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Increased ATP-sensitive K+ channel expression during acute glucose deprivation
BBRC Biochemical and Biophysical Research Communications 348 (2006) 1123–1131 www.elsevier.com/locate/ybbrc
Increased ATP-sensitive K+ channel expression during acute glucose deprivation q Andrew J. Smith a, Christopher J. Partridge a, Aruna Asipu b, Lindsey A. Mair a, Malcolm Hunter a, Asipu Sivaprasadarao a,* b
a Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK Leeds Institute of Molecular Medicine, St. James’s University Hospital, University of Leeds, Leeds LS9 7TF, UK
Received 27 July 2006 Available online 4 August 2006
Abstract ATP-sensitive potassium (KATP) channels play a central role in glucose-stimulated insulin secretion (GSIS) by pancreatic b-cells. Activity of these channels is determined by their open probability (Po) and the number of channels present in a cell. Glucose is known to reduce Po, but whether it also affects the channel density is unknown. Using INS-1 model b-cell line, we show that the expression of KATP channel subunits, Kir6.2 and SUR1, is high at low glucose, but declines sharply when the ambient glucose concentration exceeds 5 mM. In response to glucose deprivation, channel synthesis increases rapidly by up-regulating translation of existing mRNAs. The effects of glucose deprivation could be mimicked by pharmacological activation of 5 0 -AMP-activated protein kinase with 5-aminoimidazole-4-carboxamide ribonucleotide and metformin. Pancreatic b-cells which have lost their ability for GSIS do not show such changes implicating a possible (patho-)physiological link between glucose-regulated KATP channel expression and the capacity for normal GSIS. 2006 Elsevier Inc. All rights reserved. Keywords: ATP-sensitive potassium channel; KATP channels; Insulin secretion; AMP-activated protein kinase; AMPK; Glucose sensing; Glucose-stimulated insulin secretion
Pancreatic b-cells possess the remarkable ability to promptly sense changes in blood glucose levels and to secrete the appropriate amount of insulin, a process termed glucose stimulated insulin secretion (GSIS) [1]. According to the accepted model of GSIS, raised blood glucose levels lead to an increased influx of glucose into the pancreatic b-cell which in turn leads to increased metabolism and a rise in the ratio of cytoplasmic [ATP] to [ADP]. The rise in [ATP]/[ADP] ratio causes the closure of ATP-sensitive potassium (KATP) channels, leading to membrane depolarisation, opening of depolarisation activated voltage-gated
q
Abbreviations: KATP channel, ATP-sensitive potassium channel; GSIS, glucose stimulated insulin secretion; AMPK, 5 0 -AMP-activated protein kinase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; SUR1, sulphonylurea receptor 1; ABC, ATP-binding cassette; PNDM, permanent neonatal diabetes mellitus. * Corresponding author. Fax: +44 0 113 3434228. E-mail address: [email protected] (A. Sivaprasadarao). 0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.07.170
calcium channels, and influx of Ca2+ into the cell [2]. The resultant rise in intracellular Ca2+ promotes the fusion of insulin granules with the plasma membrane and release of insulin into the blood stream [3]. The importance of KATP channels in normal GSIS is underscored by the discovery of numerous genetic mutations of both Kir6.2 and SUR1 which lead to inherited disease states characterised by disrupted insulin secretion [4,5]. The pancreatic KATP channel is octameric, comprising four copies each of the two subunits, Kir6.2 and the sulphonylurea receptor1 (SUR1) [6]. Kir6.2 is a member of the inwardly rectifying potassium channel family and forms the K+ conducting pore of the channel [7,8]. SUR1 is a member of the ATP-binding cassette (ABC) transporter family [9], which confers nucleotide sensitivity on the channel through its nucleotide-binding folds, NBF1 and NBF2. In addition to these, a weak but a functionally important inhibitory binding site for ATP is located within the Kir6.2 tetramer [8,10]. SUR1 also contains binding
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sites for therapeutic drugs such as the sulphonylureas and diazoxide [11]. Sulphonylureas inhibit the KATP channel and are used to stimulate insulin secretion in diabetic patients [5]; diazoxide activates the KATP channel and is used to suppress insulin secretion in patients suffering from hyperinsulinaemia [4]. The net activity of KATP channels in a cell is a function of the number of channels present at the cell surface (N) and their open probability (Po). Whilst there have been extensive studies on how the Po of KATP channels is controlled by the blood glucose levels, there is little information on the regulation of channel density by glucose. Moritz et al. [12] reported that prolonged (72 h) exposure of pancreatic b-cells to high glucose concentrations reduces the level of KATP channel mRNA transcripts by 70%. Under physiological situations, however, changes in blood glucose levels can occur more acutely, for example, after a meal, and thus it is important to investigate if glucose