Pancreatic -cell prosurvival effects of the incretin hormones involve post-translational modification of Kv2.

1 delayed rectifier channels S-J Kim,1,3 S B Widenmaier,1,3 W S Choi,1,4 C Nian,1 Z Ao,2 G Warnock,2 and C H S McIntosh1,* Author information Article notes Copyright and License information This article has been cited by other articles in PMC. Go to: Abstract Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the major incretin hormones that exert insulinotropic and anti-apoptotic actions on pancreatic -cells. Insulinotropic actions of the incretins involve modulation of voltage-gated potassium (Kv) channels. In multiple cell types, Kv channel activity has been implicated in cell volume changes accompanying initiation of the apoptotic program. Focusing on Kv2.1, we examined whether regulation of Kv channels in -cells contributes to the prosurvival effects of incretins. Overexpression of Kv2.1 in INS-1 -cells potentiated apoptosis in response to mitochondrial and ER stress and, conversely, co-stimulation with GIP/GLP-1 uncoupled this potentiation, suppressing apoptosis. In parallel, incretins promoted phosphorylation and acetylation of Kv2.1 via pathways involving protein kinase A (PKA)/mitogen- and stress-activated kinase-1 (MSK-1) and histone acetyltransferase (HAT)/histone deacetylase (HDAC). Further studies demonstrated that acetylation of Kv2.1 was mediated by incretin actions on nuclear/cytoplasmic shuttling of CREB binding protein (CBP) and its interaction with Kv2.1. Regulation of -cell survival by GIP and GLP-1 therefore involves post-translational modifications (PTMs) of Kv channels by PKA/MSK-1 and HAT/HDAC. This appears to be the first demonstration of modulation of delayed rectifier Kv channels contributing to the -cell prosurvival effects of incretins and of 7transmembrane G protein-coupled receptor (GPCR)-stimulated export of a nuclear lysine acetyltransferase that regulates cell surface ion channel function. Keywords: incretins, diabetes, GIP, GLP-1, -cell apoptosis, Kv channel Type 1 and Type 2 diabetes are associated with absolute or relative deficits of functional cells, and there is growing evidence that apoptosis is the main mediator of -cell death. In

view of the worldwide increase in diabetes incidence, it is important to understand the molecular mechanisms involved in -cell apoptosis and to identify the agents that can reduce or abrogate this process. The gastrointestinal tract secretes over 20 peptide hormones that regulate multiple physiological processes. Among these, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the major incretin hormones that potentiate glucosestimulated insulin secretion (GSIS) during a meal,1, 2 and exert beneficial effects on -cell proliferation and survival.2, 3, 4, 5, 6 GSIS involves closure of ATP-sensitive K+ (KATP) channels resulting in membrane depolarization, activation of voltage-dependent Ca2+ channels and increased intracellular Ca2+, followed by membrane repolarization by voltage-gated K+ (Kv) and Ca2+-sensitive K+ (KCa) channels. Incretins stimulate insulin secretion through modulation of KATP channel activity, increasing Ca2+ influx through L-type Ca2+ channels and non-selective ion channels, and releasing Ca2+ from intracellular stores, as well as potentiation of Ca2+-dependent exocytosis.2, 3, 7 These effects are mediated through activation of -cell G protein-coupled receptors (GPCRs).2, 3 Both GIP and GLP-1 stimulate adenylyl cyclase, leading to increased intracellular cyclic AMP (cAMP), and activate protein kinase A (PKA) and exchange protein directly activated by cAMP 2 (EPAC2).2, 3 Incretins activate both transcription-dependent and -independent anti-apoptotic pathways in -cells 2, 3,4, 5, 6 and significant progress has been made in elucidating the underlying mechanisms. Staurosporine (STS)-induced mitochondrial translocation of Bad and BimEL, activation of mitochondrial Bax, release of cytochrome c and caspase-3 activation were all reduced by GIP treatment of -cells.6 Incretins additionally reduce the levels of ER stressrelated factors.1, 3 The cAMP/PKA pathway has a central role in -cell prosurvival effects of GIP and GLP-1,1, 3, 5 including stimulation of CREB-mediated expression of genes such as bcl-2.5 Anti-apoptotic effects of PKB are also activated by the incretins, with suppression of Bax expression4 and phosphorylation of apoptosis signal regulating kinase 1 (ASK1), resulting in sustained suppression of p38 MAPK and Jun N-terminal kinase.6 Intriguingly, in neurons, an apoptotic surge of K+ current involves ASK1 and p38 MAPK.8, 9 Kv channels are involved in the repolarization of excitable cells, and electrophysiological studies in human and rodent pancreatic -cells have demonstrated their importance in the secretory process.10, 11Members of the Kv1, Kv2 and Kv3 families are thought to have important roles in insulin secretion by modulating the amplitude and duration of action

potentials.10, 12 Although Kv1.4 channel conductance is an important component of transient outward current (Ito), other subtypes of Kv channels contribute to delayed rectifier currents (IDR) in -cells,10, 12, 13 with Kv2.1 probably having the major role.10, 12,13 In addition to regulating cell excitability, K+ channels are involved in the initiation and progression of apoptosis in several different cell types.14, 15 Increases in both conductance and surface expression of Kv channels contribute to neuronal apoptosis. Neuroprotective effects of expressing dominant-negative Kv2.1 in cortical neurons were associated with reduced K+ current density following exposure to apoptotic stimuli, and trafficking of Kv2.1 channels to the plasma membrane was a major contributor to the initiation of apoptosis.16 In the current studies, prosurvival effects of GIP and GLP-1 were found to involve post-translational modifications (PTMs) of Kv2.1 by phosphorylation and acetylation, the latter process occurring via a novel pathway involving nuclear to cytoplasmic translocation of the lysine acetyl transferase, CREB binding protein (CBP). Go to: Results Kv channels are involved in STS- or Thapsigargin-induced -cell apoptosis and GIP/GLP-1 reduces Kv2.1-mediated apoptotic cell death The potential involvement of K+ channels in -cell apoptosis was investigated by examining the attenuation of channel conductance through treatment with the K+ channel blocker tetraethylammonium (TEA) and quantifying apoptotic cell death in response to STS, an activator of mitochondria-mediated apoptosis, or Thapsigargin (Thap), an inducer of endoplasmic reticulum (ER) stress. As shown in Figures 1a and b, TEA reduced the levels of STS- and Thap-induced apoptosis in -INS-1 (832/13) cells in a concentration-dependent manner. Additionally, TEA decreased STS-induced -INS-1 cell death (Supplementary Figure 1).

Figure 1

K+ channels contribute to STS- and Thap-induced apoptosis in INS-1 -cells (a and b). Effect of TEA on STS or Thap-induced INS-1 -cell apoptosis. -INS-1 (832/13) cells were treated with STS (100 nM, a) or Thap ... In order to examine their potential involvement in -cell apoptosis, Kv2.1, Kv1.5 and Kv3.2, channel proteins that are strongly expressed in -cells10 (S Kim et al., unpublished), were transiently overexpressed in -INS-1 cells. As shown in Supplementary Figure 2, Thapinduced -cell death was significantly increased by overexpression of Kv2.1 and Kv1.5 but not by Kv3.2. In view of the more robust induction of -cell death by Kv2.1, and evidence supporting the contribution of Kv2 channel family members to 60% of -cell IDR,12 we focused further on the apoptotic effects of Kv2.1 in -cells. As Kv2.1 expression is significantly lower in INS-1 (832/13) -cells than in primary -cells, for subsequent overexpression studies transfection conditions were optimized to mimic the Kv2.1 protein levels in human islets (Supplementary Figure 3A). This resulted in a marked potentiation of both STS- and Thap-induced -cell apoptosis (Figures 1c and d) and -cell death (Figures 1e and f). We next examined the effects of GIP and GLP-1 on Kv2.1-mediated -cell apoptosis. Incubation of cells with Thap resulted in 4.5-fold increase in apoptosis and this was reduced by 44.8% and 51.6% with GIP or GLP-1 treatment, respectively (Figure 2a). Similar results were obtained for -cell death (Figure 2b). Despite the elevated levels of apoptosis in Kv2.1overexpressing -INS-1 cells, GIP and GLP-1 were both capable of reducing apoptotic cell death to levels below those observed with pcDNA-transfected cells treated with Thap, indicating that part of their effect was mediated via effects on Kv channel surface protein levels or conductance. To establish the functional involvement of endogenously expressed Kv2.1 in apoptosis, RNA interference was employed. As shown in Figure

2c andSupplementary Figure 3B, RNAi-mediated knockdown resulted in specific reductions in Kv2.1 expression, associated with greatly reduced Thap-induced -cell apoptosis. Both GIP and GLP-1 tended to further decrease the Thap-induced -cell apoptosis, although the reduction did not reach statistical significance. Taken together, these results strongly support a role for Kv2.1 in Thap-induced apoptotic cell death and its involvement in GIP/GLP-1mediated protection.

