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Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells
Biochemical and Biophysical Research Communications xxx (2015) 1e6
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Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells Shanhua Xu a, b, Ji-Hee Kim a, Kyu-Hee Hwang a, b, Ranjan Das a, Xianglan Quan a, Tuyet Thi Nguyen a, Soo-Jin Kim a, b, Seung-Kuy Cha a, **, Kyu-Sang Park a, * a b
Department of Physiology, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea Department of Global Medical Science, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 November 2015 Accepted 4 November 2015 Available online xxx
Regulation of ATP-sensitive inwardly rectifying potassium (KATP) channel plays a critical role in metabolism-secretion coupling of pancreatic b-cells. Released insulin from b-cells inhibits insulin and glucagon secretion with autocrine and paracrine modes. However, molecular mechanism by which insulin inhibits hormone secretion remains elusive. Here, we investigated the effect of autocrine insulin on surface abundance of KATP channel in mouse clonal b-cell line, MIN6. High glucose increased plasmalemmal sulfonylurea receptor 1 (SUR1), a component of KATP channel as well as exogenous insulin treatment. SUR1 trafficking by high glucose or insulin was blocked by inhibition of phosphoinositide 3kinase (PI3K) with wortmannin. Pretreatment with brefeldin A or silencing of vesicle-associated membrane protein 2 (VAMP2) abolished insulin-mediated upregulation of surface SUR1. Functionally, glucose-stimulated cytosolic Ca2þ ([Ca2þ]i) increase was blunted by insulin or diazoxide, a KATP channel opener. Insulin-induced suppression of [Ca2þ]i oscillation was prevented by an insulin receptor blocker. These results provide a novel molecular mechanism for autocrine negative feedback regulation of insulin secretion. © 2015 Elsevier Inc. All rights reserved.
Keywords: Insulin KATP channel Ca2þ signaling Phosphoinositide-3-kinase VAMP2 MIN6 cells
1. Introduction Pancreatic endocrine cells play a major role in maintaining blood glucose homeostasis by secreting hormones such as insulin and glucagon. In b-cells, high glucose is the main stimulus for insulin exocytosis through eliciting depolarization-triggered Ca2þ entry mediated by inhibition of ATP-sensitive potassium (KATP) channel conductance. Mitochondrial ATP production and subsequent increasing cytosolic ATP/ADP ratio in b-cells is known to close KATP channel contributing to glucose-induced depolarization [1e4]. Functional KATP channel in the plasma membrane is an octameric complex which is comprised of four pore-forming Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1). Each
* Corresponding author. Department of Physiology, Yonsei University Wonju College of Medicine, Wonju, Gangwon-Do 220-701, Republic of Korea. ** Corresponding author. Department of Physiology, Yonsei University Wonju College of Medicine, Wonju, Gangwon-Do 220-701, Republic of Korea. E-mail addresses: [email protected] (S.-K. Cha), [email protected] (K.-S. Park).
subunit is matured in the endoplasmic reticulum (ER), and retained until receiving specific signal to assemble and translocate to the Golgi apparatus. Membrane localization of KATP channel is mediated by vesicle-associated trafficking from the Golgi. Functional cell surface abundance of KATP channel determines membrane excitability of b-cells and metabolism-secretion coupling. Gain-offunction mutations in KATP channel induce defects of insulin secretion causing neonatal diabetes. Conversely, loss-of-function mutations elicit persistent hyperinsulinism and profound hypoglycemia of infancy [5,6]. Recent studies revealed that glucose deprivation or leptin promotes membrane abundance of KATP channel through AMPK activation [7,8], thereby reducing the insulin secretion [9]. Insulin secreted from b-cell may act as a paracrine or an autocrine signal and regulate pancreatic endocrine cell survival [10]. Released insulin can suppress glucagon secretion from neighbored a-cell, enabling antagonistic regulation under high glucose condition [11]. However, molecular mechanism for autocrine negative feedback mechanism of insulin secretion has not been defined clearly. The accumulated studies suggest that insulin increases total KATP conductance in different type of cells through
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Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028
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S. Xu et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e6
phosphoinositide 3-kinase (PI3K) [12,13]. Furthermore, PI3Kactivating growth factors increase membrane abundance of TRPC channels by stimulating exocytosis prompting us to test the hypothesis that insulin stimulates trafficking of KATP channel via PI3K pathway [14,15]. Here we show that released insulin responded to high glucose increases cell surface abundance of KATP channel and thereby inhibits glucose-stimulated cytosolic Ca2þ ([Ca2þ]i) responses in MIN6 cells, suggesting a physiologic mechanism for autocrine regulation of insulin release.
emission: 633/nm) were obtained using a laser scanning mode confocal microscopic system (TCS SPE, Leica Microsystems GmbH, Wetzlar, Germany). DAPI (1 mg/ml, 5 min) was used for nuclear staining. 2.5. Measurement of cytosolic Ca2þ change
MIN6 cells, a mouse clonal b-cell line, gifted from Prof. Hee-Sook Jun (Gachon University, Incheon, Korea) were cultured in low glucose (5.5 mM) Dulbecco's modified Eagles medium (Catalog # 11885, Life Technologies Corporation, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 mM b-mercaptoethanol.