can have acute effects on channel density. The importance of testing such a possibility is underscored by the fact that genetic mutations in KATP channels may cause either congenital hyperinsulinism by decreasing cell surface channel density [13,14] or permanent neonatal diabetes mellitus (PNDM) by increasing cell surface channel density [15]. Here we demonstrate that glucose can exert an acute effect on the density of KATP channels in the glucose responsive model b-cell line INS-1. In response to a sudden decline in glucose concentration, expression of both the KATP channel subunits increased acutely; conversely when the cells were exposed to high glucose levels the density of the channels decreased. The effects of lowering the glucose concentration could be mimicked by application of the 5 0 AMP-activated protein kinase (AMPK) activators 5aminoimidazole-4-carboxamide ribonucleotide (AICAR), and metformin [16,17]. The effects of both glucose and AMPK activation on KATP channel synthesis are absent in non-pancreatic cells and in b-cells (Rin-m), which have lost their ability to GSIS. Taken together our data suggest that glucose-mediated changes in KATP expression may have an important role in the physiology of insulin secretion. Experimental Materials. Antibodies to Kir6.2 and SUR1 (custom-made by SigmaGenosys) were raised in rabbits against peptides EDPAEPRYRARQR RARFVSKK and EEAAESEEDDNLSSVLHQRAK, respectively [13]. Donkey anti-rabbit fluorescein isothiocyanate (FITC)-conjugated antibody was supplied by Jackson ImmunoResearch Laboratories, Inc. RPMI 1640 cell culture media and heat-inactivated fetal calf serum were both obtained from Gibco-BRL. Diazoxide, tolbutamide, 5-aminoimidazole-4carboxamide ribonucleotide (AICAR) and metformin were obtained from Sigma, UK. Cell culture. INS-1 cells (passage 102–120) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 10 mM Hepes, 2 mM glutamine, 1 mM sodium pyruvate, 50 lM bmercaptoethanol, 100 U ml1 penicillin, and 100 lg ml1 streptomycin, at 37 C in a humidified atmosphere of air/5% CO2. RIN-m cells (ATCC No. CRL-2057) were grown as for INS-1 cells, but in a medium lacking Hepes
and b-mercaptoethanol. HEK293 cells stably expressing mouse Kir6.2 and hamster SUR1 were cultured as described previously [13]. Immunocytochemistry. INS-1 were cells grown on poly-L-lysine-coated glass coverslips and treated as required (see figure legends). The cells were rinsed in phosphate-buffered saline (PBS) (mM: NaCl, 137; KCl, 2.6; Na2HPO4, 1.0; and KH2PO4, 1.76; pH 7.4), fixed in 2% PFA for 15 min at room temperature, and then permeabilised in 50:50 mixture of acetone and methanol (pre-chilled to 20 C) for 15 min at 4 C. Cells were then blocked in 5% goat serum in PBS for 1 h, before extensive washing in PBS, followed by incubation in primary antibody for 2 h at room temperature. Primary antibodies used were rabbit anti-SUR1 (1:300 dilution) and rabbit anti-Kir6.2 (1:300 dilution). All antibodies were diluted in 5% goat serum in PBS. Cells were then washed extensively in PBS before incubation with FITC-conjugated donkey anti-rabbit IgG secondary antibody (1:100 dilution) for an hour at room temperature. Following extensive washing with PBS, cells were mounted onto microscope slides with VECTASHIELD (Vector Laboratories, CA) and air sealed with nail varnish. Cells were viewed on a Leica-NT laser scanning confocal microscope equipped with a 488 nm laser excitation filter for FITC. Fluorescent images were visualised under an oil-immersion 63· objective using the appropriate laser. Quantification of fluorescence intensity was performed using ImageJ software. Briefly, each individual cell was outlined by hand and the mean pixel intensity of the selected area was calculated. The mean pixel intensity of each cell was then normalised to the mean value of appropriate control cells (stated in the figure legends). The normalised data were then expressed as mean values ± SEM. Statistical significance was examined using Student’s t test. Northern analysis. Isolation of total RNA and Northern analysis were performed as described previously [18]. A 663 bp long SUR1 cDNA probe was amplified from total RNA isolated from INS-1 cells by reverse transcription PCR using 5 0 -AGAAGACACGCAGGAAGGAA-3 0 and 5 0 -CTGGAAGGCTGTTCCAGAAG-3 0 as primers. Actin probe was prepared as described before [18]. The hybridised RNA species were identified by phosphorimaging. The intensity of the bands was quantified using the Quantity 1 software (Bio-Rad). The density of SUR1 mRNA was normalised to that of actin bands. Electrophysiological measurement of whole cell currents. Whole cell currents from INS-1 cells, grown overnight in RPMI containing either 3 mM or 25 mM glucose, were recorded using the patch–clamp technique [19]. The pipette solution for whole cell recordings comprised (mM): 140 KCl, 0.6 MgCl2, 1.03 CaCl2, 10 EGTA, 10 Hepes, 0.1 MgATP, and 0.1 ADP (adjusted to pH 7.2). The bath solution comprised (mM): 122.5 NaCl, 5.0 KCl, 1.0 MgCl2, 1.0 CaCl2, and 10.0 Hepes (pH 7.4). This bath solution was supplemented with either 25 mM D-glucose or 3 mM D-glucose + 22 mM D-sorbitol as required. Currents were measured during a voltage ramp (100 mV to +100 mV) from a holding potential of 0 mV and normalised to the membrane capacitance. Diazoxide (200 lM) was perfused to activate the channel and tolbutamide (100 lM) to block the activated currents. Insulin assays. INS-1 cells grown in a 24-well plate were incubated in 200 ll KRB–Hepes buffer supplemented with either 3 mM or 25 mM glucose for 1 h. Aliquots of the supernatant were used to assay for the insulin content by radioimmunoassay using a kit from ICN Biomedicals Ltd, UK, according to the instructions of the manufacturer. Protein content of the detergent (0.5% Triton X-100 and 0.1% SDS) extracts of cells from each well was estimated by the bicinchoninic acid method. The insulin secretion data were normalised to the protein content of the cell.
Results The density of KATP channels in INS-1 cells is dependent on the glucose concentration of the medium To investigate if glucose regulates the density of KATP channels, we have incubated the pancreatic b-cell line INS-1 in media supplemented with varying concentrations
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of glucose for 2 h. The cells were then immunolabelled with anti-Kir6.2 or anti-SUR1 antibodies and examined by confocal microscopy. The specificity of each antibody for its target antigen has previously been demonstrated [13]. Fig. 1A shows that the expression of both the KATP channel subunits is high at and below 5 mM glucose, but declines as the ambient glucose concentration is raised to 11 mM and beyond. A similar level of expression was observed for both subunits at all glucose concentrations, suggesting that the changes had occurred in a stoichiometric fashion allowing the formation of fully assembled channels. Measurement of the average pixel intensity of labelled cells revealed that fluorescence in cells incubated in 0.5 mM glucose was 2.5-fold higher compared to those cells incubated in 25 mM glucose (Fig 1B). Substitution of mannitol, a non-metabolisable glucose derivative, failed to show such an effect (Fig. 1C), indicating that the effect is not due to changes in the osmolarity of the medium. Reducing the glucose concentration causes an acute increase in the density of KATP channels We next asked whether the observed dependence of KATP channel density on glucose concentration is due to an
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increase in the synthesis of channel subunits when exposed to low glucose or due to an increase in channel degradation in high glucose. For this INS-1 cells were incubated in high (25 mM) or low (3 mM) glucose medium overnight to allow the channel density to reach steady-state before changing to media containing low (3 mM) and high (25 mM) glucose concentrations, respectively. At different time intervals the cells were fixed and stained for SUR1 to follow the resulting changes in channel density. The results show that the density of SUR1 increases acutely when the cells were shifted from a high to low glucose (Fig. 2A, top panels; Fig. 2B), with a noticeable increase within 15 min. This increase in fluorescence appeared uniformly throughout the cytoplasm. In contrast, the fluorescence decreases comparatively more slowly when the cells were switched from low to high glucose (Fig. 2A, bottom panels; Fig. 2C), the decrease being more prominent from the perinuclear region of the cell. Quantitative analysis of the fluorescence intensity revealed that significant increase (1.4-fold; p < 0.05) in channel expression occurs as early as 15 min following the shift from high to low glucose; after 10 h the fluorescence intensity was 2.7-fold greater compared to the zero time point (Fig. 2B). In contrast, it takes 2 h for a significant decrease in fluorescence to be observed following the shift from low to high glu-
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Fig. 1. The density of KATP channels in INS-1 cells decreases when the glucose concentration of the bathing medium exceeds the physiological range. (A) Effect of medium glucose concentration on the expression of KATP channel subunits. INS-1 cells were incubated for 2 h in RPMI medium supplemented with indicated concentrations of glucose. The cells were then fixed, permeabilised, and immunostained for Kir6.2 (upper panel) or SUR1 (lower panels) as described in Experimental. Representative images taken from one dataset of three using identical settings are shown. (B) The average pixel intensity of the fluorescence of cells incubated for 2 h in either 0.5 mM glucose (light grey) or 25 mM glucose (dark grey) was calculated. The data represent means ± SEM normalised to the mean of 0.5 mM glucose values. *p < 0.01 (Student’s two-tailed t test). n = number of individual cells examined for fluorescence intensity from three separate experiments. (C) The decline in the channel density is not due to increased osmolarity. INS-1 cells exposed to either 3 mM glucose or 3 mM glucose + 22 mM mannitol for 2 h were stained for SUR1. Representative images taken using identical settings are shown. Scale bars = 10 lm.