Figure 2 GIP and GLP-1 reduce apoptosis potentiated by Kv2.1 overexpression. (a) Effects of GIP and GLP-1 on Thap-induced INS-1 -cell apoptosis. INS-1 (832/13) -cells were transfected as described above and treated with Thap (1 ... GIP and GLP-1 increase phosphorylation and acetylation of Kv2.1 in pancreatic -cells In earlier studies, both GIP11 and GLP-110 were found to increase Kv channel phosphorylation. Recently, reversible protein lysine acetylation has also been demonstrated to be a common PTM of non-nuclear proteins.17 As GIP and GLP-1 were both shown to be capable of stimulating histone acetylation,18 the effects of these incretins on levels of phosphorylated and acetylated Kv2.1 were examined. Transfected INS-1 -cells were treated with GIP or GLP-1 and solubilized extracts immunoprecipitated with phosphoserine/threonine or acetyl-lysine antibodies, followed by immunoblotting with Kv2.1 antibody. GIP (142, 100 nM) and GLP-1 (736, 100 nM) increased phosphorylation (Figures 3a and b) and acetylation (Figures 3c and d) of Kv2.1, whereas truncated forms of the peptides, GIP (1930) and GLP-1 (936), that do not exhibit insulinotropic activity, had no effect. These results indicate that the incretin hormones are capable of increasing both phosphorylation and acetylation of Kv2.1 protein in pancreatic -cells.

Figure 3

GIP and GLP-1 increase phosphorylation and acetylation of Kv2.1. (ad) Effect of GIP/GLP1 on phosphorylation and acetylation of Kv2.1. INS-1 -cells were transfected with Kv2.1 and incubated with 100 nM of indicated peptides, ... PKA/mitogen- and stress-activated kinase-1 (MSK-1) and histone acetyltransferase (HAT)/histone deacetylase (HDAC) enzymes are involved in incretin-mediated PTM of Kv2.1 and promotion of -cell survival Signaling modules potentially involved in promoting PTM of Kv2.1 channels were next examined, initially focusing on pathways previously demonstrated to mediate incretin actions.2, 3 GIP and GLP-1-stimulated Kv2.1 phosphorylation was greatly reduced or ablated by H-89 (10 M), an inhibitor of both PKA and MSK-1, as well as the specific competitive inhibitor of cAMP binding to the regulatory subunit of PKA, Rp-cAMPs (200 M) (Figure 4a). Acetylation of Kv2.1 in response to GIP and GLP-1 was also greatly reduced by H-89 and Rp-cAMPs, as well as by an inhibitor of HAT (HAT inhibitor II (HATII); 40 M). Conversely, the HDAC inhibitor, Trichostatin A (TSA; 300 nM), mimicked the effects of GIP and GLP-1 on the acetylation of Kv2.1 (Figure 4b), as well as potentiated the level of incretin-induced Kv2.1 acetylation. As expected, neither the HAT nor the HDAC inhibitor affected Kv channel phosphorylation (Figure 4a). To determine whether phosphorylation and acetylation of Kv2.1 contribute to GIP and GLP-1-mediated -cell survival, Thap-induced cell apoptosis was determined in the absence or presence of PKA/MSK-1, PKA and HAT/HDAC inhibitors. As shown in Figure 4c, incretin-mediated -cell survival was greatly reduced by H-89, Rp-cAMPs and HAT inhibitor II. In contrast, the HDAC inhibitor potentiated the effects of GIP and GLP-1 on -cell survival. Together, these results demonstrate that pathways involving PKA/MSK-1 and HAT/HDAC are involved in incretinmediated PTMs of Kv2.1 associated with -cell survival.

Figure 4 PKA/MSK-1 and HAT/HDAC are involved in incretin-mediated PTMs of Kv 2.1 and -cell survival. (a and b) Effect of inhibiting PKA/MSK-1 or HAT/HDAC on PTMs of Kv 2.1. INS-1 -cells were transfected with Kv2.1 and stimulated with 100 nM ... GIP and GLP-1 regulate the nuclear/cytoplasmic localization of CBP In view of the recent demonstration that cytoplasmic CBP was capable of acetylating the prolactin receptor19 and the relative selectivity of HAT inhibitor II for CBP/p300, we examined the effects of GIP and GLP-1 on the cellular distribution of CBP. As shown in Figures 5a, b and e, GIP or GLP-1 treatment of INS-1 -cells decreased nuclear, and increased cytoplasmic, localization of CBP. These responses were apparent 15 min after initiation of GIP or GLP-1 treatment and thereafter sustained to 120 min, returning towards basal by 1200 min. Subsequent studies demonstrated a GIP/GLP-1 concentration dependence for nuclear/cytoplasmic shuttling of CBP (Figures 5c, d and f) and observed similar responses with human pancreatic islets (Figures 5g and h). Immunocytochemical staining of INS-1 cells confirmed the effects of incretins on nuclear CBP translocation detected by western blotting (Figure 5i). On the other hand, GIP and GLP-1 were without effect on the nuclear/cytoplasmic distribution of p300 (Figures 5j and k), a CREB-related adaptor protein that shares high sequence homology with CBP and exerts both common and unique transcriptional activities.20

Figure 5 GIP and GLP-1 export nuclear CBP to cytoplasm. (ad) Time course (a and b) and concentrationresponse effect (c and d) of GIP/GLP-1 on nuclear (a and c) and cytoplasmic (b and d) CBP. INS-1 -cells were treated for the indicated ... CBP mediates GIP and GLP-1-induced acetylation of Kv2.1, resulting in channel internalization

As GIP and GLP-1 increased the cytoplasmic localization of CBP, its role in GIP and GLP-1mediated acetylation of Kv2.1 was examined. As shown in Figure 6a, in coimmunoprecipitation experiments, proteinprotein interactions between CBP and Kv2.1 were observed in GIP or GLP-1-treated human islets. To establish the functional importance of CBP in GIP/GLP-1-mediated regulation of Kv2.1, RNA interference was employed. RNAimediated knockdown resulted in greatly reduced nuclear CBP expression in INS-1 -cells (Figure 6b). Co-immunoprecipitation experiments demonstrated that CBP knockdown almost completely ablated GIP or GLP-1-induced lysine acetylation of Kv2.1 (Figure 6c) and greatly reduced proteinprotein interactions between CBP and Kv2.1 (Figure 6d). Translocation of CBP from the nucleus is therefore likely to be an important mediator of GIP- and GLP-1induced lysine acetylation of Kv2.1.

Figure 6 CBP is responsible for GIP/GLP-1-mediated acetylation of Kv2.1. (a) Co-

immunoprecipitation (Co-IP). Human islets were treated with GIP or GLP-1 (100 nM) for 1 h. Cytoplasmic extracts were isolated from each sample and IP with CBP followed ... GIP-induced phosphorylation of Kv1.4 was previously demonstrated to result in endocytosis,11 and Kv2.1 trafficking to the plasma membrane was shown to be responsible for increases in K+ currents associated with neuronal apoptosis.16 We therefore determined whether GIP/GLP-1-mediated regulation of Kv2.1 involves channel protein internalization. Incubating cells with proteinase K randomly cleaves the extracellular regions of proteins, thus producing digested forms, whereas intracellular protein is protected. Using this technique, surface membrane expression levels of Kv2.1 in human islets were shown to decrease in response to treatment with GIP (142, 100 nM) or GLP-1 (736, 100 nM) (Figure 6e). As cellular K+ efflux has been shown to be a central step for the progression and completion of apoptotic cell death in numerous cell types, incretin-mediated Kv2.1 PTM and internalization are likely to be critical for -cell survival.

Go to: Discussion Although a role for Kv channels in the regulation of membrane repolarization and insulin secretion in pancreatic -cells is established, their involvement in -cell apoptosis has not been previously reported. It has been demonstrated in several cell types that apoptotic cell shrinkage (apoptotic volume decrease; AVD) occurs early in the process, before ultrastructural and biochemical changes, such as DNA fragmentation, cytochrome c release and caspase 3 activation,14 and is a prerequisite for completion of programmed cell death. Ionic mechanisms involved in AVD are complex, but K+ is the major cytoplasmic cation responsible for maintaining cell volume14, 21, 22 and efflux of K+ is a critical component of the AVD.21 Indeed, activities of a number of caspases and endonucleases are suppressed at normal intracellular K+ levels and one of the main early events in apoptosis results from decreases in its intracellular concentration, allowing their activation.22 A number of different K+ channels have been implicated in AVD, including Kv family members,14, 16, 21, 22 and treatment with the K+ channel blockers TEA and 4-AP inhibits apoptosis in various cell types.21 In earlier studies, GIP and GLP-1 receptor activation was demonstrated to potentiate GSIS by modulating Kv channels,11, 23, 24, 25 and both incretins exert beneficial effects on -cell survival.2, 3, 4,5, 6 The current study was therefore initiated in order to test the hypothesis that GIP's and GLP-1's actions on Kv channels are linked to effects on -cell survival. In rodent primary -cells, >80% of the outward K+ currents are generated by channels that are sensitive to the blocker TEA.24 Treatment of -INS-1 cells with TEA greatly reduced apoptosis induced by STS or Thap treatment (Figures 1a and b), suggesting the involvement of K+ channels. In addition to direct actions on K+ channels, TEA has been suggested to act on other cation channels in HeLa cells and by reducing cytochrome c release from mitochondria,26 and we cannot rule out their contribution to -cell responses. Kv2.1 has an important role in -cell repolarization.12, 23 Additionally, the cytoplasmic Cterminus of Kv2.1 binds to syntaxin 1A in -cells and their interaction is involved in channel gating, trafficking and insulin exocytosis.27, 28 Multifunctional roles have also been shown in other cell types, and Kv2.1 facilitation of secretory vesicle recruitment in neuroendocrine cells,29 and the clustering of cell surface Kv2.1 channels in hippocampal neurons30 were shown to be independent from K+ conductance. In view of its additional pro-apoptotic role in

