucose. Loss of syntaxin 1A from lipid rafts may affect its association with insulin granule release sites. In order to study the plasma membrane distribution of syntaxin 1A with greater resolution, we expressed EGFP-tagged syntaxin 1A (syx-EGFP) in INS-1 cells, together with a granule marker, NPY-cherry, and imaged living cells in total internal reflection mode (TIRF). Cells cultured in 11 mM glucose exhibited clusters of syx-EGFP at the plasma membrane (Fig. 4A), similar to those observed previously with immuno-staining techniques [23,24]. A subset of these clusters also colocalized with secretory granules. However, when cells were cultured in 28 mM glucose, syx-EGFP was more diffusely localized throughout the membrane (Fig. 4C). Furthermore, the intensity of these clusters was clearly diminished (Fig. 4B vs. D). For each cell we quantified these clusters by measuring the excess syx-EGFP at randomly chosen granules (see Methods), and normalized to the cell’s overall brightness as a measure of expression level. On average, in cells cultured at 11 mM glucose, the plasma membrane beneath granules contained 12.5 ± 2.0% more syx-EGFP, compared with the surrounding area (933 granules in 44 cells) (Fig. 4E). Cells that had been cultured in 28 mM glucose were studied in parallel, and the corresponding value was less than half (5.7 ± 1.5%; 696 granules in 36 cells, p < 0.01 vs. 11 mM), consistent with reduced binding of syntaxin 1A to granule-associated rafts. This average loss of syntaxin 1A from granule sites also corresponded to fewer granules colocalizing with a syntaxin cluster (30 ± 4% and 44 ± 3% for cells cultured at 28 and 11 mM glucose, respectively; 36 and 44 cells, p < 0.001) (Fig. 4F). In keeping with a
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Fig. 3. Effect of high glucose upon lipid raft integrity. INS-1 cells were incubated for 72 h in either 11 mM or 28 mM glucose, then resuspended in lysis buffer (1% Triton X100, 25 mM Tris–HCl, 1 mM EDTA, 150 mM NaCl, 1 mM NaVO4, 1 mM NaF) and homogenized by passing 8 through 25 gauge syringe needle. Eight fractions were generated per gradient, following centrifugation down a three-step discontinuous OptiPrep gradient. (A) Cholesterol concentration was quantified for each fraction generated, following extraction with chloroform/Triton X-100. OD was measured at 570 nm to quantify cholesterol concentration. (B) Fractions were separated on 10% SDS gel, then immunoblotted with anti-flotillin antibody and detected using ECL chemiluminescence. (C) Fractions were dotted directly onto PVDF membrane, then immunoblotted with anti-cholera toxin B subunit peroxidase conjugate and detected using ECL chemiluminescence. (D) Fractions were separated on 10% SDS gel, then immunoblotted with antisyntaxin 1A antibody and detected using ECL chemiluminescence.
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Fig. 4. Accumulation of syx-EGFP at granules. (A) TIRF images of a cell cultured in 11 mM glucose and expressing both syx-EGFP and NPY-cherry. The inset shows an enlarged area of the same cell. (B) Average image constructed from small syx-EGFP images centered at granule sites. All identified granules locations from one coverslip each are included in the average. (C) As in A, but cultured in 28 mM glucose. (D) As in B, but cultured in 28 mM glucose. (E) Colocalization of syx-EGFP with granules was measured following location of granules by eye. The circle represents the fluorescence at the site of the granule while the annulus represents the local background onto which the cluster is superimposed. The annulus value was subtracted from that of the circle to yield the excess syx-EGFP fluorescence beneath a granule. Bargraph shows mean and standard error of the excess syx-EGFP at granules in cells cultured at 11 and 28 mM glucose. (F) Granules were counted automatically with an in-house algorithm written as MetaMorph journal. The image was subjected to morphological tophat, H-Dome, and dilate operations, before internal thresholding and counting of objects. A threshold was set by the operator once for the entire data set. Bargraph shows mean and standard error of granule numbers associated with syntaxin clusters. (G) Bargraph shows mean and standard error of granules per unit area of plasma membrane. *p < 0.05; **p < 0.01; ***p < 0.001.