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Fig. 2. Density of KATP channels in INS-1 cells increases rapidly during glucose deprivation. (A) INS-1 cells were incubated overnight (16 h) in media supplemented with 25 mM glucose (top panel) or 3 mM glucose (bottom panel) before switching to 3 mM or 25 mM glucose, respectively. Following incubation for the indicated times in the new media, the cells were fixed and immunolabelled with anti-SUR1 antibodies. Representative confocal images taken using identical settings are shown. (B, C) The average pixel intensity of the fluorescence of cells switched from 25 mM to 3 mM glucose (B) or 3 mM to 25 mM glucose (C) were calculated. The data represent means ± SEM normalized to the mean of 0 min values. *p < 0.01 (Student’s two-tailed t test). Scale bars = 10 lm.
cose with an eventual decrease of 60% after 10 h (Fig. 2C). By this time the intensity of the cells was similar to that of cells incubated in the continuous presence of high glucose. A similar pattern was observed for the Kir6.2 subunit (data not shown). Stimulation of KATP channel expression during glucose deficiency occurs via increased translation Various pharmacological agents were used to investigate whether the increase in protein synthesis was due to an increase in the rate of transcription or translation. Fig. 3A shows that inhibitors of transcription, a-amanitin and actinomycin D, both failed to prevent the increase in the expression of SUR1 during a shift to lower glucose concentrations. By contrast, cycloheximide, an inhibitor of translation, prevented the increase in the expression of SUR1. These data suggest that glucose deficiency has little effect on the transcription of the SUR1 (ABCC8) gene but appears to increase the translation of SUR1 mRNA. We performed Northern analysis on RNA isolated from cells exposed to 3 mM glucose and 25 mM glucose for 2 h (Figs. 3B and C) and the data (Fig. 3C) show no significant difference (p > 0.05) in the SUR1 mRNA content (expressed as means ± SEM of SUR1/actin ratio) of cells treated with 3 mM glucose (3.00 ± 0.27) from that of 25 mM glucose
(3.14 ± 0.4). The lack of glucose-dependent changes in mRNA levels during short-term exposure is consistent with other reports [20,21]. Electrophysiological measurements reveal no significant differences in the cell surface channel density As mentioned above the increased fluorescence appeared uniformly throughout the cytoplasm. As a result it is not clear if the increase in channel synthesis is accompanied by a corresponding increase in the channel density at the cell surface. Both antibodies are directed to cytoplasmic domains of their respective subunits making them unsuitable for selective labelling of cell surface channels. For this reason, we have determined the cell surface channel density by measuring whole cell currents by the patch– clamp technique. KATP channel currents were maximally activated by application of diazoxide and inhibited by application of tolbutamide (Figs. 4A and B). The diazoxide-stimulated, tolbutamide-sensitive currents were normalised to the capacitance of the cell (which gives a measure of cell surface area) to obtain a measure of the channel density at the cell surface. Fig. 4C shows that the density of functional channels in cells incubated in 3 mM glucose (636 ± 125 pA/pF; n = 7) is approximately 30% higher than that of cells in 25 mM glucose (428 ± 70
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Fig. 3. The induction of KATP channel synthesis during glucose deprivation is due to an increase in translation. (A) Inhibitors of translation, but not transcription, prevent the induction of KATPchannel synthesis. INS-1 cells were pre-incubated for 4 h in 25 mM glucose (in RPMI) supplemented with either a-amanitin (5 lg/ml), actinomycin-D (Act-D, 1 lg/ml) or cycloheximide (CHX, 25 lg/ml) followed by a further 2 h incubation in medium containing 3 mM glucose, supplemented with the relevant pharmacological agent. Cells were then fixed, permeabilised and immunostained for SUR1. For comparison, cells incubated in 25 mM glucose alone were also stained in a parallel experiment. Representative confocal images taken using identical settings are shown. (B, C) Lack of effect of glucose concentration on SUR1 mRNA. Northern blots of total RNA isolated from INS-1 cells incubated in 3 mM (lanes 1 and 2) or 25 mM (lanes 3 and 4) glucose, showing the SUR1 and actin bands; data generated using RNA isolated from two separate flasks of cells are shown for each glucose concentration. (C) Means ± SEM (n = 4) of SUR1 mRNA, normalised to actin mRNA band intensity; p > 0.05 (Student’s two-tailed t-test). Scale bars = 10 lm.