neurons, we focused on the potential role of Kv2.1 in -cell apoptosis and the ability of GIP and GLP-1 to modulate its action. Expression of Kv2.1 protein in -INS-1 cells, at levels similar to those found in human -cells, strongly potentiated apoptotic responses to both STS and Thap (Figures 1cf, 2a and b), whereas RNAi-mediated knockdown of endogenous Kv2.1 attenuated Thap-induced apoptosis (Figure 2c). These findings supported a role for Kv2.1 in -cell apoptosis. Modulation of channel conductance has been considered to be the major effect of GLP-1 receptor activation on -cell Kv2.1.24, 25 However, the pro-apoptotic effects of Kv2.1 overexpression in -INS-1 cells were strongly reduced by both incretin hormones (Figure 2a). A number of voltage-gated K+channel proteins are regulated by serine/threonine phosphorylation,11, 25, 31 and both phosphorylation25 and SUMOylation32 of Kv2.1 modulate channel conductance in pancreatic -cells. In mass spectrometry-based assays multiple phosphorylation sites were identified in Kv2.131 and, among these, p38 MAPK-mediated phosphorylation of Kv2.1 at Ser800 is involved in Kv2.1 trafficking to the plasma membrane during neuronal apoptosis.33 An Src family tyrosine kinase was shown to constitutively activate Kv2.1,34 and Tyr124 of Kv2.1, a target for Src kinase, is critical for the neuronal apoptotic surge.35 Additionally, downregulation of Kv2.1, via the cAMP/PKA signaling pathway, reduced apoptosis in cerebellar granular neurons.36 Lysine acetylation of proteins has recently been identified as a PTM of widespread importance, linked to the modulation of cytoskeleton dynamics, endocytosis, autophagy and energy metabolism,20, 37 and >80 transcription factors and nuclear regulators, as well as various cytoplasmic proteins, have been identified as acetyltransferase targets.38 As both GIP and GLP-1 act via stimulation of protein kinase cascades1, 2, 3 and also increase histone acetylation,18 the potential involvement of both PTMs of Kv2.1 was investigated and shown to occur in INS-1 -cells (Figures 3ad). Incretin-mediated phosphorylation of Kv2.1 was greatly reduced by H-89 and Rp-cAMPs, whereas inhibitors of HAT/HDAC were without effect (Figures 4a and b). On the other hand, acetylation of Kv2.1 was greatly reduced by H89, Rp-cAMPs and HAT/HDAC inhibitors. Although Rp-cAMPs is a relatively specific inhibitor of PKA, H-89 also strongly inhibits both GIP and GLP-1 stimulation of phosphorylation of MSK-1 at Ser376, mediated by PKA, and MSK-1 enzymatic activity in INS-1 -cells.18 It is therefore plausible that both PKA and MSK-1 are involved in incretinmediated PTMs of Kv channels. As incretin-mediated -cell survival was greatly reduced by HAT inhibitor II and the HDAC inhibitor potentiated the effects of GIP/GLP-1 on -cell

survival (Figure 4c), without affecting incretin-mediated Ser/Thr phosphorylation of Kv2.1 (Figure 4a), acetylation seems to be the more critical incretin-mediated PTM of Kv2.1 associated with -cell survival. However, it is still possible that PKA-mediated phosphorylation is a prerequisite step for acetylation of Kv2.1, as incretin-mediated acetylation of Kv2.1 was greatly reduced by H-89 and Rp-cAMPs treatment (Figure 4b). Cytosolic acetyltransferase and deacetylase enzymes have not been extensively characterized. However, dimerization of the prolactin receptor was recently shown to be stimulated through acetylation by CBP/p300,19 key regulators of CREB-mediated gene transcription, and both GIP and GLP-1 stimulate expression of multiple proteins via PKA activation of CREBrelated pathways,1, 2, 3, 5 as well as by stimulating histone lysine acetylation.18 We therefore investigated CBP and/or p300 involvement in GIP/GLP-1-stimulated lysine acetylation of Kv2.1. Surprisingly, GIP and GLP-1 induced extensive export of nuclear CBP to the cytoplasm (Figures 5a and b), whereas p300 was relatively unaffected, and both incretins also increased proteinprotein interactions between CBP and Kv2.1 (Figure 6a). RNAi-mediated knockdown of CBP resulted in disruption of these interactions, as well as reduced acetylated Kv2.1 levels (Figures 6c and d), suggesting that CBP is a major acetyltransferase responsible for Kv2.1 acetylation. At present, nothing is known about the mechanisms underlying transport of acetylases or deacetylases between cytosolic and nuclear compartments. However, increased cytosolic levels of CBP were detected within 15 min of stimulation and levels were increased for up to 2 h. Changes in the relative balance between acetylation and deacetylation have previously been shown to significantly impact on pancreatic -cell death: inhibition of HDACs preventing cytokine-induced -cell apoptosis and impairing -cell function.39, 40 As cellular K+ efflux is a critical step in the apoptotic cell death program, incretin-mediated PTMs of Kv2.1 and associated internalization (Figure 6e) are therefore likely to be important for -cell survival. In summary, we have shown that GIP and GLP-1 modulate both phosphorylation and acetylation of Kv2.1, resulting in reduced surface expression, and that PKA/MSK-1 and HAT/HDAC are involved in this process. The resultant decrease in K+ efflux is likely an important factor in incretin-mediated prosurvival effects. However, both incretins also modulate the activities of intracellular mediators of apoptosis, including Ask1, Bax and Bad, as well as cytochrome c release. As trafficking of CBP to the plasma membrane appears to be a major contributor to acetylation of Kv2.1, developing a better understanding of its regulation and the implications for regulation of gene expression is clearly important. This

also appears to be the first example of GPCR-stimulated export of a nuclear lysine acetylase that regulates cell surface ion channel function. Altogether, our findings reveal an intriguing scenario whereby the release of nutrient-sensitive gut hormones couples to both functional and survival potentiating mechanisms in the -cell via a signaling axis converging on voltage-sensitive ion channels that function to restore homeostasis following cellular excitation. Go to: Materials and Methods -INS-1 cell culture and transient transfections -INS-1 cells (clone 832/13) were kindly provided by Dr. CB Newgard (Duke University, Durham, NC, USA). Cells were cultured in 11 mM glucose RPMI 1640 (Sigma-Aldrich, Oakville, ON, Canada) supplemented with 2 mM glutamine, 50 M -mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 10% fetal bovine serum, 100 units/ml penicillin Gsodium and 100 g/ml streptomycin sulfate. Cell passages 4575 were used. Transient transfections were performed with Kv1.5, Kv2.1 or Kv3.2 or pcDNA3 plasmids using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Islet isolation and primary cell culture Human islets were isolated from the pancreas of five adult organ donors using collagenase duct perfusion, dissociation and density gradient purification at the Ike Barber Islet Transplantation Laboratory (VGH, Vancouver, BC, Canada). The Research Ethics Board of UBC provided ethics approval. Cell death assays and apoptosis -INS-1 cells were treated with STS (100 nM) or Thap (1 M) for 6 h and cell death determined by counting propidium iodide-positive nuclei and total cell number measured by counting Hoechst 33342-positive nuclei.6 Fluorescent signals were quantified with highthroughput imaging systems, CellomicsArrayscan V automated imager or Cellomics KineticScan (Cellomics Inc., Pittsburgh, PA, USA), and percent of cell death calculated. Detection of apoptotic cells was performed with the APOPercentage apoptosis assay kit (Biocolor Ltd, Belfast, Northern Ireland), based on specific uptake of dye by an apoptotic

flip-flop' mechanism. APOPercentage dye was added to cells 30 min before completion of the apoptotic inducer/incretin incubation. Quantitative analysis was performed using a colorimetric assay following release of dye from the apoptotic cells (representative images for APOPercentage dye labelled cells can be found in Supplementary Figure 4). Cell fractionations Nuclear/cytoplasmic extracts were prepared from cells as described previously.4, 5, 18 Briefly, cells were washed with PBS, and scraped with 200 l ice-cold buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P40 and protease inhibitors). Following centrifugation, supernatants (cytoplasmic extracts) were collected and resulting pellets re-suspended in 20 l buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 20% glycerol and protease inhibitors) and incubated on ice for 10 min. After clarification by centrifugation, supernatants (nuclear extracts) were collected and subjected to western blot analysis. Histone and -actin were used as nuclear and non-nuclear markers, respectively, to establish lack of cross-contamination between fractions (Supplementary Figure 5). Western blot analysis Protein samples were separated on a 15% sodium dodecyl sulfate (SDS)/polyacrylamide gel and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Mississauga, ON, Canada). Probing of the membranes was performed with Kv2.1, phospho-Ser/Thr, acetylLys, CBP, p300, histone or -actin antibodies (Cell Signaling Technology, Beverly, MA, USA; Santa Cruz Biotechnology, Santa Cruz, CA, USA; Sigma-Aldrich; Novus Biologicals, Littleton, CO, USA). Immunoreactive bands were visualized by enhanced

chemiluminescence (Millipore, Billerica, MA, USA) using horseradish peroxidaseconjugated IgG secondary antibodies. Co-immunoprecipitation (Co-IP) For the data presented in Figure 2, total cellular extracts were prepared following experimental treatments and immunoprecipitated with phospho-serine/threonine, acetyllysine or Kv2.1 antibodies using Dynabead protein A (Invitrogen). Precipitated products were resolved by SDS-PAGE and probed with antibodies against Kv2.1, phospho-serine/threonine or acetyl-lysine.