Intracellular Ca2þ concentration ([Ca2þ]i) measurement was previously described [17]. Briefly, MIN6 cells cultured on coverslips were loaded with Fura-2/AM in a dark room for 40 min at room temperature. After dye loading, cells were washed using a low glucose KRB solution (in mM: 140 NaCl, 3.6 KCl, 0.5 NaH2PO4, 0.5 MgSO4, 1.5 CaCl2, 10 HEPES, 2 NaHCO3, 2.8 glucose, pH 7.4) and transferred to a perfusion chamber on an inverted microscope and alternately excited at 340 nm and 380 nm by a monochromatic light source (Lamda DG-4; Sutter, Novato, CA, USA). Fluorescence images were captured at 510 nm with an intensified CCD camera (Cascade; Roper, Duluth, GA, USA) and the ratio of fluorescence intensities (F340/F380) reflecting [Ca2þ]i was analyzed by using MetaFluor 6.3 software (Molecular Devices, Sunnyvale, CA, USA).
2.2. DNA constructs and materials
2.6. Data analysis
Complementary DNAs for GFP-tagged Kir6.2 and SUR1 were transfected using FuGENE-HD reagent (Roche, Somerville, NJ, USA) according to the manufacturer's instructions. Non-targeting control oligonucleotide (sc37007) and siRNA against mouse VAMP2 (M041975-01) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Dharmacon (Chicago, IL, USA), respectively. Transfection of siRNA oligonucleotide was performed using DharmaFECT-1 siRNA transfection reagent (Thermo Scientific, Lafayette, CO, USA) according to the manufacturer's instructions. Unless otherwise noted, all reagents and drugs were purchased from SigmaeAldrich Co. (St. Louis, MO, USA). Fura2/AM and GSK1838705 were obtained from Molecular Probes® (Invitrogen, Carlsbad, CA, USA) and Tocris Bioscience (Bristol, UK), respectively.
Data analysis was performed using the Prism software (ver6, GraphPad Software, San Diego, CA, USA). Statistical comparisons between two groups of data were made using a two-tailed unpaired Student's t-test. Multiple comparisons were performed using oneway ANOVA followed by Tukey's multiple comparison tests. p values less than 0.05 and 0.01 were considered significant for single and multiple comparisons, respectively. Data were presented as mean ± SEM.
2. Materials and methods 2.1. Cell cultures
2.3. Western blot and cell-surface biotinylation assay Western blotting and cell surface biotinylation assay were described previously [16]. The primary antibodies against surface and total SUR1 (1:500 and 1:1000, Catalog #ab32844, Abcam, Cambridge, UK), phosphorylated and total AKT (1:2000, #9275, #9271 or #9272, Cell signaling technology, Danvers, MA, USA), bactin (1:5000, #ab6276, Abcam), and VAMP2 (1:2000, #ab181869, Abcam) were used. Membranes were incubated for 1 h at room temperature in horseradish peroxidase (HRP)-conjugated secondary antibody against either mouse or rabbit IgG (Catalog # 31450 and 31460, Thermo Fisher Scientific Inc.). The bands in the immunoblots were detected and quantified using Biospectrum-600 Imaging System (UVP, Upland, CA, USA) and Image J software (ver. 1.32j, NIH, USA). 2.4. Morphological analysis of plasma membrane translocation To observe the translocation of KATP channel to the plasma membrane, HEK293 cells were transfected with a GFP-tagged Kir6.2 with SUR1 using the FuGENE® HD transfection reagent (Promega, Madison, WI, USA). After 48 h of culture, cells were further incubated for 15 min with wheat germ agglutinin Alexa Fluor 633 (10 mg/ml, Molecular Probes, Thermo Fisher Scientific Inc.) to stain the plasma membrane and fixed with 4% paraformaldehyde in PBS. Microscopic fluorescence images for GFP (excitation/emission: 488/535 nm) and Alexa Fluor 633 (excitation/
3. Results 3.1. Glucose and exogenous insulin stimulates translocation of KATP channel to the plasma membrane Pancreatic b-cells sense blood glucose followed by insulin secretion which is triggered by [Ca2þ]i increase. While the mechanism of glucose-stimulated insulin release is well established, however, its negative feedback mechanism remains elusive. Multiple studies demonstrate that insulin increases total KATP conductance. Thus, we examined whether released insulin evoked by high glucose stimulates cell surface abundance of KATP channel in mouse clonal b-cell line, MIN6. To allow for studying the effect of insulin, we first examined the effect of serum and glucose deprivation to exclude the effect of insulin and growth factors in culture medium. The basal Akt phosphorylation, a downstream effector of insulin, was disappeared after 2 h deprivation of serum and glucose (data not shown). Therefore, we used 2 h of glucose and serum deprivation in following experiments throughout this study. By using biotinylation assay, we observed that glucose stimulated the cell surface abundance of SUR1, a component of KATP channel in a dose- and time-dependent manner (Fig. 1A and B). Application of insulin also increased cell membrane abundance of SUR1 and insulin stimulation was blunted by pretreatment of GSK1838705, an insulin receptor blocker (Fig. 1C). To confirm the translocation of KATP channel, we further investigated membrane abundance of channel regulated by insulin using heterologous expression of GFPtagged Kir6.2 in HEK293 cells. Consistent with biotinylation assay, insulin increased plasma membrane localization of GFP-Kir6.2 (Fig. 1D) supporting that insulin stimulates membrane residence of KATP channel. Taken together, these results suggest that either endogenous or exogenous insulin augments membrane abundance of KATP channel by acting at insulin receptor in MIN6 cells.
Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028
S. Xu et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e6
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Fig. 1. Glucose and Endogenous insulin increases cell surface abundance of SUR1, a KATP channel component. Effect of dose- (A) and time-dependent (B) increase of abundance of SUR1 on the cell surface (s-SUR) and in cell lysates (t-SUR). Cell-surface abundance was determined by biotinylation assay. The abundance of b-actin in lysates served as a loading control. All data are presented as mean ± S.E.M. Bands were quantified by densitometry. * denotes p < 0.05. (C) Treatment of exogenous insulin (1 mM, for 10 min) increased cell surface abundance of SUR1, which was blunted by an insulin receptor blocker, GSK1838705 (GSK, 2 nM) (N ¼ 3). Data are presented as mean ± S.E.M. Bands were quantified by densitometry. * denotes p < 0.05. (D) Insulin increased plasma membrane localization of Kir6.2. HEK293 cells expressing GFP-tagged Kir6.2 and SUR1 were incubated with vehicle or insulin (100 nM, for 2 h), followed by co-stained with wheat germ agglutinin Alexa Fluor 633 (WGA) as a plasma membrane fluorescence marker.
3.2. Insulin stimulates VAMP-2-associated exocytosis of KATP channel via PI3K-Akt pathway Activation of PI3K by growth factors such as IGF1 increases cell surface abundance of channel proteins by stimulating their exocytosis [14,15]. We tested hypothesis that insulin activates PI3K, as the major downstream effector of insulin receptor, to promote exocytosis of KATP channel. Glucose- or insulin-stimulated cell surface abundance of SUR1 was prevented by pretreatment of PI3K inhibitor wortmannin (Fig. 2A and B). It has been reported that insulin increases KATP conductance via ERK signaling in the dorsal vagal complex to inhibit glucose production [18]. In order to exclude the involvement of other signaling activated by insulin, we examined insulin-stimulated ERK signaling in MIN6 cell. As shown in Fig. 2C, we could not detect the phosphorylation of ERK1/2 by short-term exposure of either high glucose (for 30 min, data not shown) or exogenous insulin (for 10 min, Fig. 2C). These results imply that PI3K pathway, but not ERK, is the major downstream signaling of insulin receptor activation, participating in insulin's action on cell surface abundance of KATP channel. To demonstrate the increase in exocytosis of KATP channel by insulin, we applied brefeldin A (BFA) known to inhibit protein transport from the ER to the Golgi. Blocking exocytosis with BFA blunted insulin-stimulated membrane abundance of SUR1 (Fig. 3A). Next, we found that insulin-induced membrane delivery of SUR1 was blocked by knockdown of VAMP2 (vesicle-associated
membrane protein 2), a v-SNARE (Fig. 3B), indicating that translocation of KATP channel to plasma membrane is VAMP2-mediated exocytosis in MIN6 cells. 3.3. Suppression of glucose-stimulate cytosolic calcium oscillation by insulin Increased KATP channel density and total KATP conductance can restrain depolarization-induced [Ca2þ]i oscillation and metabolism-secretion coupling of pancreatic b-cells. We recorded [Ca2þ]i changes in MIN6 cells using a fluorescence probe Fura-2. Consistent with the notion, we found that blockade of insulin receptors by GSK1838705 augmented glucose-induced [Ca2þ]i response, which is more prominent at late period (10e15 min) of glucose stimulation (Fig. 4A and B). Acute exposure (5 min) to exogenous insulin suppressed [Ca2þ]i response to glucose, which is similar to the effect of diazoxide, a KATP channel opener (Fig. 4C and D). Insulin action on [Ca2þ]i signaling was completely reversed by GSK1838705 pretreatment. These results demonstrate that released insulin from MIN6 cells inhibits glucose-stimulated [Ca2þ]i signaling as a feedback regulation. 4. Discussion It is well established that reduced KATP conductance by nutrients elicits depolarization-triggered Ca2þ entry leading to insulin
Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028
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Fig. 2. Insulin increases cell surface abundance of KATP channel through PI3K pathway. (A) Effect of pretreatment of a PI3K inhibitor, wortmannin (WMN, 10 nM, for 30 min) on glucose-stimulated cell-surface abundance of SUR1. (B) Insulin-induced SUR1 translocation was blunted by pretreatment of WMN. (C) Insulin treatment for 30 min did not activates ERK1/2 signaling (N ¼ 3). Data are presented as mean ± SEM, and * denote p < 0.05.