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Fig. 4. The density of KATP channels at the cell surface does not appear to be affected by glucose concentration. (A, B) KATP channel currents measured from INS-1 cells cultured in 3 mM glucose. Currents were measured in the whole cell patch–clamp configuration. Data points represent currents measured at 0 mV during perfusion of bath solution alone or bath solution supplemented with diazoxide (D) or tolbutamide (T), as indicated by horizontal bars. (B) Current–voltage relationships measured during the application of diazoxide or tolbutamide, corresponding to time points (gray filled circles) labelled 1 and 2 in (A). (C) Histogram of means ± SEM of currents from cells cultured overnight in 3 and 25 mM glucose as indicated.
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pA/pF; n = 8); however, the difference is not significant (p = 0.16).
Glucose mediated changes in the KATP channel density are dependent on the ability of the cells for GSIS
The increase in KATP channel expression in low glucose may involve the activation of AMPK
We next asked if the glucose-regulation of channel expression is specific to pancreatic b-cells. We tested the effect of glucose concentration on HEK cells heterologously expressing Kir6.2 and SUR1, and found that glucose has no effect on channel density (Fig. 6A). We then investigated whether glucose-regulated channel expression is dependent on the ability of the pancreatic b-cell for GSIS. For this we utilised Rin-m cells, another model pancreatic b-cell line which have lost their ability for GSIS and tested them in parallel with INS-1 cells. Whilst INS-1 cells exposed to 25 mM glucose secrete 10-fold more insulin (p < 0.01; Student’s t test) than cells incubated in 3 mM glucose, the late passage Rin-m cells failed to elicit GSIS (Fig. 6B, C). When examined for glucose effects on KATP channel density, INS-1 cells showed increased channel density in low glucose relative to high glucose as has been shown in Fig. 1, but Rin-m cells did not show such differences (Fig. 6A). These data suggest that the effect of glucose on the KATP channel density is not only b-cell specific but more importantly, depends on the ability of the cells for normal GSIS.
One consequence of glucose starvation is decreased metabolism and an increase in the intracellular levels of ADP and AMP potentially leading to the activation of AMPK [22,23]. Indeed, it has been shown that in pancreatic b-cells the activity of AMPK increases with the decrease in ambient glucose concentration [24,25]. To address a possible role for AMPK activation in mediating glucose-regulated KATP channel expression, we tested the ability of two activators of AMPK, AICAR [16] and metformin [17,26], to mediate changes in channel expression. Both compounds activate AMPK regardless of ambient glucose concentration and would thus mimic the effects of glucose deprivation leading to increased KATP channel expression even at high ambient glucose concentrations. Both AICAR and metformin enhanced the expression of KATP channels in cells incubated in high (25 mM) glucose to the same level as in cells subjected to glucose deprivation (3 mM) (Fig. 5). By contrast, AICAR and metformin caused no apparent increase in channel expression in cells incubated in 3 mM glucose, which is consistent with the expectation that in low glucose AMPK would be already fully activated. Thus glucose starvation appears to induce expression of KATP channels via activation of AMPK.
Discussion We have investigated the effect of ambient glucose concentration on the density of KATP channels in the rat pancreatic INS-1 b-cell line. We found that in response to a decrease in ambient glucose concentration, synthesis
Fig. 5. Pharmacological activation of AMPK mimics the effect of low glucose on the expression of KATP channels incubated in high glucose. INS-1 cells were incubated for 2 h in media containing either 3 mM or 25 mM glucose supplemented with AICAR (0.5 mM) or metformin (0.5 mM) as required. The cells were then fixed and immunolabelled using anti-SUR1 antibodies. Representative confocal images taken from one dataset of three using identical settings are shown. Scale bars = 10 lm.