role of syntaxin 1A in docking, these cells tended to have fewer granules near the plasma membrane (0.54 ± 0.05 vs. 0.67 ± 0.04 granules/lm2 (p < 0.05) at 28 and 11 mM glucose) (Fig. 4G), in agreement with previous reports [24]. Interestingly, it has recently been observed that cholesterol-dependent SNAP-25 clustering is also essential for insulin secretion [25]. However, to our knowledge, our data provides the first evidence of a general requirement for lipid raft-dependent t-SNARE clustering that is sensitive to extracellular glucose environment. In conclusion, our data shows high glucose reduces membrane cholesterol content, with the resulting perturbation of plasma membrane lipid raft nanodomains leading to a reduction in syntaxin 1A-granule interactions. Importantly, interactions that do still take place are likely to occur at areas of the plasma membrane where there is an absence of clustering together of all key exitosome complex members. Under these circumstances the fidelity of the exocytotic machinery would be compromised, leading to the failure of b-cells to secrete adequate amounts of insulin in response to subsequent secretory stimuli. We suggest that the resulting inhibition in insulin secretion may lead to the breakdown in glucose homeostasis which results in the development of T2D.
Acknowledgments We are grateful to Shanta Persaud and Peter Jones (Diabetes Research Group, King’s College London) for provision of isolated rat islets. This research was supported by grant awards from Barts and The London Charitable Foundation (RAC 408 and RAB/06/ PHD/12). The authors declare that they have no conflict of interest. References [1] B.L. Wajchenberg, D-cell failure in diabetes and preservation by clinical treatment, Endocrine Rev. 28 (2007) 187–218. [2] G.C. Webb, M.S. Akbar, C. Zhao, D.F. Steiner, Expression profiling of pancreatic b-cells: glucose regulation of secretory and metabolic pathway genes, Proc. Natl. Acad. Sci. USA 97 (2000) 5773–5778. [3] F. Schuit, D. Flamez, A. De Vos, D. Pipeleers, Glucose-regulated gene expression maintaining the glucose-responsive state of b-cells, Diabetes 51 (2002) S326– S332. [4] P.M. Jones, F.M. Mann, S.J. Persaud, C.P.D. Wheeler-Jones, Mastoparan stimulates insulin secretion from pancreatic beta-cells at a late stage in the secretory pathway, Mol. Cell Endocrinol. 94 (1993) 97–104. [5] C. Marshall, G.A. Hitman, C.J. Partridge, A. Clark, H. Ma, T.R. Shearer, M.D. Turner, Evidence that an isoform of calpain-10 is a regulator of exocytosis in pancreatic b-cells, Mol. Endocrinol. 19 (2005) 213–224.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
High extracellular glucose inhibits exocytosis through disruption of syntaxin 1A-containing lipid rafts Sangeeta Somanath a, Sebastian Barg b, Catriona Marshall a, Christopher J. Silwood a, Mark D. Turner a,* a
Centre for Diabetes, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Whitechapel, London E1 2AT, UK b Department of Medical Cell Biology, Biomedical Center, Uppsala University, 751 23 Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 20 August 2009 Available online 28 August 2009 Keywords: Hyperglycemia Cholesterol Insulin secretion SNAREs
a b s t r a c t Diabetes is characterized by high blood glucose which eventually impairs the secretion of insulin. Glucose directly affects cholesterol biosynthesis and may in turn affect cellular structures that depend on the sterol, including lipid rafts that help organize the secretory apparatus. Here, we investigated the long-term effects of glucose upon lipid rafts and secretory granule dynamics in pancreatic b-cells. Raft fractions, identified by the presence of GM1 and flotillin, contained characteristically high levels of cholesterol and syntaxin 1A, the t-SNARE which tethers granules to the plasma membrane. Seventy-two hours exposure to 28 mM glucose resulted in 30% reduction in membrane cholesterol, with consequent redistribution of raft markers and syntaxin 1A throughout the plasma membrane. Live cell imaging indicated loss of syntaxin 1A from granule docking sites, and fewer docked granules. In conclusion, glucose-mediated inhibition of cholesterol biosynthesis perturbs lipid raft stability, resulting in a loss of syntaxin 1A from granule docking sites and inhibition of insulin secretion. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Type 2 diabetes (T2D) is characterized by failure to control glucose homeostasis, and numerous diabetic complications are attributable to exposure of tissues to high glucose. Glucotoxicity leads to a gradual decline in both b-cell function and b-cell mass [1]. Pancreatic b-cells also undergo multiple changes in gene expression in response to either acute or chronic high glucose. The functions of these genes are widespread and affect metabolism, signaling, transcription, oligonucleotide splicing, cell cycle, apoptosis, and the secretory pathway [2,3]. However, the precise mechanisms by which high glucose inhibits insulin secretion still remain poorly understood. Materials and methods Materials. Anti-3-hydroxy-methylglutaryl coenzyme A reductase antibody was obtained from Millipore (Consett, UK), anti-flotillin antibody from BD Biosciences (Franklin Lakes, NJ, USA), anti-mouse secondary antibody from Dako Cytomation Ltd. (Ely, UK), and antirabbit secondary antibody from Bio-Rad Laboratories (Hemel * Corresponding author. Address: Centre for Diabetes, Blizard Institute for Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, 4 Newark Street, Whitechapel, London E1 2AT, UK. Fax: +44 (0) 20 7882 2186. E-mail address: [email protected] (M.D. Turner). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.08.126
Hempstead, UK). Unless otherwise stated, all other reagents were obtained from Sigma–Aldrich Company Ltd. (Poole, UK). Cell culture and insulin secretion. INS-1 b-cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum, and either 11 mM or 28 mM glucose, for 72 h. Media was also supplemented for 1 h with soluble cholesterol, where indicated. Cells were then washed with Krebs–Ringer (KRH) solution (125 mM NaCl, 1.2 mM KH2PO4, 2 mM MgSO4, 1 mM CaCl2, 1.67 mM glucose, 0.1% BSA, 25 mM HEPES; pH 7.4) and incubated for 30 min in KRH ±60 mM KCl. Supernatant was collected and insulin concentration determined following ELISA kit protocols (Mercodia; Uppsala, Sweden). Cellular insulin content. INS-1 cells were grown in RPMI-1640 media supplemented with glucose, in parallel to those used for secretion experiments. Media was removed and 500 ll of extraction solution containing 1.5% hydrochloric acid, 18.5% distilled water, 80% ethanol added. After 24 h at 4 °C, 500 ll 0.1 M sodium hydroxide was added to neutralize the samples, then insulin content assayed using standard ELISA protocols (Mercodia; Uppsala, Sweden). Cholesterol quantitation. Islets were isolated from Wistar rats following previously published protocols [4]. Islets were lysed with 200 ll of RIPA buffer. INS-1 membrane and cytosol fractions were isolated from cell lysates as detailed previously [5]. Two hundred microliters of chloroform–1% Triton X-100 was added to each sample, spun for 15 min at 14,000 rpm, and the lower organic phase collected. Samples were air dried at 50 °C for 30 min, then vacuum
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dried for a further 45 min. The resulting pellet was dissolved in 150 ll of cholesterol reaction buffer. Fifty microliters of reaction mix (46 ll reaction buffer, 2 ll cholesterol probe, 2 ll enzyme) was added to 50 ll sample and incubated at 37 °C for 60 min. OD was measured at 570 nm. Discontinuous density fractionation. INS-1 cells were grown in RPMI-1640, plus 11.7 mM or 28 mM glucose, for 72 h, then resuspended in lysis buffer (1% Triton X-100, 25 mM Tris–HCl, 1 mM EDTA, 150 mM NaCl, 1 mM NaVO4, 1 mM NaF) plus Complete protease inhibitor (Roche Diagnostics, Basel, Switzerland). Cells were homogenized by passing 8 through a 25 gauge syringe needle, then samples centrifuged at 500g for 10 min at 4 °C to remove