secretion in pancreatic b-cells, indicating that KATP channel functions as a prime regulator of insulin release and metabolic sensor. However, the physiological role of secreted insulin acting on b-cell as autocrine hormone and negative feedback mechanism on insulin secretion remain largely elusive. The presented data in this study provide compelling evidence suggesting that autocrine insulin responded to high glucose suppresses insulin release as a negative feedback mechanism via stimulating surface abundance of KATP channel. Either autocrine or exogenous insulin suppresses the [Ca2þ]i response to glucose similar to that of KATP channel opener suggest that released insulin from b-cells attenuates further insulin granule exocytosis by impeding glucose-stimulated depolarization due to increase of total KATP conductance. Insulin increases GLUT4 translocation to the plasma membrane in adipocyte and muscle through PI3K-AKT pathway [19]. The fusion of GLUT4 with the plasma membrane is mediated by a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that consists of 23 kDa synaptosomalassociated protein (SNAP23), syntaxin4, and VAMP2 [20,21]. The increase of cell surface abundance of KATP channel by insulin may be caused by increased exocytosis and/or decreased endocytosis of the channel. Blocking exocytosis by brefeldin A (BFA) or knockdown of SNARE protein VAMP2 largely prevented the effect of insulin on cell surface abundance of SUR1 suggesting the major action of insulin on SUR1 may be by blocking exocytosis rather than endocytosis. On the other hand, the regulation of KATP channel conductance by insulin can be mediated via alteration of intrinsic channel properties such as single channel open probability. Insulin activates WNK1 kinase promoting phosphoinositide [4,5]bisphosphate (PIP2) resynthesis [22], which is crucial for KATP
channel activation. Thus, it is also conceivable that insulin may stimulate KATP channel via WNK1-promoting PIP2 resynthesis. Future studies will investigate the hypothesis that insulin inhibits endocytosis of KATP channel and stimulates PIP2 resynthesis activating channel open probability. Our results that PI3K-activation by insulin stimulates VAMP2dependent cell surface trafficking of KATP channel support the notion that increased residence time of KATP channel at plasma membrane may serve as negative feedback mechanism of insulin secretion in b-cell. Released insulin may function as not only autocrine hormone but paracrine hormone acting on other endocrine cells in the islet. Indeed, KATP channel is also expressed in acells and regulates glucagon secretion. Thus, physiological role and underlying mechanism of insulin-stimulation of KATP channel acting on a-cells in glucagon secretion awaits future investigation. Intriguingly, we found that MIN6 cells did not sensitively respond to exogenous insulin under normoglycemic (5.5 mM) condition, indicating marked desensitization of insulin signaling (data not shown). Under glucose-deprived condition, insulin elicited prominent activation of downstream signaling in those cells. These findings suggest that insulin accumulation even under normal glucose level can inhibit further insulin response due to receptor downregulation. This could be noteworthy information to investigators who perform experiments to detect an insulin response from MIN6 cells. However, this may not be the case of in vivo pancreatic islets, in where insulin and glucagon are secreted as a pulsatile manner [23,24]. Therefore, spontaneous insulin secretion might be washed away rapidly and hardly lead to insulin resistance of endocrine cells. Insulin resistance is a general feature in type 2 diabetic patients
Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028
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Fig. 3. Insulin triggers VAMP2-dependent exocytosis of SUR1. Pretreatment of brefeldin A (BFA) (A) and silencing of vesicle-associated membrane protein 2 (VAMP2) using siRNA (siVAMP2) (B) inhibits insulin-increased SUR1 membrane abundance. Cell-surface abundance of SUR1 was assayed by biotinylation (N ¼ 3). Non-targeting siRNA oligo used as siRNA control (siCon). Data are presented as mean ± SEM, and * denote p < 0.05.
Fig. 4. Insulin suppresses glucose-stimulated cytosolic Ca2þ increase. Cytosolic Ca2þ change was detected by fluorescence imaging system using Fura-2 dye. (A~B) Effects of GSK1838705 (GSK, 2 nM), an insulin receptor blocker, on cytosolic Ca2þ increase were analyzed by calculating area under the curve during early (0e5 min) and late period (10e15 min) after glucose incubation (N ¼ 4). (C~D) Glucose-stimulated Ca2þ increase was blunted by pretreatment of insulin (Ins) and a KATP channel activator, diazoxide (Diaz). Suppression of Ca2þ response by insulin was reverted by GSK (N ¼ 4). All data are presented as mean ± SEM, and * denote p < 0.05.
Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028
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as well as animal disease models. Sustained hyperinsulinemic condition aggravates downregulation of insulin receptor, which in turn further demands more insulin secretion. In pancreatic b-cell, desensitization to insulin response may occur due to excessive and continuous autocrine action. As a consequence of desensitization, reduced KATP conductance may contribute to Ca2þ overload and cytotoxicity. In conclusion, we propose that novel mechanism of autocrine action of insulin stabilizing membrane potential via increasing cell surface abundance of KATP channel, which participates in the physiologic feedback regulation to avoid sustained insulin secretion, but also may involve in pathologic processes such as diabetogenesis. Acknowledgments We are grateful to Prof. Hee-Sook Jun (Gachon University of Medicine and Science, Incheon, Korea) for providing MIN6 cells. This work was supported by the grant from the National Research Foundation (NRF-2013R1A1A4A01010780 and NRF-2010-0024789) and Yonsei University Future-leading Research Initiative of 2014 [2014-22-0127].