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Fig. 6. Glucose mediated regulation of the density of KATP channels is dependent on the ability of b-cells for GSIS. (A) Cell specific effect of glucose. HEK cells stably co-transfected with SUR1 and Kir6.2, Rin-m cells and INS-1 cells were incubated in low (0.5 mM) and high (25 mM) glucose for 2 h before immunostaining for SUR1. Representative confocal images taken from one dataset of three are shown. The inset panel for high glucose INS-1 cells (bottom right panel) shows the same field of view as the main picture but with increased brightness and contrast to show that cells are present in the field of view. (B, C) Glucose induced insulin secretion from INS-1 (B) and Rin-m (C) cells. Cells were incubated in 3 mM or 25 mM glucose for 2 h and the amount of insulin secreted into the medium was assayed. The data represent means ± SEM of four independent experiments. Scale bars = 10 lm.
of KATP channels is acutely stimulated and that this increase in channel synthesis occurs via increased translation. The effect of glucose deprivation can be mimicked by the pharmacological stimulation of AMPK by AICAR and metformin. More importantly, the glucose-mediated change in channel expression is dependent on the ability of b-cells for GSIS, indicating that glucose induced changes in channel density have potential (patho-)physiological significance. Immunolabelling experiments revealed that the density of KATP channel subunits, Kir6.2 and SUR1, is inversely dependent on ambient glucose concentration (Fig. 1). These changes appear to result from a stimulation of channel expression in low glucose and suppression in high glucose media. The induction of channel expression following the sudden switch from a high to low glucose medium was quite rapid with a significant (1.4-fold; p < 0.05) increase occurring within only 15 min (Fig. 2). This increase was due to increased translation of pre-existing mRNA and not due to an increase in gene transcription (Fig. 3). In this regard it resembles proinsulin whose expression is stimulated by glucose [27] or the iron homeostatic proteins transferrin and ferritin whose expression is determined by iron availability [28,29]. Since translation alone can produce a much faster response compared to transcription-coupled translation, the finding that glucose-mediated changes in
channel expression is regulated at the translational level seems consistent with the speed with which the b-cells responded to glucose depravation (Fig. 2). Several studies have investigated the effect of either acute or chronic exposure to elevated glucose levels on KATP channel mRNA expression [12,20,21]. It appears that short-term exposure to high glucose (<24 h) has no measurable effect on the level of either Kir6.2 or SUR1 mRNA [20,21]. By contrast, more prolonged exposure to high glucose has been shown to significantly reduce mRNA levels in both INS1 cells [12] and in b-cells isolated from mice with induced diabetes [30]. Taken together with the published data, it seems that the channel density is regulated at the translational level to produce a rapid response and at the transcriptional level to maintain a sustained response. In contrast to the marked differences in the total density of the channels between low and high glucose treated cells, patch–clamp-data (Fig. 4) showed only a small (30%) difference in the density of functional channels at the plasma membrane. This lack of correlation between functional data and the immunofluorescence data suggests that there may be other mechanisms that control the number of functional channels at the cell surface. Emerging evidence suggests that the cell surface density of KATP channels is subjected to downregulation by endocytic mechanisms [15,31]. Consistent with this idea in cells exposed to high
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glucose the fluorescence appears to be more prominent at the periphery of cells in the form of puncta (Figs. 1–3) which may represent endocytic/exocytic vesicles. We have demonstrated that the effect of glucose deprivation could be mimicked by the pharmacological activators of AMPK, AICAR, and metformin (Fig. 5). These two compounds activate AMPK by different mechanisms [16,17,26], yet produce the same stimulatory effect on channel expression. Since both compounds are able to maximally stimulate KATP channel expression it is likely that the response is mediated via AMPK activation and not simply because of another drug-mediated non-specific mechanism. The finding that AMPK activation mimics the effect of glucose deprivation on KATP channel synthesis is interesting because previous studies have established a clear relationship between the activity of AMPK in pancreatic b-cells and ambient glucose concentration [24,25] as well as between AMPK activity and insulin secretion [24,32]. With the decrease in glucose concentration, the activity of AMPK increases and insulin secretion is suppressed [24,32]. Although these studies suggest that glucose deprivation inhibits insulin secretion by activating AMPK, the mechanism by which AMPK activation suppresses insulin secretion remained unclear. Our present data suggest that the suppression of insulin secretion by AMPK may be mediated in part by the upregulation of KATP channel expression by AMPK; greater channel numbers might be expected to have an increased capacity for maintaining a sustained membrane hyperpolarisation thus more effectively suppressing insulin secretion. Another important finding is that cells which have lost the ability for GSIS have also lost their ability to induce KATP channel expression during glucose starvation. Salt et al. [24] reported that the late passage b-cell lines which have lost their ability for GSIS also fail to show glucosemediated changes in AMPK activity. Taken together these findings suggest that glucose-mediated regulation of KATP channel expression (via AMPK) has an essential role in GSIS. Since loss of GSIS is the hallmark of type2 diabetes, loss of glucose mediated changes in AMPK activity, and hence of KATP channel expression, may be another contributing factor in type-2 diabetes mellitus. In this context, it is also interesting to note that metformin has been shown to restore a normal secretory pattern in rat pancreatic islets whose function has been impaired by chronic exposure to high glucose concentrations [33,34]; this useful property may be related to the ability of this anti-diabetic drug to induce KATP channel expression via AMPK activation. Acknowledgments The work was supported by the Medical Research Council, UK. A.S. is supported by Emma and Leslie Reid Scholarship. We thank Dr. C.B. Wollheim and Dr. P. Maechler for kindly providing INS-1 cells.