Confocal microscopy -INS-1 cells were treated with GIP or GLP-1 (100 nM) for 1 h. Immunocytochemical staining was performed using antibodies against CBP, and visualized with Texas Red dyeconjugated anti-rabbit secondary antibody (Molecular Probes, Invitrogen Canada, Burlington, ON, Canada). Cell nuclei were counterstained with DAPI (4,6 -diamino-2-phenylindole). Stained cells were imaged using a Nikon confocal microscope (Nikon Canada, Mississauga, ON, Canada). All imaging data were analyzed using EZC1 software (Nikon). RNA interference knockdown of CBP and Kv2.1 -INS-1 cells were transfected with MISSION siRNA for CBP (SASI_Rn01_00079791, Sigma-Aldrich) or Kv2.1 (SI01527631, Qiagen Canada, Toronto, ON, Canada) and incubated for 72 h. The level of reduction in CBP and Kv2.1 protein expression was determined by western blot hybridization using antibodies against CBP, Kv2.1, histone and -actin. Proteinase K digestion experiments For proteinase K digestion,11 human islets incubated with 100 nM of each peptide were washed three times with ice-cold PBS and incubated with 10 mM HEPES, 150 mM NaCl and 2 mM CaCl2 (pH 7.4) with 200 g/ml proteinase K at 37 C for 30 min. Cells were harvested and proteinase K digestion quenched with ice-cold PBS containing 6 mM phenylmethylsulfonyl fluoride and 25 mM EDTA. This was followed by SDS-PAGE and immunoblotting, and probing of membranes with antibodies against Kv channels and tubulin. Statistical analysis Data are expressed as meansS.E.M. with numbers of individual experiments presented in figure legends. Significance was tested using analysis of variance (ANOVA) with Newman Keuls post hoc test (P<0.05). Go to: Acknowledgments These studies were generously supported by funding from the Canadian Institutes of Health Research (CIHR), Canadian Diabetes Association and the Michael Smith Foundation for

Health Research (MSFHR) (to CHSMc), from the Canadian Foundation for Innovation, MSFHR, PA Woodward Foundation and the Ike Barber Diabetes Research Endowment (to GW) and from MSFHR (Graduate fellowship) (to SW). We would like to thank Dr. C B Newgard (Duke University Medical Center, Durham, NC, USA) for INS-1 cells (clone 832/13). Go to: Glossary ANOVA analysis of variance AVD apoptotic volume decrease cAMP cyclic AMP GIP glucose-dependent insulinotropic polypeptide GLP-1 glucagon-like peptide 1 GPCR G protein-coupled receptor GSIS glucose-stimulated insulin secretion HAT histone acetyltransferase HDAC histone deacetylase KATP ATP-sensitive potassium channel Kv voltage-gated potassium channel MSK-1 mitogen- and stress-activated kinase-1 PKA

protein kinase A PTM(s) post-translational modification(s) STS staurosporine TEA tetraethylammonium Thap thapsigargin TSA Trichostatin A Go to: Notes The authors declare no conflict of interest. Go to: Footnotes Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd) Edited by JA Cidlowski Go to: Supplementary Material Supplementary Figures 15 Click here for additional data file.(221K, pdf) Supplementary Figure Legends Click here for additional data file.(37K, doc) Go to:

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ancreatic - sel efek prosurvival dari hormon incretin melibatkan modifikasi pasca - translasi Kv2.1 saluran penyearah tertunda

SJ Kim , 1,3 SB Widenmaier , 1,3 WS Choi , 1,4 C Nian , 1 Z Ao , 2 G Warnock , 2 dan CHS McIntosh1 , * Informasi penulis catatan Pasal Informasi Hak Cipta dan Lisensi Artikel ini telah dikutip oleh artikel lainnya di PMC . Pergi ke : abstrak Insulinotropic glukosa tergantung polipeptida ( GIP ) dan glucagon - like peptide - 1 ( GLP 1 ) adalah hormon incretin utama yang mengerahkan insulinotropic dan tindakan anti apoptosis pada pankreas - sel . Tindakan insulinotropic dari incretins melibatkan modulasi kalium tegangan-gated ( Kv ) saluran . Dalam beberapa jenis sel , aktivitas saluran Kv telah terlibat dalam sel perubahan volume yang menyertai dimulainya program apoptosis . Berfokus pada Kv2.1 , kami memeriksa apakah regulasi saluran Kv di - sel berkontribusi terhadap efek prosurvival dari incretins . Ekspresi dari Kv2.1 di INS - 1 - sel potentiated apoptosis dalam respon terhadap stres mitokondria dan ER dan, sebaliknya , co- stimulasi dengan GIP/GLP-1 uncoupled potensiasi ini , apoptosis menekan . Secara paralel , incretins dipromosikan fosforilasi dan asetilasi Kv2.1 melalui jalur yang melibatkan protein kinase A ( PKA ) / mitogen - dan stres - diaktifkan kinase - 1 ( MSK - 1 ) dan asetiltransferase histon ( HAT ) / histon deacetylase ( HDAC ) . Penelitian lebih lanjut menunjukkan bahwa asetilasi Kv2.1 dimediasi oleh aksi incretin pada nuklir / sitoplasma bolak protein mengikat CREB ( CBP ) dan interaksi dengan Kv2.1 . Peraturan hidup - sel dengan GIP dan GLP - 1 karena melibatkan modifikasi pasca-translasi ( PTM ) saluran Kv oleh PKA/MSK-1 dan HAT / HDAC . Hal ini tampaknya menjadi demonstrasi pertama dari modulasi tertunda penyearah Kv saluran berkontribusi terhadap efek prosurvival - sel incretins dan 7 - transmembran G protein - coupled receptor ( GPCR ) - merangsang ekspor dari lisin asetiltransferase nuklir yang mengatur ion permukaan sel fungsi saluran.

Kata kunci : incretins , diabetes , GIP , GLP - 1 , apoptosis - sel , Kv saluran Diabetes tipe 1 dan tipe 2 berhubungan dengan defisit absolut atau relatif fungsional - sel , dan ada semakin banyak bukti bahwa apoptosis adalah mediator utama kematian - sel . Mengingat peningkatan di seluruh dunia dalam insiden diabetes , penting untuk memahami mekanisme molekuler yang terlibat dalam apoptosis - sel dan untuk mengidentifikasi agen yang dapat mengurangi atau membatalkan proses ini .

Saluran pencernaan mengeluarkan lebih dari 20 hormon peptida yang mengatur banyak proses fisiologis . Di antaranya , insulinotropic glukosa tergantung polipeptida ( GIP ) dan glucagon - like peptide - 1 ( GLP - 1 ) adalah hormon utama incretin yang mempotensiasi glukosa merangsang sekresi insulin ( GSIS ) selama makan , 1 , 2 dan memberi efek menguntungkan pada proliferasi - sel dan survival.2 , 3 , 4 , 5 , 6 GSIS melibatkan penutupan K ATP - sensitif + ( KATP ) saluran mengakibatkan depolarisasi membran , aktivasi tegangan tergantung Ca2 + channel dan meningkatkan intraseluler Ca2 + , diikuti oleh repolarisasi membran dengan tegangan-gated K + ( Kv ) dan Ca2 + - sensitif K + ( KCA ) saluran . Incretins merangsang sekresi insulin melalui modulasi KATP aktivitas saluran , meningkatkan Ca2 + masuknya melalui L - type Ca2 + saluran dan saluran ion non - selektif , dan melepaskan Ca2 + dari toko intraseluler , serta potensiasi dari Ca2 + -tergantung exocytosis.2 , 3 , 7 ini efek dimediasi melalui aktivasi reseptor - sel G protein-coupled ( GPCRs ) .2 , 3 Kedua GIP dan GLP - 1 menstimulasi adenilat siklase , yang menyebabkan peningkatan siklik AMP intraseluler ( cAMP ) , dan mengaktifkan protein kinase A ( PKA ) dan pertukaran protein langsung diaktifkan oleh cAMP 2 ( EPAC2 ) .2 , 3