nuclei and cell debris. A three-step discontinuous OptiPrep gradient was prepared utilizing 40%, 30%, and 0% OptiPrep. Gradients were centrifuged at 17,000g for 2 h at 4 °C in a Beckman XL-70 ultracentrifuge, using a swinging 55-Ti rotor. Eight fractions were generated per gradient. An aliquot was kept for Western blotting while the rest were freeze dried and cholesterol assay performed as per protocol. Western and dot blotting. INS-1 cell homogenates (after normalizing samples for cellular protein content), or gradient fractions, were boiled in Laemmli sample buffer and separated on 10% SDS–PAGE gels. Protein was transferred onto PVDF membrane using a Hoefer TE 70 semi-dry transfer unit (Amersham Pharmacia, Little Chalfont, UK). In the case of GM1, fractions were dotted directly onto PVDF membrane and allowed to dry. Protein was detected using anti-3-hydroxy-methylglutaryl coenzyme A reductase, anti-flotillin and anti-syntaxin 1 primary antibodies, and either anti-mouse or anti-rabbit HRP-conjugated secondary
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antibody, or cholera toxin B subunit peroxidase conjugate. Antibody binding was detected with ECL Plus (Amersham Pharmacia; Little Chalfont, UK) and visualized with Hyperfilm ECL (Amersham Pharmacia; Little Chalfont, UK). TIRF. INS-1 cells were cultured in 11 or 28 mM glucose for 72 h. On day two, cells were plated onto polylysine coated coverslips and transfected with 1 lg syntaxin 1A-EGFP and 0.3 lg NPY-cherry using Lipofectamine 2000 (Invitrogen; Paisley, UK). On day 3, cells were imaged in a custom-built lens-type total internal reflection (TIRF) microscope with a 100/1.45 objective (Carl Zeiss MicroImaging; Thornwood, NY, USA). Excitation was at 473 and 555 nm (CrystaLaser; Reno, NV, USA). The emission light was separated onto two halves of an EMCCD camera (Roper Cascade 512B) using an image splitter (Photometrics; Tucson, AZ, USA) and emission filters (ET520/40 and ET630/75; both from Chroma Technology; Rockingham, VT, USA). Images shown are averages of 1 s movies acquired at 50 Hz and 20 ms exposure at 100 nm/pixel. Average fluorescence was read automatically by a macro written as MetaMorph journal in (1) a circle of 0.7 lm diameter and (2) in a surrounding annulus with an outer diameter of 1.1 lm. The circle represents fluorescence at the site of the granule, while the annulus represents local background. The annulus value was subtracted from that of the circle to yield the excess syx-EGFP fluorescence beneath a granule. Granules were counted automatically with an in-house algorithm written as MetaMorph journal. Briefly, the image was subjected to morphological tophat, H-Dome, and dilate operations, before internal thresholding and counting of objects. A threshold was set by the operator once for the entire data set.
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Fig. 1. Effect of high glucose on b-cell cholesterol content. (A) INS-1 cells were cultured for 72 h in RPMI-1640 media containing either 11 mM or 28 mM glucose. Cell homogenates were boiled in SDS dissociation buffer, then separated on 10% SDS gel, immunoblotted with anti-3-hydroxy-methylglutaryl coenzyme A reductase antibody and detected using ECL chemiluminescence. (B) Rat islets were isolated by collagenase digestion and incubated for 72 h in either 11 mM or 28 mM glucose. Cholesterol concentration was determined following extraction with chloroform/Triton X-100. OD was measured at 570 nm to quantify cholesterol concentration. (C) INS-1 membrane and cytosol fractions were isolated by differential centrifugation. Cholesterol concentration was determined following extraction with chloroform/Triton X-100. OD was measured at 570 nm to quantify cholesterol concentration.
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Statistical analysis. Bar graphs represent mean ± SEM (n = 5 or more). Comparisons of parameters between the different media conditions were by one-way ANOVA, followed by Dunnett t posthoc test (2-way).