References [1] C.B. Newgard, J.D. McGarry, Metabolic coupling factors in pancreatic beta-cell signal transduction, Annu. Rev. Biochem. 64 (1995) 689–719. [2] C.G. Nichols, KATP channels as molecular sensors of cellular metabolism, Nature 440 (2006) 470–476. [3] P.E. MacDonald, L. Eliasson, P. Rorsman, Calcium increases endocytotic vesicle size and accelerates membrane fission in insulinsecreting INS-1 cells, J. Cell Sci. 118 (2005) 5911–5920. [4] M.J. Dunne, K.E. Cosgrove, R.M. Shepherd, A. Aynsley-Green, K.J. Lindley, Hyperinsulinism in infancy: from basic science to clinical disease, Physiol. Rev. 84 (2004) 239–275. [5] F.M. Ashcroft, ATP-sensitive potassium channelopathies: focus on insulin secretion, J. Clin. Invest. 115 (2005) 2047–2058. [6] L. Aguilar-Bryan, J. Bryan, Molecular biology of adenosine triphosphate-sensitive potassium channels, Endocr. Rev. 20 (1999) 101–135. [7] N. Inagaki, T. Gonoi, J.P. Clement, N. Namba, J. Inazawa, G. Gonzalez, L. Aguilar-Bryan, S. Seino, J. Bryan, Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor, Science 270 (1995) 1166–1170. [8] S.J. Tucker, F.M. Gribble, C. Zhao, S. Trapp, F.M. Ashcroft, Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor, Nature 387 (1997) 179– 183. [9] L. Aguilar-Bryan, C.G. Nichols, S.W. Wechsler, J.P. Clement, A.E. Boyd III, G. Gonzalez, H. Herrera-Sosa, K. Nguy, J. Bryan, D.A. Nelson, Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion, Science 268 (1995) 423–426. [10] J.F. Antcliff, S. Haider, P. Proks, M.S. Sansom, F.M. Ashcroft, Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit, EMBO J. 24 (2005) 229– 239. [11] A.P. Babenko, G. Gonzalez, J. Bryan, Pharmaco-topology of sulfonylurea receptors. Separate domains of the regulatory subunits of KATP channel isoforms are required for selective interaction with K+ channel openers, J. Biol. Chem. 275 (2000) 717–720. [12] W. Moritz, C.A. Leech, J. Ferrer, J.F. Habener, Regulated expression of adenosine triphosphate-sensitive potassium channel subunits in pancreatic beta-cells, Endocrinology 142 (2001) 129–138. [13] C.J. Partridge, D.J. Beech, A. Sivaprasadarao, Identification and pharmacological correction of a membrane trafficking defect associated with a mutation in the sulfonylurea receptor causing familial hyperinsulinism, J. Biol. Chem. 276 (2001) 35947–35952. [14] E.A. Cartier, L.R. Conti, C.A. Vandenberg, S.L. Shyng, Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy, Proc. Natl. Acad. Sci. USA 98 (2001) 2882–2887. [15] J. Mankouri, T.K. Taneja, A.J. Smith, S. Ponnambalam, A. Sivaprasadarao, Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP-sensitive potassium channels, EMBO J., in press. [16] N. Henin, M.F. Vincent, G. Van Den Berghe, Stimulation of rat liver AMP-activated protein kinase by AMP analogues, Biochim. Biophys. Acta 1290 (1996) 197–203. [17] G. Zhou, R. Myers, Y. Li, Y. Chen, X. Shen, J. Fenyk-Melody, M. Wu, J. Ventre, T. Doebber, N. Fujii, N. Musi, M.F. Hirshman, L.J. Goodyear, D.E. Moller, Role of AMP-activated protein kinase in mechanism of metformin action, J. Clin. Invest. 108 (2001) 1167– 1174. [18] A. Asipu, A.L. Mellor, G.E. Blair, The specificity of the myelin basic protein gene promoter studied in transgenic mice, Biochem. Biophys. Res. Commun. 288 (2001) 809–818. [19] J. Harvey, S.C. Hardy, M.L. Ashford, Dual actions of the metabolic inhibitor, sodium azide on K(ATP) channel currents in the rat CRIG1 insulinoma cell line, Br. J. Pharmacol. 126 (1999) 51–60.