Incretins mengaktifkan kedua jalur anti -apoptosis transkripsi - dependent dan independen dalam - sel 2 , 3 , 4 , 5 , 6 dan kemajuan yang signifikan telah dibuat dalam menjelaskan mekanisme yang mendasari . Staurosporine ( STS ) yang disebabkan translokasi mitokondria aktivasi buruk dan BimEL , mitokondria Bax , pelepasan sitokrom c dan caspase - 3 aktivasi semua dikurangi dengan pengobatan GIP dari - cells.6 incretins tambahan mengurangi tingkat ER faktor stres terkait .1 , 3 cAMP / PKA jalur memiliki peran sentral dalam efek prosurvival - sel GIP dan GLP - 1 , 1 , 3 , 5 termasuk stimulasi ekspresi CREB - dimediasi gen seperti bcl -2.5 efek Anti - apoptosis PKB juga diaktifkan oleh incretins , dengan

penekanan Bax expression4 dan fosforilasi sinyal apoptosis mengatur kinase 1 ( ASK1 ) , sehingga penekanan berkelanjutan dari p38 MAPK dan Jun N - terminal kinase.6 Menariknya , dalam neuron , lonjakan apoptosis K + saat ini melibatkan ASK1 dan p38 MAPK.8 , 9

Saluran Kv terlibat dalam repolarisasi sel bersemangat , dan studi elektrofisiologi di pankreas - sel manusia dan tikus telah menunjukkan pentingnya mereka dalam sekresi process.10 , 11 Anggota KV1 , KV2 dan Kv3 keluarga diperkirakan memiliki peran penting dalam sekresi insulin oleh modulasi amplitudo dan durasi tindakan potentials.10 , 12 Meskipun Kv1.4 saluran konduktansi merupakan komponen penting dari transien luar saat ini ( Ito ) , subtipe lain saluran Kv berkontribusi tertunda penyearah arus ( IDR) dalam - sel , 10 , 12 , 13 dengan Kv2.1 mungkin memiliki role.10 utama , 12 , 13 Selain mengatur rangsangan sel , K + channel yang terlibat dalam inisiasi dan perkembangan apoptosis dalam beberapa sel yang berbeda types.14 , 15 Peningkatan baik konduktansi dan ekspresi permukaan saluran Kv berkontribusi terhadap apoptosis neuronal . Efek saraf mengungkapkan dominan -negatif Kv2.1 di neuron kortikal yang dikaitkan dengan penurunan K + rapat arus setelah terekspos terhadap rangsangan apoptosis , dan perdagangan Kv2.1 saluran ke membran plasma merupakan penyumbang utama terhadap inisiasi apoptosis.16 Dalam studi saat ini , efek prosurvival GIP dan GLP - 1 ditemukan untuk melibatkan modifikasi pasca-translasi ( PTM ) dari Kv2.1 oleh fosforilasi dan asetilasi , proses yang terakhir terjadi melalui jalur baru yang melibatkan nuklir untuk translokasi sitoplasma dari lisin asetil transferase , binding protein CREB ( CBP ) .

Pergi ke : hasil Saluran Kv terlibat dalam STS - atau Thapsigargin -induced apoptosis - sel dan GIP/GLP-1 mengurangi kematian sel apoptosis Kv2.1 - dimediasi

Potensi keterlibatan K + saluran dalam apoptosis - sel diselidiki dengan memeriksa redaman saluran konduktansi melalui pengobatan dengan K + channel blocker tetraetilamonium ( TEA ) dan mengukur kematian sel apoptosis dalam menanggapi STS ,

penggerak mitokondria -mediated apoptosis , atau Thapsigargin ( Thap ) , inducer dari retikulum endoplasma ( ER ) stres . Seperti ditunjukkan dalam Gambar 1a dan b , TEA mengurangi tingkat STS - dan Thap -induced apoptosis pada - INS - 1 ( 832/13 ) sel dalam cara yang tergantung konsentrasi . Selain itu , TEA menurun - 1 - INS kematian sel STS induced ( Tambahan Gambar 1 ) .

Gambar 1 K + channel berkontribusi untuk STS - dan Thap -induced apoptosis INS - 1 - sel ( a dan b ) . Pengaruh TEA pada STS atau Thap - induksi apoptosis INS - 1 - sel . - INS - 1 ( 832/13 ) sel diobati dengan STS ( 100nm , a) atau Thap ... Dalam rangka untuk memeriksa potensi keterlibatan mereka dalam apoptosis sel - , Kv2.1 , Kv1.5 dan Kv3.2 , protein channel yang kuat diekspresikan dalam - cells10 ( S Kim et al . , Tidak diterbitkan ) , yang transiently diekspresikan dalam - INS - 1 sel . Seperti ditunjukkan dalam Tambahan Gambar 2 , kematian - sel Thap diinduksi secara signifikan meningkat berlebih dari Kv2.1 dan Kv1.5 tetapi tidak Kv3.2 . Dalam pandangan induksi lebih kuat kematian - sel dengan Kv2.1 , dan bukti yang mendukung kontribusi KV2 saluran anggota keluarga untuk ~ 60 % dari Rp - sel , 12 kami fokus lebih pada efek apoptosis Kv2.1 di - sel . Seperti Kv2.1 ekspresi secara signifikan lebih rendah di INS - 1 ( 832/13 ) - sel daripada di primer - sel , untuk studi berlebih kondisi transfeksi berikutnya yang dioptimalkan untuk meniru kadar protein Kv2.1 di pulau manusia ( Tambahan Gambar 3A ) . Ini mengakibatkan potensiasi ditandai dari kedua STS - dan Thap -induced apoptosis - sel (Angka 1c dan d ) dan kematian - sel (Angka 1e dan f ) .

Kami selanjutnya meneliti efek dari GIP dan GLP - 1 pada Kv2.1 - dimediasi apoptosis sel . Inkubasi sel dengan Thap mengakibatkan ~ 4,5 kali lipat peningkatan apoptosis dan ini berkurang sebesar 44,8 % dan 51,6 % dengan GIP atau GLP - 1 pengobatan, masing-masing ( Gambar 2a ) . Hasil yang sama diperoleh kematian - sel ( Gambar 2b ) . Meskipun kadar apoptosis pada Kv2.1 - overexpressing - INS - 1 sel , GIP dan GLP - 1 keduanya mampu mengurangi kematian sel apoptosis ke tingkat bawah yang diamati dengan sel pcDNA -

transfected diobati dengan Thap , menunjukkan bahwa bagian dari efeknya dimediasi melalui efek pada saluran tingkat protein permukaan Kv atau konduktansi . Untuk membangun keterlibatan fungsional endogen menyatakan Kv2.1 dalam apoptosis , interferensi RNA dipekerjakan . Seperti ditunjukkan dalam Gambar 2c dan Tambahan Gambar 3B , RNAi dimediasi knockdown menghasilkan pengurangan spesifik dalam ekspresi Kv2.1 , terkait dengan sangat berkurang Thap - induksi apoptosis -sel. Kedua GIP dan GLP - 1 cenderung lebih menurunkan Thap - induksi apoptosis sel - , meskipun penurunan tersebut tidak bermakna secara statistik . Secara keseluruhan, hasil ini sangat mendukung peran Kv2.1 dalam kematian sel apoptosis Thap - diinduksi dan keterlibatannya dalam perlindungan GIP/GLP-1-mediated .

Gambar 2 GIP dan GLP - 1 mengurangi apoptosis potensial oleh Kv2.1 berlebih . ( a) Pengaruh GIP dan GLP - 1 pada Thap - induksi apoptosis INS - 1 - sel . INS - 1 ( 832/13 ) - sel transfected seperti yang dijelaskan di atas dan diobati dengan Thap ( 1 ... GIP dan GLP - 1 meningkatkan fosforilasi dan asetilasi Kv2.1 di pankreas - sel

Dalam studi sebelumnya, baik GIP11 dan GLP - 110 ditemukan untuk meningkatkan fosforilasi saluran Kv . Baru-baru ini , reversibel protein asetilasi lisin juga telah ditunjukkan untuk menjadi PTM umum proteins.17 non-nuklir Sebagai GIP dan GLP - 1 keduanya terbukti mampu menstimulasi asetilasi histon , 18 efek incretins ini pada tingkat terfosforilasi dan Kv2.1 asetat diperiksa . Transfected INS - 1 - sel diobati dengan GIP atau GLP - 1 dan ekstrak dilarutkan immunoprecipitated dengan antibodi phospho-serine/threonine atau asetil lisin , diikuti oleh imunoblotting dengan Kv2.1 antibodi . GIP ( 1-42 , 100nm ) dan GLP - 1 ( 7-36 , 100nm ) peningkatan fosforilasi (Angka 3a dan b ) dan asetilasi (Angka 3c dan d ) dari Kv2.1 , sedangkan bentuk terpotong dari peptida , GIP ( 19 -30 ) dan GLP - 1 ( 9-36 ) , yang tidak menunjukkan aktivitas insulinotropic , tidak berpengaruh . Hasil ini menunjukkan bahwa hormon incretin mampu meningkatkan baik fosforilasi dan asetilasi Kv2.1 protein dalam pankreas - sel .