of INS-1 b-cells in 28 mM glucose, we observed a significant reduction in 3-hydroxy-methylglutaryl coenzyme A reductase (Fig. 1A). Densitometry analysis of films from 6 independent experiments showed that there was 69.0 ± 8.2% reduction (p < 0.01) in 3-hydroxy-methylglutaryl coenzyme A reductase protein. In order to determine the effect of high glucose upon islet cholesterol content, we isolated rat islets and exposed them to either 11 or 28 mM glucose for 72 h. Exposure to 28 mM glucose reduced total islet cholesterol content by 23.2 ± 3.8% (Fig. 1B). However islets consist of multiple cell types, and it is also not clear to what extent glucose would affect metabolic cholesterol in the cytosol, or membraneassociated cholesterol. This is particularly important, given that high serum cholesterol can inhibit insulin secretion through downregulation of b-cell metabolism [19]. Following 72 h exposure of INS-1 b-cells to 28 mM glucose we observed a 30.5 ± 5.9% reduction in membrane cholesterol, and a 25.7 ± 6.3% inhibition in cytosolic cholesterol (Fig. 1C). In order to investigate the effect of glucose upon b-cell exocytosis, without the consequences of altered glucose metabolism, we cultured INS-1 cells in either 11 mM or 28 mM glucose for 72 h, then depolarized cells with 60 mM KCl in the presence of 1.67 mM glucose. We observed 72.7 ± 6.7% inhibition of KCl-stimulated insulin secretion from cells which had been pre-incubated in 28 mM glucose (Fig. 2A). Interestingly, when cells were incubated in media containing 28 mM glucose which was also supplemented with soluble cholesterol for 1 h immediately prior to stimulation (Fig. 2A), we observed a concentration-dependent reduction in glucose-mediated inhibition of stimulated insulin secretion. In particular, we observed a reduction to only 27.9 ± 2.5% inhibition from cells incubated with 5 mM cholesterol. Crucially, cells are extremely sensitive to the extracellular choles-
Results and discussion Lipid rafts are specialized membrane nanodomains enriched with glycosphingolipids and cholesterol. The resultant tight packing of these acyl lipid chains is thought to slow lateral protein diffusion rates [6–8]. Interestingly, cholesterol depletion inhibits a number of fundamental intracellular transport steps, including both endocytosis [9,10] and exocytosis [11,12]. Exocytosis is the final step in the secretory pathway, and is mediated by soluble N ethyl maleimide sensitive fusion protein attachment receptor (SNARE) complex proteins. SNAREs form helical structures that together form helical bundles between granules/vesicles (v-SNAREs) and target membranes (t-SNAREs) to facilitate membrane fusion [13,14]. In addition to having a highly enriched t-SNARE content, lipid rafts also serve to compartmentalize the predominant voltage-gated Ca2+ and K+ channels that mediate stimulus-secretion coupling in b-cells [15]. By concentrating these molecules into discreet areas of the plasma membrane where insulin-containing secretory granules are docked [16,17], the exocytotic machinery becomes effectively clustered into multiprotein exitosome complexes [18]. It has previously been shown that 24 h exposure of MIN6 b-cells to high glucose resulted in a 3.5-fold decrease in expression of 3hydroxy-methylglutaryl coenzyme A reductase [2], a key enzyme in cholesterol biosynthesis. Similarly, following 72 h incubation
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Fig. 2. Effect of high glucose on insulin secretion, insulin content and cell viability. (A) INS-1 cells were incubated for 72 h in media supplemented either 11 mM or 28 mM glucose. Media was also supplemented for 1 h with soluble cholesterol where indicated. Cells were then washed three times, and incubated for 30 min in Krebs–Ringer solution ±60 mM KCl. Supernatant was collected and insulin concentration determined using rat insulin ELISA kit (Mercodia, Uppsala, Sweden). Cellular protein content of lysed cells was quantified, and used to normalize secretion data. (B) INS-1 cells were cultured for 72 h in RPMI-1640 media containing either 11 or 28 mM glucose. Media was removed from the wells, and intracellular insulin extracted using hydrochloric acid/ethanol. Insulin content was assayed using standard ELISA protocols. Total protein content was quantified to normalize insulin values obtained. (C) INS-1 cells were incubated in either 11 or 28 mM glucose. Cell viability was determined based on Trypan blue exclusion from nuclei of intact cells.