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Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells Shanhua Xu a, b, Ji-Hee Kim a, Kyu-Hee Hwang a, b, Ranjan Das a, Xianglan Quan a, Tuyet Thi Nguyen a, Soo-Jin Kim a, b, Seung-Kuy Cha a, **, Kyu-Sang Park a, * a b
Department of Physiology, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea Department of Global Medical Science, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 November 2015 Accepted 4 November 2015 Available online xxx
Regulation of ATP-sensitive inwardly rectifying potassium (KATP) channel plays a critical role in metabolism-secretion coupling of pancreatic b-cells. Released insulin from b-cells inhibits insulin and glucagon secretion with autocrine and paracrine modes. However, molecular mechanism by which insulin inhibits hormone secretion remains elusive. Here, we investigated the effect of autocrine insulin on surface abundance of KATP channel in mouse clonal b-cell line, MIN6. High glucose increased plasmalemmal sulfonylurea receptor 1 (SUR1), a component of KATP channel as well as exogenous insulin treatment. SUR1 trafficking by high glucose or insulin was blocked by inhibition of phosphoinositide 3kinase (PI3K) with wortmannin. Pretreatment with brefeldin A or silencing of vesicle-associated membrane protein 2 (VAMP2) abolished insulin-mediated upregulation of surface SUR1. Functionally, glucose-stimulated cytosolic Ca2þ ([Ca2þ]i) increase was blunted by insulin or diazoxide, a KATP channel opener. Insulin-induced suppression of [Ca2þ]i oscillation was prevented by an insulin receptor blocker. These results provide a novel molecular mechanism for autocrine negative feedback regulation of insulin secretion. © 2015 Elsevier Inc. All rights reserved.
Keywords: Insulin KATP channel Ca2þ signaling Phosphoinositide-3-kinase VAMP2 MIN6 cells
1. Introduction Pancreatic endocrine cells play a major role in maintaining blood glucose homeostasis by secreting hormones such as insulin and glucagon. In b-cells, high glucose is the main stimulus for insulin exocytosis through eliciting depolarization-triggered Ca2þ entry mediated by inhibition of ATP-sensitive potassium (KATP) channel conductance. Mitochondrial ATP production and subsequent increasing cytosolic ATP/ADP ratio in b-cells is known to close KATP channel contributing to glucose-induced depolarization [1e4]. Functional KATP channel in the plasma membrane is an octameric complex which is comprised of four pore-forming Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1). Each
* Corresponding author. Department of Physiology, Yonsei University Wonju College of Medicine, Wonju, Gangwon-Do 220-701, Republic of Korea. ** Corresponding author. Department of Physiology, Yonsei University Wonju College of Medicine, Wonju, Gangwon-Do 220-701, Republic of Korea. E-mail addresses: [email protected] (S.-K. Cha), [email protected] (K.-S. Park).
subunit is matured in the endoplasmic reticulum (ER), and retained until receiving specific signal to assemble and translocate to the Golgi apparatus. Membrane localization of KATP channel is mediated by vesicle-associated trafficking from the Golgi. Functional cell surface abundance of KATP channel determines membrane excitability of b-cells and metabolism-secretion coupling. Gain-offunction mutations in KATP channel induce defects of insulin secretion causing neonatal diabetes. Conversely, loss-of-function mutations elicit persistent hyperinsulinism and profound hypoglycemia of infancy [5,6]. Recent studies revealed that glucose deprivation or leptin promotes membrane abundance of KATP channel through AMPK activation [7,8], thereby reducing the insulin secretion [9]. Insulin secreted from b-cell may act as a paracrine or an autocrine signal and regulate pancreatic endocrine cell survival [10]. Released insulin can suppress glucagon secretion from neighbored a-cell, enabling antagonistic regulation under high glucose condition [11]. However, molecular mechanism for autocrine negative feedback mechanism of insulin secretion has not been defined clearly. The accumulated studies suggest that insulin increases total KATP conductance in different type of cells through
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Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028
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phosphoinositide 3-kinase (PI3K) [12,13]. Furthermore, PI3Kactivating growth factors increase membrane abundance of TRPC channels by stimulating exocytosis prompting us to test the hypothesis that insulin stimulates trafficking of KATP channel via PI3K pathway [14,15]. Here we show that released insulin responded to high glucose increases cell surface abundance of KATP channel and thereby inhibits glucose-stimulated cytosolic Ca2þ ([Ca2þ]i) responses in MIN6 cells, suggesting a physiologic mechanism for autocrine regulation of insulin release.
emission: 633/nm) were obtained using a laser scanning mode confocal microscopic system (TCS SPE, Leica Microsystems GmbH, Wetzlar, Germany). DAPI (1 mg/ml, 5 min) was used for nuclear staining. 2.5. Measurement of cytosolic Ca2þ change
MIN6 cells, a mouse clonal b-cell line, gifted from Prof. Hee-Sook Jun (Gachon University, Incheon, Korea) were cultured in low glucose (5.5 mM) Dulbecco's modified Eagles medium (Catalog # 11885, Life Technologies Corporation, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 mM b-mercaptoethanol.