Gambar 3 GIP dan GLP - 1 meningkatkan fosforilasi dan asetilasi Kv2.1 . ( a- d ) Pengaruh GIP/GLP-1 pada fosforilasi dan asetilasi Kv2.1 . INS - 1 - sel transfected dengan Kv2.1 dan diinkubasi dengan 100nm peptida ditunjukkan , ... PKA/mitogen- dan stres - diaktifkan kinase - 1 ( MSK - 1 ) dan asetiltransferase histon ( HAT ) / histon deacetylase ( HDAC ) enzim yang terlibat dalam incretin - dimediasi PTM dari Kv2.1 dan promosi hidup - sel

Signaling modul berpotensi terlibat dalam mempromosikan PTM dari Kv2.1 saluran yang selanjutnya diperiksa , awalnya berfokus pada jalur sebelumnya menunjukkan untuk menengahi incretin actions.2 , 3 GIP dan GLP - 1 - dirangsang Kv2.1 fosforilasi sangat berkurang atau ablated oleh H - 89 ( 10m ) , penghambat kedua PKA dan MSK - 1 , serta inhibitor kompetitif spesifik cAMP mengikat subunit regulasi PKA , Rp - kamp ( 200M ) ( Gambar 4a ) . Asetilasi Kv2.1 dalam menanggapi GIP dan GLP - 1 juga sangat dikurangi dengan H - 89 dan Rp - kamp , serta dengan inhibitor HAT ( HAT inhibitor II ( HATII ) ; 40M ) . Sebaliknya, inhibitor HDAC , Trichostatin A ( TSA , 300nm ) , menirukan efek GIP dan GLP - 1 pada asetilasi Kv2.1 ( Gambar 4b ) , serta potentiated tingkat incretin -induced asetilasi Kv2.1 . Seperti yang diharapkan , baik HAT maupun inhibitor HDAC terpengaruh Kv saluran fosforilasi ( Gambar 4a ) . Untuk menentukan apakah fosforilasi dan asetilasi Kv2.1 berkontribusi GIP dan GLP - 1 - dimediasi hidup - sel , Thap -induced apoptosis sel ditentukan dengan atau tanpa kehadiran PKA/MSK-1 , PKA dan HAT / HDAC inhibitor . Seperti ditunjukkan dalam Gambar 4c , kelangsungan hidup - sel incretin - mediated sangat dikurangi dengan H - 89 , Rp - kamp dan HAT inhibitor II . Sebaliknya, inhibitor HDAC potentiated efek GIP dan GLP - 1 pada kelangsungan hidup - sel . Bersama-sama , hasil ini menunjukkan bahwa jalur yang melibatkan PKA/MSK-1 dan HAT / HDAC terlibat dalam PTM incretin - dimediasi Kv2.1 terkait dengan kelangsungan hidup - sel .

Gambar 4 PKA/MSK-1 dan HAT / HDAC terlibat dalam PTM incretin - dimediasi Kv 2.1 dan kelangsungan hidup sel - . ( a dan b ) Pengaruh PKA/MSK-1 menghambat atau HAT / HDAC pada PTM dari Kv 2.1 . INS - 1 - sel transfected dengan Kv2.1 dan dirangsang dengan 100nm ... GIP dan GLP - 1 mengatur lokalisasi nuklir / sitoplasma CBP

Dalam pandangan demonstrasi terbaru yang sitoplasma CBP mampu acetylating prolaktin receptor19 dan selektivitas relatif HAT inhibitor II CBP/p300 , kami meneliti efek GIP dan GLP - 1 pada distribusi seluler CBP . Seperti ditunjukkan pada Gambar 5a , b dan e , GIP atau GLP - 1 pengobatan INS - 1 - sel menurun nuklir , dan peningkatan sitoplasma , lokalisasi CBP . Respon ini adalah 15 menit jelas setelah inisiasi GIP atau GLP - 1 pengobatan dan selanjutnya dipertahankan untuk 120 menit , kembali menuju basal oleh 1200min . Penelitian selanjutnya menunjukkan ketergantungan konsentrasi GIP/GLP-1 untuk nuklir / sitoplasma bolak CBP (Angka 5c , d dan f ) dan mengamati tanggapan serupa dengan pulau pankreas manusia (Angka 5g dan h ) . Pewarnaan imunositokimia INS - 1 - sel menegaskan efek dari incretins pada CBP translokasi nuklir terdeteksi oleh western blotting ( Gambar 5i ) . Di sisi lain , GIP dan GLP - 1 yang tanpa efek pada distribusi nuklir / sitoplasma dari P300 (Angka 5j dan k ) , protein adaptor CREB terkait yang berbagi homologi urutan tinggi dengan CBP dan diberikannya baik kegiatan transkripsi umum dan unik . 20

Gambar 5 GIP dan GLP - 1 ekspor nuklir CBP ke sitoplasma . ( a- d ) Waktu kursus ( a dan b ) dan konsentrasi - respon efek ( c dan d ) dari GIP/GLP-1 pada nuklir (a dan c ) dan sitoplasma ( b dan d ) CBP . INS - 1 - sel dirawat karena ditunjukkan ... CBP menengahi GIP dan GLP - 1 -induced asetilasi Kv2.1 , mengakibatkan saluran internalisasi

Sebagai GIP dan GLP - 1 meningkatkan lokalisasi sitoplasma dari CBP , perannya dalam GIP dan GLP - 1 - dimediasi asetilasi Kv2.1 diperiksa . Seperti ditunjukkan dalam Gambar 6a , dalam percobaan co- immunoprecipitation , interaksi protein - protein antara CBP dan Kv2.1 diamati pada GIP atau pulau manusia GLP - 1 -diobati. Untuk menetapkan pentingnya fungsional CBP dalam peraturan GIP/GLP-1-mediated dari Kv2.1 , interferensi RNA dipekerjakan . RNAi - dimediasi knockdown menghasilkan sangat berkurang ekspresi CBP nuklir INS - 1 - sel ( Gambar 6b ) . Percobaan Co - immunoprecipitation menunjukkan bahwa CBP knockdown hampir sepenuhnya ablated GIP atau GLP - 1 -induced asetilasi lisin dari Kv2.1 ( Gambar 6c ) dan sangat mengurangi interaksi protein - protein antara CBP dan Kv2.1 (Gambar 6d ) . Translokasi CBP dari inti karena itu mungkin menjadi mediator penting GIP - dan GLP - 1 -induced asetilasi lisin dari Kv2.1 .

Gambar 6 CBP bertanggung jawab untuk asetilasi GIP/GLP-1-mediated dari Kv2.1 . (a ) Co immunoprecipitation ( Co - IP ) . Pulau manusia diobati dengan GIP atau GLP - 1 ( 100nm ) untuk 1h . Ekstrak sitoplasma diisolasi dari sampel masing-masing dan IP dengan CBP diikuti ... GIP - diinduksi fosforilasi Kv1.4 sebelumnya menunjukkan hasil dalam endositosis , 11 dan Kv2.1 perdagangan ke membran plasma terbukti bertanggung jawab untuk peningkatan K + arus yang berhubungan dengan saraf apoptosis.16 Oleh karena itu kami menentukan apakah GIP/GLP- peraturan 1 - dimediasi Kv2.1 melibatkan saluran protein internalisasi . Sel diinkubasi dengan proteinase K acak membelah daerah ekstraseluler protein , sehingga menghasilkan bentuk dicerna , sedangkan protein intraseluler dilindungi . Dengan menggunakan teknik ini , permukaan membran tingkat ekspresi Kv2.1 di pulau manusia yang ditampilkan untuk mengurangi sebagai respon terhadap pengobatan dengan GIP ( 1-42 , 100nm ) atau GLP - 1 ( 7-36 , 100nm ) (Gambar 6e ) . Sebagai seluler K + penghabisan telah terbukti menjadi langkah utama bagi perkembangan dan penyelesaian kematian sel apoptosis pada jenis sel banyak, PTM Kv2.1 incretin - dimediasi dan internalisasi cenderung penting untuk kelangsungan hidup sel - .

Pergi ke : diskusi Meskipun peran saluran Kv dalam regulasi repolarisasi membran dan sekresi insulin dalam pankreas - sel didirikan , keterlibatan mereka dalam apoptosis sel - belum pernah dilaporkan sebelumnya . Ini telah dibuktikan dalam beberapa jenis sel yang penyusutan sel apoptosis (penurunan volume yang apoptosis , AVD ) terjadi di awal proses , sebelum perubahan ultrastruktur dan biokimia , seperti fragmentasi DNA , sitokrom c rilis dan caspase 3 aktivasi , 14 dan merupakan prasyarat untuk penyelesaian kematian sel terprogram . Mekanisme ionik yang terlibat dalam AVD sangat kompleks , tapi K + adalah kation sitoplasma utama yang bertanggung jawab untuk menjaga sel volume14 , 21 , 22 dan penghabisan dari K + merupakan komponen penting dari AVD.21 Memang , kegiatan sejumlah caspases dan endonuklease ditekan pada K intraseluler yang normal + tingkat dan salah satu peristiwa awal utama dalam hasil apoptosis dari penurunan konsentrasi intraseluler , yang memungkinkan mereka activation.22 sejumlah berbeda K + channel telah terlibat dalam AVD , termasuk anggota keluarga Kv , 14 , 16 , 21 , 22 dan pengobatan dengan K + channel blocker TEA dan 4 - AP menghambat apoptosis dalam berbagai sel types.21

Dalam studi sebelumnya, GIP dan GLP - 1 reseptor aktivasi ditunjukkan untuk mempotensiasi GSIS oleh saluran modulasi Kv , 11 , 23 , 24 , 25 dan kedua incretins memberi efek menguntungkan pada - sel survival.2 , 3 , 4 , 5 , 6 Oleh karena itu penelitian ini dimulai dalam rangka untuk menguji hipotesis bahwa GIP dan tindakan GLP -1 pada saluran Kv terkait dengan efek pada kelangsungan hidup - sel . Dalam tikus primer - sel , > 80 % dari K + luar arus yang dihasilkan oleh saluran yang sensitif terhadap blocker TEA.24 Pengobatan - INS - 1 sel dengan TEH sangat berkurang apoptosis diinduksi oleh STS atau pengobatan Thap ( Gambar 1a dan b ) , menunjukkan keterlibatan K + channel . Selain tindakan langsung pada K + channel , TEA telah disarankan untuk bertindak pada saluran kation lain dalam sel HeLa dan dengan mengurangi sitokrom c rilis dari mitokondria , 26 dan kita tidak bisa mengesampingkan kontribusi mereka terhadap respon - sel .