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terol concentration, as exposure to 12.5 mM cholesterol resulted in 99.0 ± 2.0% inhibition of simulated secretion. This latter observation is also consistent with the recent report of inhibition of insulin secretion from islets exposed to 10 mM extracellular cholesterol [19]. One of the genes sensitive to extracellular glucose concentration is that encoding insulin itself [20,21], and glucotoxicity has previously been shown to partially deplete b-cell insulin content [22]. We also found that INS-1 cells cultured for 72 h in 28 mM glucose had a 35.4 ± 1.2% reduction in cellular insulin content relative to control (Fig. 2B). This was not a result of glucotoxic cell death, as we observed no significant effect on cell viability (Fig. 2C). Although insulin content was partially depleted by high glucose, this alone cannot explain the much larger inhibition in insulin secretion. Moreover, recent work has also shown that high glucose does not deplete b-cells of exocytosis-competent insulin-containing secretory granules [22]. Our observations are therefore indicative of an effect of glucose upon a late exocytotic step. In order to test this hypothesis we investigated whether glucose could affect secretion by altering the distribution of either syntaxin 1A, which is highly enriched within lipid nanodomains [11,15], or the widely utilized lipid raft markers, flotillin and GM1. As lipid raft fractions are resistant to solubilization by the detergent Triton X-100, we exploited this characteristic, along with their buoyancy on sucrose gradients, to purify raft fractions. At 11 mM glucose, cholesterol, flotillin, and GM1 concentrations were highest in fractions 2–3 from discontinuous density fractionation (Fig. 3), indicative of their presence in lipid rafts. Although cells incubated in 28 mM glucose showed a reduction in cholesterol content in all sub-cellular
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fractions (Fig. 3A), there was a marked redistribution of the raft markers, flotillin and GM1 (Fig. 3B and C), and syntaxin 1A (Fig. 3D) to non-raft fractions in cells cultured in 28 mM glucose. Loss of syntaxin 1A from lipid rafts may affect its association with insulin granule release sites. In order to study the plasma membrane distribution of syntaxin 1A with greater resolution, we expressed EGFP-tagged syntaxin 1A (syx-EGFP) in INS-1 cells, together with a granule marker, NPY-cherry, and imaged living cells in total internal reflection mode (TIRF). Cells cultured in 11 mM glucose exhibited clusters of syx-EGFP at the plasma membrane (Fig. 4A), similar to those observed previously with immuno-staining techniques [23,24]. A subset of these clusters also colocalized with secretory granules. However, when cells were cultured in 28 mM glucose, syx-EGFP was more diffusely localized throughout the membrane (Fig. 4C). Furthermore, the intensity of these clusters was clearly diminished (Fig. 4B vs. D). For each cell we quantified these clusters by measuring the excess syx-EGFP at randomly chosen granules (see Methods), and normalized to the cell’s overall brightness as a measure of expression level. On average, in cells cultured at 11 mM glucose, the plasma membrane beneath granules contained 12.5 ± 2.0% more syx-EGFP, compared with the surrounding area (933 granules in 44 cells) (Fig. 4E). Cells that had been cultured in 28 mM glucose were studied in parallel, and the corresponding value was less than half (5.7 ± 1.5%; 696 granules in 36 cells, p < 0.01 vs. 11 mM), consistent with reduced binding of syntaxin 1A to granule-associated rafts. This average loss of syntaxin 1A from granule sites also corresponded to fewer granules colocalizing with a syntaxin cluster (30 ± 4% and 44 ± 3% for cells cultured at 28 and 11 mM glucose, respectively; 36 and 44 cells, p < 0.001) (Fig. 4F). In keeping with a
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Fig. 3. Effect of high glucose upon lipid raft integrity. INS-1 cells were incubated for 72 h in either 11 mM or 28 mM glucose, then resuspended in lysis buffer (1% Triton X100, 25 mM Tris–HCl, 1 mM EDTA, 150 mM NaCl, 1 mM NaVO4, 1 mM NaF) and homogenized by passing 8 through 25 gauge syringe needle. Eight fractions were generated per gradient, following centrifugation down a three-step discontinuous OptiPrep gradient. (A) Cholesterol concentration was quantified for each fraction generated, following extraction with chloroform/Triton X-100. OD was measured at 570 nm to quantify cholesterol concentration. (B) Fractions were separated on 10% SDS gel, then immunoblotted with anti-flotillin antibody and detected using ECL chemiluminescence. (C) Fractions were dotted directly onto PVDF membrane, then immunoblotted with anti-cholera toxin B subunit peroxidase conjugate and detected using ECL chemiluminescence. (D) Fractions were separated on 10% SDS gel, then immunoblotted with antisyntaxin 1A antibody and detected using ECL chemiluminescence.