Intracellular Ca2þ concentration ([Ca2þ]i) measurement was previously described [17]. Briefly, MIN6 cells cultured on coverslips were loaded with Fura-2/AM in a dark room for 40 min at room temperature. After dye loading, cells were washed using a low glucose KRB solution (in mM: 140 NaCl, 3.6 KCl, 0.5 NaH2PO4, 0.5 MgSO4, 1.5 CaCl2, 10 HEPES, 2 NaHCO3, 2.8 glucose, pH 7.4) and transferred to a perfusion chamber on an inverted microscope and alternately excited at 340 nm and 380 nm by a monochromatic light source (Lamda DG-4; Sutter, Novato, CA, USA). Fluorescence images were captured at 510 nm with an intensified CCD camera (Cascade; Roper, Duluth, GA, USA) and the ratio of fluorescence intensities (F340/F380) reflecting [Ca2þ]i was analyzed by using MetaFluor 6.3 software (Molecular Devices, Sunnyvale, CA, USA).
2.2. DNA constructs and materials
2.6. Data analysis
Complementary DNAs for GFP-tagged Kir6.2 and SUR1 were transfected using FuGENE-HD reagent (Roche, Somerville, NJ, USA) according to the manufacturer's instructions. Non-targeting control oligonucleotide (sc37007) and siRNA against mouse VAMP2 (M041975-01) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Dharmacon (Chicago, IL, USA), respectively. Transfection of siRNA oligonucleotide was performed using DharmaFECT-1 siRNA transfection reagent (Thermo Scientific, Lafayette, CO, USA) according to the manufacturer's instructions. Unless otherwise noted, all reagents and drugs were purchased from SigmaeAldrich Co. (St. Louis, MO, USA). Fura2/AM and GSK1838705 were obtained from Molecular Probes® (Invitrogen, Carlsbad, CA, USA) and Tocris Bioscience (Bristol, UK), respectively.
Data analysis was performed using the Prism software (ver6, GraphPad Software, San Diego, CA, USA). Statistical comparisons between two groups of data were made using a two-tailed unpaired Student's t-test. Multiple comparisons were performed using oneway ANOVA followed by Tukey's multiple comparison tests. p values less than 0.05 and 0.01 were considered significant for single and multiple comparisons, respectively. Data were presented as mean ± SEM.
2. Materials and methods 2.1. Cell cultures
2.3. Western blot and cell-surface biotinylation assay Western blotting and cell surface biotinylation assay were described previously [16]. The primary antibodies against surface and total SUR1 (1:500 and 1:1000, Catalog #ab32844, Abcam, Cambridge, UK), phosphorylated and total AKT (1:2000, #9275, #9271 or #9272, Cell signaling technology, Danvers, MA, USA), bactin (1:5000, #ab6276, Abcam), and VAMP2 (1:2000, #ab181869, Abcam) were used. Membranes were incubated for 1 h at room temperature in horseradish peroxidase (HRP)-conjugated secondary antibody against either mouse or rabbit IgG (Catalog # 31450 and 31460, Thermo Fisher Scientific Inc.). The bands in the immunoblots were detected and quantified using Biospectrum-600 Imaging System (UVP, Upland, CA, USA) and Image J software (ver. 1.32j, NIH, USA). 2.4. Morphological analysis of plasma membrane translocation To observe the translocation of KATP channel to the plasma membrane, HEK293 cells were transfected with a GFP-tagged Kir6.2 with SUR1 using the FuGENE® HD transfection reagent (Promega, Madison, WI, USA). After 48 h of culture, cells were further incubated for 15 min with wheat germ agglutinin Alexa Fluor 633 (10 mg/ml, Molecular Probes, Thermo Fisher Scientific Inc.) to stain the plasma membrane and fixed with 4% paraformaldehyde in PBS. Microscopic fluorescence images for GFP (excitation/emission: 488/535 nm) and Alexa Fluor 633 (excitation/
3. Results 3.1. Glucose and exogenous insulin stimulates translocation of KATP channel to the plasma membrane Pancreatic b-cells sense blood glucose followed by insulin secretion which is triggered by [Ca2þ]i increase. While the mechanism of glucose-stimulated insulin release is well established, however, its negative feedback mechanism remains elusive. Multiple studies demonstrate that insulin increases total KATP conductance. Thus, we examined whether released insulin evoked by high glucose stimulates cell surface abundance of KATP channel in mouse clonal b-cell line, MIN6. To allow for studying the effect of insulin, we first examined the effect of serum and glucose deprivation to exclude the effect of insulin and growth factors in culture medium. The basal Akt phosphorylation, a downstream effector of insulin, was disappeared after 2 h deprivation of serum and glucose (data not shown). Therefore, we used 2 h of glucose and serum deprivation in following experiments throughout this study. By using biotinylation assay, we observed that glucose stimulated the cell surface abundance of SUR1, a component of KATP channel in a dose- and time-dependent manner (Fig. 1A and B). Application of insulin also increased cell membrane abundance of SUR1 and insulin stimulation was blunted by pretreatment of GSK1838705, an insulin receptor blocker (Fig. 1C). To confirm the translocation of KATP channel, we further investigated membrane abundance of channel regulated by insulin using heterologous expression of GFPtagged Kir6.2 in HEK293 cells. Consistent with biotinylation assay, insulin increased plasma membrane localization of GFP-Kir6.2 (Fig. 1D) supporting that insulin stimulates membrane residence of KATP channel. Taken together, these results suggest that either endogenous or exogenous insulin augments membrane abundance of KATP channel by acting at insulin receptor in MIN6 cells.
Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028
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Fig. 1. Glucose and Endogenous insulin increases cell surface abundance of SUR1, a KATP channel component. Effect of dose- (A) and time-dependent (B) increase of abundance of SUR1 on the cell surface (s-SUR) and in cell lysates (t-SUR). Cell-surface abundance was determined by biotinylation assay. The abundance of b-actin in lysates served as a loading control. All data are presented as mean ± S.E.M. Bands were quantified by densitometry. * denotes p < 0.05. (C) Treatment of exogenous insulin (1 mM, for 10 min) increased cell surface abundance of SUR1, which was blunted by an insulin receptor blocker, GSK1838705 (GSK, 2 nM) (N ¼ 3). Data are presented as mean ± S.E.M. Bands were quantified by densitometry. * denotes p < 0.05. (D) Insulin increased plasma membrane localization of Kir6.2. HEK293 cells expressing GFP-tagged Kir6.2 and SUR1 were incubated with vehicle or insulin (100 nM, for 2 h), followed by co-stained with wheat germ agglutinin Alexa Fluor 633 (WGA) as a plasma membrane fluorescence marker.
3.2. Insulin stimulates VAMP-2-associated exocytosis of KATP channel via PI3K-Akt pathway Activation of PI3K by growth factors such as IGF1 increases cell surface abundance of channel proteins by stimulating their exocytosis [14,15]. We tested hypothesis that insulin activates PI3K, as the major downstream effector of insulin receptor, to promote exocytosis of KATP channel. Glucose- or insulin-stimulated cell surface abundance of SUR1 was prevented by pretreatment of PI3K inhibitor wortmannin (Fig. 2A and B). It has been reported that insulin increases KATP conductance via ERK signaling in the dorsal vagal complex to inhibit glucose production [18]. In order to exclude the involvement of other signaling activated by insulin, we examined insulin-stimulated ERK signaling in MIN6 cell. As shown in Fig. 2C, we could not detect the phosphorylation of ERK1/2 by short-term exposure of either high glucose (for 30 min, data not shown) or exogenous insulin (for 10 min, Fig. 2C). These results imply that PI3K pathway, but not ERK, is the major downstream signaling of insulin receptor activation, participating in insulin's action on cell surface abundance of KATP channel. To demonstrate the increase in exocytosis of KATP channel by insulin, we applied brefeldin A (BFA) known to inhibit protein transport from the ER to the Golgi. Blocking exocytosis with BFA blunted insulin-stimulated membrane abundance of SUR1 (Fig. 3A). Next, we found that insulin-induced membrane delivery of SUR1 was blocked by knockdown of VAMP2 (vesicle-associated
membrane protein 2), a v-SNARE (Fig. 3B), indicating that translocation of KATP channel to plasma membrane is VAMP2-mediated exocytosis in MIN6 cells. 3.3. Suppression of glucose-stimulate cytosolic calcium oscillation by insulin Increased KATP channel density and total KATP conductance can restrain depolarization-induced [Ca2þ]i oscillation and metabolism-secretion coupling of pancreatic b-cells. We recorded [Ca2þ]i changes in MIN6 cells using a fluorescence probe Fura-2. Consistent with the notion, we found that blockade of insulin receptors by GSK1838705 augmented glucose-induced [Ca2þ]i response, which is more prominent at late period (10e15 min) of glucose stimulation (Fig. 4A and B). Acute exposure (5 min) to exogenous insulin suppressed [Ca2þ]i response to glucose, which is similar to the effect of diazoxide, a KATP channel opener (Fig. 4C and D). Insulin action on [Ca2þ]i signaling was completely reversed by GSK1838705 pretreatment. These results demonstrate that released insulin from MIN6 cells inhibits glucose-stimulated [Ca2þ]i signaling as a feedback regulation. 4. Discussion It is well established that reduced KATP conductance by nutrients elicits depolarization-triggered Ca2þ entry leading to insulin
Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028
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Fig. 2. Insulin increases cell surface abundance of KATP channel through PI3K pathway. (A) Effect of pretreatment of a PI3K inhibitor, wortmannin (WMN, 10 nM, for 30 min) on glucose-stimulated cell-surface abundance of SUR1. (B) Insulin-induced SUR1 translocation was blunted by pretreatment of WMN. (C) Insulin treatment for 30 min did not activates ERK1/2 signaling (N ¼ 3). Data are presented as mean ± SEM, and * denote p < 0.05.