Kv2.1 memiliki peran penting dalam - sel repolarization.12 , 23 Selain itu, sitoplasma C terminus Kv2.1 mengikat syntaxin 1A di - sel dan interaksi mereka yang terlibat dalam saluran gating , perdagangan dan insulin exocytosis.27 , 28 peran multifungsi juga telah ditunjukkan dalam jenis sel lain , dan Kv2.1 fasilitasi sekretori perekrutan vesikula dalam sel neuroendokrin , 29 dan pengelompokan permukaan sel Kv2.1 saluran dalam hippocampal neurons30 ditunjukkan untuk merdeka dari K + konduktansi . Dalam pandangan tambahan peran pro - apoptosis pada neuron , kami fokus pada peran potensial Kv2.1 dalam apoptosis - sel dan kemampuan GIP dan GLP - 1 untuk memodulasi aksinya . Ekspresi Kv2.1 protein dalam - INS - 1 sel , pada tingkat yang sama dengan yang ditemukan pada manusia - sel , sangat potentiated respon apoptosis untuk kedua STS dan Thap (Angka 1c - f , 2a dan b ) , sedangkan RNAi - dimediasi knockdown endogen Kv2.1 dilemahkan Thap -induced apoptosis ( Gambar 2c ) . Temuan ini didukung peran Kv2.1 dalam apoptosis -sel.

Modulasi saluran konduktansi telah dianggap sebagai pengaruh besar dari GLP - 1 reseptor aktivasi pada - sel Kv2.1.24 , 25 Namun, efek pro - apoptosis Kv2.1 berlebih di - INS - 1 sel sangat dikurangi dengan kedua hormon incretin ( Gambar 2a ) . Sejumlah K tegangan gated channel + protein diatur oleh serin / treonin fosforilasi , 11 , 25 , 31 dan kedua phosphorylation25 dan SUMOylation32 dari Kv2.1 memodulasi saluran konduktansi di pankreas - sel . Dalam tes berbasis spektrometri massa beberapa situs fosforilasi diidentifikasi pada Kv2.131 dan , di antaranya , p38 MAPK fosforilasi - dimediasi Kv2.1 pada Ser800 terlibat dalam perdagangan Kv2.1 ke membran plasma selama saraf apoptosis.33 Sebuah keluarga Src tirosin kinase terbukti konstitutif mengaktifkan Kv2.1 , 34 dan Tyr124 dari Kv2.1 , target untuk Src kinase , sangat penting untuk neuronal apoptosis surge.35 Selain itu , downregulation Kv2.1 , melalui cAMP / PKA sinyal jalur , mengurangi apoptosis pada cerebellar granular neurons.36

Asetilasi Lysine protein baru-baru ini telah diidentifikasi sebagai PTM penting luas , terkait dengan modulasi dinamika sitoskeleton , endositosis , autophagy dan metabolisme energi , 20 , 37 dan > 80 faktor transkripsi dan regulator nuklir , serta berbagai protein sitoplasma , memiliki telah diidentifikasi sebagai asetiltransferase targets.38 karena kedua GIP dan GLP 1 bertindak melalui stimulasi protein kinase cascades1 , 2 , 3 dan juga meningkatkan asetilasi histon , 18 keterlibatan potensi kedua PTM dari Kv2.1 diselidiki dan terbukti terjadi di INS -1

 - sel (Angka 3a - d ) . Incretin - dimediasi fosforilasi Kv2.1 sangat berkurang oleh H - 89 dan Rp - kamp , sedangkan inhibitor HAT / HDAC yang tanpa efek (Angka 4a dan b ) . Di sisi lain , asetilasi Kv2.1 sangat berkurang oleh H - 89 , Rp - kamp dan HAT / inhibitor HDAC . Meskipun Rp - kamp adalah inhibitor yang relatif spesifik PKA , H - 89 juga sangat menghambat baik GIP dan GLP - 1 stimulasi fosforilasi MSK - 1 pada Ser376 , dimediasi oleh PKA , dan MSK - 1 aktivitas enzimatik di INS - 1 - cells.18 Oleh karena itu masuk akal bahwa kedua PKA dan MSK - 1 terlibat dalam PTM incretin - dimediasi saluran Kv . Karena kelangsungan hidup - sel incretin - dimediasi sangat berkurang oleh HAT inhibitor II dan inhibitor HDAC potentiated efek pada kelangsungan hidup GIP/GLP-1 - sel ( Gambar 4c ) , tanpa mempengaruhi incretin - dimediasi Ser / Thr fosforilasi dari KV2 . 1 ( Gambar 4a ) , asetilasi tampaknya menjadi lebih kritis PTM incretin - dimediasi Kv2.1 terkait dengan kelangsungan hidup - sel . Namun, masih mungkin bahwa fosforilasi PKA dimediasi adalah langkah prasyarat untuk asetilasi Kv2.1 , sebagai incretin - dimediasi asetilasi Kv2.1 sangat berkurang oleh H - 89 dan pengobatan Rp - kamp ( Gambar 4b ) .

Enzim asetiltransferase dan deacetylase sitosol belum secara ekstensif ditandai . Namun , dimerisasi reseptor prolaktin baru-baru ini terbukti dirangsang melalui asetilasi oleh CBP/p300 , 19 regulator kunci dari transkripsi gen CREB - dimediasi , dan kedua GIP dan GLP - 1 menstimulasi ekspresi beberapa protein melalui PKA aktivasi jalur CREB terkait , 1 , 2 , 3 , 5 serta dengan merangsang histon lisin acetylation.18 oleh karena itu kami menyelidiki CBP dan / atau P300 keterlibatan dalam GIP/GLP-1-stimulated asetilasi lisin dari Kv2.1 . Anehnya , GIP dan GLP - 1 diinduksi ekspor ekstensif nuklir CBP ke sitoplasma (Angka 5a dan b ) , sedangkan P300 adalah relatif tidak terpengaruh , dan kedua incretins juga meningkat interaksi protein - protein antara CBP dan Kv2.1 ( Gambar 6a ) . RNAi dimediasi knockdown CBP mengakibatkan terganggunya interaksi ini, serta mengurangi tingkat asetat Kv2.1 (Angka 6c dan d ) , menunjukkan bahwa CBP adalah asetiltransferase jawab utama untuk asetilasi Kv2.1 . Saat ini, tidak ada yang diketahui tentang mekanisme yang mendasari transportasi acetylases atau deacetylases antara kompartemen sitosolik dan nuklir . Namun, peningkatan kadar sitosol CBP terdeteksi dalam 15 menit rangsangan dan tingkat meningkat hingga 2h . Perubahan dalam keseimbangan relatif antara asetilasi dan deasetilasi sebelumnya telah terbukti berdampak signifikan pada kematian - sel pankreas : penghambatan HDACs mencegah sitokin - induksi apoptosis - sel dan kecacatan - sel function.39 , 40 Sebagai seluler K + penghabisan adalah langkah penting dalam program

kematian sel apoptosis , PTM incretin - dimediasi Kv2.1 dan terkait internalisasi ( Gambar 6e ) karena itu mungkin penting untuk kelangsungan hidup sel - .

Singkatnya , kami telah menunjukkan bahwa GIP dan GLP - 1 memodulasi baik fosforilasi dan asetilasi Kv2.1 , sehingga mengurangi ekspresi permukaan , dan bahwa PKA/MSK-1 dan HAT / HDAC terlibat dalam proses ini . Penurunan dalam K + penghabisan kemungkinan merupakan faktor penting dalam efek prosurvival incretin - dimediasi . Namun , kedua incretins juga memodulasi aktivitas mediator intraseluler apoptosis , termasuk Ask1 , Bax dan Bad , serta sitokrom c rilis . Sebagai perdagangan CBP ke membran plasma tampaknya menjadi kontributor utama asetilasi Kv2.1 , mengembangkan pemahaman yang lebih baik peraturan dan implikasinya terhadap regulasi ekspresi gen jelas penting . Hal ini juga tampaknya menjadi contoh pertama dari ekspor GPCR - dirangsang dari Acetylase lisin nuklir yang mengatur permukaan sel ion fungsi saluran. Secara keseluruhan , temuan kami menunjukkan skenario menarik dimana pelepasan nutrisi sensitif usus hormon pasangan untuk mekanisme potentiating baik fungsional dan kelangsungan hidup dalam - sel melalui sumbu sinyal berkumpul di saluran ion tegangan - sensitif yang berfungsi untuk mengembalikan homeostasis setelah eksitasi seluler .