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Fig. 4. Accumulation of syx-EGFP at granules. (A) TIRF images of a cell cultured in 11 mM glucose and expressing both syx-EGFP and NPY-cherry. The inset shows an enlarged area of the same cell. (B) Average image constructed from small syx-EGFP images centered at granule sites. All identified granules locations from one coverslip each are included in the average. (C) As in A, but cultured in 28 mM glucose. (D) As in B, but cultured in 28 mM glucose. (E) Colocalization of syx-EGFP with granules was measured following location of granules by eye. The circle represents the fluorescence at the site of the granule while the annulus represents the local background onto which the cluster is superimposed. The annulus value was subtracted from that of the circle to yield the excess syx-EGFP fluorescence beneath a granule. Bargraph shows mean and standard error of the excess syx-EGFP at granules in cells cultured at 11 and 28 mM glucose. (F) Granules were counted automatically with an in-house algorithm written as MetaMorph journal. The image was subjected to morphological tophat, H-Dome, and dilate operations, before internal thresholding and counting of objects. A threshold was set by the operator once for the entire data set. Bargraph shows mean and standard error of granule numbers associated with syntaxin clusters. (G) Bargraph shows mean and standard error of granules per unit area of plasma membrane. *p < 0.05; **p < 0.01; ***p < 0.001.
role of syntaxin 1A in docking, these cells tended to have fewer granules near the plasma membrane (0.54 ± 0.05 vs. 0.67 ± 0.04 granules/lm2 (p < 0.05) at 28 and 11 mM glucose) (Fig. 4G), in agreement with previous reports [24]. Interestingly, it has recently been observed that cholesterol-dependent SNAP-25 clustering is also essential for insulin secretion [25]. However, to our knowledge, our data provides the first evidence of a general requirement for lipid raft-dependent t-SNARE clustering that is sensitive to extracellular glucose environment. In conclusion, our data shows high glucose reduces membrane cholesterol content, with the resulting perturbation of plasma membrane lipid raft nanodomains leading to a reduction in syntaxin 1A-granule interactions. Importantly, interactions that do still take place are likely to occur at areas of the plasma membrane where there is an absence of clustering together of all key exitosome complex members. Under these circumstances the fidelity of the exocytotic machinery would be compromised, leading to the failure of b-cells to secrete adequate amounts of insulin in response to subsequent secretory stimuli. We suggest that the resulting inhibition in insulin secretion may lead to the breakdown in glucose homeostasis which results in the development of T2D.
Acknowledgments We are grateful to Shanta Persaud and Peter Jones (Diabetes Research Group, King’s College London) for provision of isolated rat islets. This research was supported by grant awards from Barts and The London Charitable Foundation (RAC 408 and RAB/06/ PHD/12). The authors declare that they have no conflict of interest. References [1] B.L. Wajchenberg, D-cell failure in diabetes and preservation by clinical treatment, Endocrine Rev. 28 (2007) 187–218. [2] G.C. Webb, M.S. Akbar, C. Zhao, D.F. Steiner, Expression profiling of pancreatic b-cells: glucose regulation of secretory and metabolic pathway genes, Proc. Natl. Acad. Sci. USA 97 (2000) 5773–5778. [3] F. Schuit, D. Flamez, A. De Vos, D. Pipeleers, Glucose-regulated gene expression maintaining the glucose-responsive state of b-cells, Diabetes 51 (2002) S326– S332. [4] P.M. Jones, F.M. Mann, S.J. Persaud, C.P.D. Wheeler-Jones, Mastoparan stimulates insulin secretion from pancreatic beta-cells at a late stage in the secretory pathway, Mol. Cell Endocrinol. 94 (1993) 97–104. [5] C. Marshall, G.A. Hitman, C.J. Partridge, A. Clark, H. Ma, T.R. Shearer, M.D. Turner, Evidence that an isoform of calpain-10 is a regulator of exocytosis in pancreatic b-cells, Mol. Endocrinol. 19 (2005) 213–224.
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