secretion in pancreatic b-cells, indicating that KATP channel functions as a prime regulator of insulin release and metabolic sensor. However, the physiological role of secreted insulin acting on b-cell as autocrine hormone and negative feedback mechanism on insulin secretion remain largely elusive. The presented data in this study provide compelling evidence suggesting that autocrine insulin responded to high glucose suppresses insulin release as a negative feedback mechanism via stimulating surface abundance of KATP channel. Either autocrine or exogenous insulin suppresses the [Ca2þ]i response to glucose similar to that of KATP channel opener suggest that released insulin from b-cells attenuates further insulin granule exocytosis by impeding glucose-stimulated depolarization due to increase of total KATP conductance. Insulin increases GLUT4 translocation to the plasma membrane in adipocyte and muscle through PI3K-AKT pathway [19]. The fusion of GLUT4 with the plasma membrane is mediated by a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that consists of 23 kDa synaptosomalassociated protein (SNAP23), syntaxin4, and VAMP2 [20,21]. The increase of cell surface abundance of KATP channel by insulin may be caused by increased exocytosis and/or decreased endocytosis of the channel. Blocking exocytosis by brefeldin A (BFA) or knockdown of SNARE protein VAMP2 largely prevented the effect of insulin on cell surface abundance of SUR1 suggesting the major action of insulin on SUR1 may be by blocking exocytosis rather than endocytosis. On the other hand, the regulation of KATP channel conductance by insulin can be mediated via alteration of intrinsic channel properties such as single channel open probability. Insulin activates WNK1 kinase promoting phosphoinositide [4,5]bisphosphate (PIP2) resynthesis [22], which is crucial for KATP
channel activation. Thus, it is also conceivable that insulin may stimulate KATP channel via WNK1-promoting PIP2 resynthesis. Future studies will investigate the hypothesis that insulin inhibits endocytosis of KATP channel and stimulates PIP2 resynthesis activating channel open probability. Our results that PI3K-activation by insulin stimulates VAMP2dependent cell surface trafficking of KATP channel support the notion that increased residence time of KATP channel at plasma membrane may serve as negative feedback mechanism of insulin secretion in b-cell. Released insulin may function as not only autocrine hormone but paracrine hormone acting on other endocrine cells in the islet. Indeed, KATP channel is also expressed in acells and regulates glucagon secretion. Thus, physiological role and underlying mechanism of insulin-stimulation of KATP channel acting on a-cells in glucagon secretion awaits future investigation. Intriguingly, we found that MIN6 cells did not sensitively respond to exogenous insulin under normoglycemic (5.5 mM) condition, indicating marked desensitization of insulin signaling (data not shown). Under glucose-deprived condition, insulin elicited prominent activation of downstream signaling in those cells. These findings suggest that insulin accumulation even under normal glucose level can inhibit further insulin response due to receptor downregulation. This could be noteworthy information to investigators who perform experiments to detect an insulin response from MIN6 cells. However, this may not be the case of in vivo pancreatic islets, in where insulin and glucagon are secreted as a pulsatile manner [23,24]. Therefore, spontaneous insulin secretion might be washed away rapidly and hardly lead to insulin resistance of endocrine cells. Insulin resistance is a general feature in type 2 diabetic patients
Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028
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Fig. 3. Insulin triggers VAMP2-dependent exocytosis of SUR1. Pretreatment of brefeldin A (BFA) (A) and silencing of vesicle-associated membrane protein 2 (VAMP2) using siRNA (siVAMP2) (B) inhibits insulin-increased SUR1 membrane abundance. Cell-surface abundance of SUR1 was assayed by biotinylation (N ¼ 3). Non-targeting siRNA oligo used as siRNA control (siCon). Data are presented as mean ± SEM, and * denote p < 0.05.
Fig. 4. Insulin suppresses glucose-stimulated cytosolic Ca2þ increase. Cytosolic Ca2þ change was detected by fluorescence imaging system using Fura-2 dye. (A~B) Effects of GSK1838705 (GSK, 2 nM), an insulin receptor blocker, on cytosolic Ca2þ increase were analyzed by calculating area under the curve during early (0e5 min) and late period (10e15 min) after glucose incubation (N ¼ 4). (C~D) Glucose-stimulated Ca2þ increase was blunted by pretreatment of insulin (Ins) and a KATP channel activator, diazoxide (Diaz). Suppression of Ca2þ response by insulin was reverted by GSK (N ¼ 4). All data are presented as mean ± SEM, and * denote p < 0.05.
Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028
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as well as animal disease models. Sustained hyperinsulinemic condition aggravates downregulation of insulin receptor, which in turn further demands more insulin secretion. In pancreatic b-cell, desensitization to insulin response may occur due to excessive and continuous autocrine action. As a consequence of desensitization, reduced KATP conductance may contribute to Ca2þ overload and cytotoxicity. In conclusion, we propose that novel mechanism of autocrine action of insulin stabilizing membrane potential via increasing cell surface abundance of KATP channel, which participates in the physiologic feedback regulation to avoid sustained insulin secretion, but also may involve in pathologic processes such as diabetogenesis. Acknowledgments We are grateful to Prof. Hee-Sook Jun (Gachon University of Medicine and Science, Incheon, Korea) for providing MIN6 cells. This work was supported by the grant from the National Research Foundation (NRF-2013R1A1A4A01010780 and NRF-2010-0024789) and Yonsei University Future-leading Research Initiative of 2014 [2014-22-0127].
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Please cite this article in press as: S. Xu, et al., Autocrine insulin increases plasma membrane KATP channel via PI3K-VAMP2 pathway in MIN6 cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.11.028