Pergi ke : Bahan dan Metode - INS - 1 kultur sel dan transfections transien

- INS - 1 sel ( clone 832/13 ) yang baik yang diberikan oleh Dr CB Newgard ( Duke University , Durham , NC , USA ) . Sel dikultur dalam glukosa 11mm RPMI 1640 ( Sigma Aldrich , Oakville , ON , Kanada ) ditambah dengan 2mm glutamin , 50M mercaptoethanol , 10mm HEPES , 1mM natrium piruvat , 10 % serum janin sapi , 100units/ml penisilin G - natrium dan 100g/ml streptomisin sulfat . Sel bagian 45-75 digunakan . Transfections Transient dilakukan dengan Kv1.5 , Kv2.1 atau Kv3.2 atau pcDNA3 plasmid menggunakan Lipofectamine 2000 reagen ( Invitrogen , Carlsbad , CA , USA) .

Isolasi pulau kecil dan kultur sel primer

Pulau manusia diisolasi dari pankreas dari lima donor organ dewasa menggunakan kolagenase perfusi saluran, disosiasi dan kepadatan gradien pemurnian di Ike Barber Islet Transplantasi Laboratorium ( VGH , Vancouver , BC , Canada ) . Penelitian Etika Dewan UBC diberikan persetujuan etika .

Tes kematian sel dan apoptosis

- INS - 1 sel diperlakukan dengan STS ( 100nm ) atau Thap ( 1M ) untuk kematian 6h dan sel ditentukan dengan menghitung propidium inti iodida - positif dan jumlah sel diukur dengan menghitung Hoechst sinyal Fluorescent 33342 - positif nuclei.6 yang diukur dengan sistem high -throughput pencitraan , CellomicsArrayscan V otomatis imager atau Cellomics KineticScan ( Cellomics Inc , Pittsburgh , PA , USA) , dan persen kematian sel dihitung . Deteksi sel apoptosis dilakukan dengan APOPercentage apoptosis assay kit ( Biocolor Ltd , Belfast , Irlandia Utara ) , berdasarkan penyerapan spesifik pewarna dengan mekanisme apoptosis ' flip-flop ' . APOPercentage pewarna ditambahkan ke sel 30menit sebelum selesainya inkubasi inducer / incretin apoptosis . Analisis kuantitatif dilakukan dengan menggunakan alat tes kolorimetri berikut rilis pewarna dari sel apoptosis ( gambar representatif untuk APOPercentage sel berlabel pewarna dapat ditemukan dalam Tambahan Gambar 4 ) .

fractionations sel

Ekstrak nuklir / sitoplasma telah disiapkan dari sel seperti yang dijelaskan previously.4 , 5 , 18 Secara singkat , sel-sel dicuci dengan PBS , dan tergores dengan penyangga dingin 200l A ( 10mm HEPES pH 7,9 , 10mm KCl , 1.5mm MgCl2 , 1mM EDTA , 1mM dithiothreitol ,

0,1 % Nonidet P40 dan protease inhibitor ) . Setelah sentrifugasi , supernatan ( ekstrak sitoplasma ) dikumpulkan dan menghasilkan pelet kembali ditangguhkan di 20l penyangga B ( HEPES 20mm , pH 7,9 , 400mm NaCl , 1mM EDTA , 1mM dithiothreitol , 20 % gliserol dan protease inhibitor ) dan diinkubasi pada es selama 10 menit . Setelah klarifikasi dengan sentrifugasi , supernatan ( ekstrak nuklir ) dikumpulkan dan sasaran analisis western blot . Histon dan - aktin digunakan sebagai penanda nuklir dan non-nuklir , masing-masing, untuk membangun kurangnya kontaminasi silang antara fraksi ( Tambahan Gambar 5 ) .

Analisis Western blot

Sampel protein dipisahkan pada 15 % natrium dodesil sulfat ( SDS ) / gel poliakrilamid dan ditransfer ke membran nitroselulosa ( Bio - Rad Laboratories , Mississauga , ON , Kanada ) . Probing dari membran dilakukan dengan Kv2.1 , phospho-Ser/Thr , asetil - Lys , CBP , P300 , histon atau antibodi ( Cell Technology Signaling , Beverly , MA , USA - aktin , Santa Cruz Bioteknologi , Santa Cruz , CA , Amerika Serikat; Sigma - Aldrich , Novus Biologicals , Littleton , CO , USA ) . Band immunoreactive yang divisualisasikan dengan kemiluminesensi ( Millipore , Boston, MA , USA ) menggunakan horseradish peroksidase terkonjugasi antibodi sekunder IgG .

Co- immunoprecipitation ( Co - IP )

Untuk data yang disajikan pada Gambar 2 , jumlah ekstrak seluler disusun mengikuti pengobatan eksperimental dan immunoprecipitated dengan phospho-serine/threonine , antibodi asetil - lisin atau Kv2.1 menggunakan protein Dynabead A ( Invitrogen ) . Produk diendapkan diselesaikan dengan SDS -PAGE dan diperiksa dengan antibodi terhadap Kv2.1 , phospho-serine/threonine atau asetil - lisin .

confocal microscopy

 - INS - 1 sel diperlakukan dengan GIP atau GLP - 1 ( 100nm ) untuk 1h . Pewarnaan immunocytochemical dilakukan dengan menggunakan antibodi terhadap CBP , dan divisualisasikan dengan Texas Red dye - terkonjugasi antibodi sekunder anti - kelinci ( Probe Molekuler , Invitrogen Kanada , Burlington , ON , Kanada ) . Inti sel yang counterstained dengan DAPI ( 4 ' ,6 - diamino - 2 - phenylindole ) . Stained sel dicitrakan menggunakan Nikon confocal mikroskop ( Nikon Kanada , Mississauga , ON , Kanada ) . Semua data pencitraan dianalisis dengan menggunakan software EZC1 ( Nikon ) .

Interferensi RNA knockdown CBP dan Kv2.1

- INS - 1 sel transfected dengan MISI siRNA untuk CBP ( SASI_Rn01_00079791 , Sigma Aldrich ) atau Kv2.1 ( SI01527631 , Qiagen Kanada , Toronto , ON , Kanada ) dan diinkubasi selama 72 jam . Tingkat penurunan CBP dan Kv2.1 ekspresi protein ditentukan oleh hibridisasi western blot menggunakan antibodi terhadap CBP , Kv2.1 , histon dan - aktin .

Proteinase K percobaan pencernaan

Untuk proteinase K pencernaan , 11 pulau manusia diinkubasi dengan 100nm setiap peptida dicuci tiga kali dengan dingin PBS dan diinkubasi dengan HEPES 10mm , 150mm NaCl dan 2mm CaCl2 ( pH 7,4 ) dengan 200g/ml proteinase K pada 37 C selama 30 menit . Sel dipanen dan proteinase K pencernaan dipadamkan dengan es dingin PBS yang mengandung fluoride dan 6mm phenylmethylsulfonyl 25mm EDTA . Hal ini diikuti oleh SDS - PAGE dan imunobloting , dan probing membran dengan antibodi terhadap saluran Kv dan - tubulin .

analisis statistik

Data dinyatakan sebagai berarti S.E.M. dengan jumlah eksperimen individu disajikan dalam legenda angka . Signifikansi diuji dengan analisis varians ( ANOVA ) dengan Newman - Keuls post hoc test ( P <0,05 ) .

Pergi ke : Ucapan Terima Kasih Studi ini murah hati didukung oleh dana dari Institut Penelitian Kesehatan Kanada ( CIHR ) , Canadian Diabetes Association dan Smith Yayasan Michael Penelitian Kesehatan ( MSFHR ) ( ke CHSMc ) , dari Yayasan Kanada untuk Inovasi , MSFHR , PA Woodward Foundation dan Ike Barber Diabetes Research Endowment ( ke GW ) dan dari MSFHR (Graduate persekutuan ) ( ke SW ) . Kami ingin berterima kasih kepada Dr CB Newgard ( Duke University Medical Center , Durham , NC , USA ) untuk INS - 1 sel ( clone 832/13 ) .

Pergi ke : glosarium ANOVAanalysis volume varianceAVDapoptotic decreasecAMPcyclic insulinotropic

AMPGIPglucose tergantung polypeptideGLP - 1glucagon - like peptide 1GPCRG proteincoupled receptorGSISglucose dirangsang insulin secretionHAThistone

acetyltransferaseHDAChistone deacetylaseKATPATP - sensitif kalium channelKvvoltage gated kalium channelMSK - 1mitogen - dan stres - diaktifkan kinase - kinase 1PKAprotein APTM ( s ) posting translasi modifikasi ( s )

STSstaurosporineTEAtetraethylammoniumThapthapsigarginTSATrichostatin A Pergi ke : Catatan Para penulis menyatakan tidak ada konflik kepentingan .

Pergi ke :

Catatan kaki Informasi Tambahan menyertai kertas pada Cell Kematian dan website Diferensiasi ( http://www.nature.com/cdd )

Diedit oleh JA Cidlowski

Pergi ke : Bahan Tambahan Angka Tambahan 1-5

Klik di sini untuk tambahan data file . ( 221K , pdf ) Tambahan Gambar Legends

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