A.J. Smith et al. / Biochemical and Biophysical Research Communications 348 (2006) 1123–1131 [20] A. Shalev, C.A. Pise-Masison, M. Radonovich, S.C. Hoffmann, B. Hirshberg, J.N. Brady, D.M. Harlan, Oligonucleotide microarray analysis of intact human pancreatic islets: identification of glucoseresponsive genes and a highly regulated TGFbeta signaling pathway, Endocrinology 143 (2002) 3695–3698. [21] H. Wang, B.R. Gauthier, K.A. Hagenfeldt-Johansson, M. Iezzi, C.B. Wollheim, Foxa2 (HNF3beta) controls multiple genes implicated in metabolism-secretion coupling of glucose-induced insulin release, J. Biol. Chem. 277 (2002) 17564–17570. [22] D.G. Hardie, Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status, Endocrinology 144 (2003) 5179–5183. [23] G.A. Rutter, G. DaSilva-Xavier, I. Leclerc, Roles of 5 0 -AMPactivated protein kinase (AMPK) in mammalian glucose homoeostasis, Biochem. J. 375 (2003) 1–16. [24] I.P. Salt, G. Johnson, S.J. Ashcroft, D.G. Hardie, AMP-activated protein kinase is activated by low glucose in cell lines derived from pancreatic beta cells, and may regulate insulin release, Biochem. J. 335 (1998) 533–539. [25] G. DaSilva-Xavier, I. Leclerc, I.P. Salt, B. Doiron, D.G. Hardie, A. Kahn, G.A. Rutter, Role of AMP-activated protein kinase in the regulation by glucose of islet beta cell gene expression, Proc. Natl. Acad. Sci. USA 97 (2000) 4023–4028. [26] S.A. Hawley, A.E. Gadalla, G.S. Olsen, D.G. Hardie, The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism, Diabetes 51 (2002) 2420–2425. [27] H. Jahr, D. Schroder, B. Ziegler, M. Ziegler, H. Zuhlke, Transcriptional and translational control of glucose-stimulated (pro)insulin biosynthesis, Eur. J. Biochem. 110 (1980) 499–505.
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Increased ATP-sensitive K+ channel expression during acute glucose deprivation q Andrew J. Smith a, Christopher J. Partridge a, Aruna Asipu b, Lindsey A. Mair a, Malcolm Hunter a, Asipu Sivaprasadarao a,* b
a Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK Leeds Institute of Molecular Medicine, St. James’s University Hospital, University of Leeds, Leeds LS9 7TF, UK
Received 27 July 2006 Available online 4 August 2006
Abstract ATP-sensitive potassium (KATP) channels play a central role in glucose-stimulated insulin secretion (GSIS) by pancreatic b-cells. Activity of these channels is determined by their open probability (Po) and the number of channels present in a cell. Glucose is known to reduce Po, but whether it also affects the channel density is unknown. Using INS-1 model b-cell line, we show that the expression of KATP channel subunits, Kir6.2 and SUR1, is high at low glucose, but declines sharply when the ambient glucose concentration exceeds 5 mM. In response to glucose deprivation, channel synthesis increases rapidly by up-regulating translation of existing mRNAs. The effects of glucose deprivation could be mimicked by pharmacological activation of 5 0 -AMP-activated protein kinase with 5-aminoimidazole-4-carboxamide ribonucleotide and metformin. Pancreatic b-cells which have lost their ability for GSIS do not show such changes implicating a possible (patho-)physiological link between glucose-regulated KATP channel expression and the capacity for normal GSIS. 2006 Elsevier Inc. All rights reserved. Keywords: ATP-sensitive potassium channel; KATP channels; Insulin secretion; AMP-activated protein kinase; AMPK; Glucose sensing; Glucose-stimulated insulin secretion
Pancreatic b-cells possess the remarkable ability to promptly sense changes in blood glucose levels and to secrete the appropriate amount of insulin, a process termed glucose stimulated insulin secretion (GSIS) [1]. According to the accepted model of GSIS, raised blood glucose levels lead to an increased influx of glucose into the pancreatic b-cell which in turn leads to increased metabolism and a rise in the ratio of cytoplasmic [ATP] to [ADP]. The rise in [ATP]/[ADP] ratio causes the closure of ATP-sensitive potassium (KATP) channels, leading to membrane depolarisation, opening of depolarisation activated voltage-gated
q
Abbreviations: KATP channel, ATP-sensitive potassium channel; GSIS, glucose stimulated insulin secretion; AMPK, 5 0 -AMP-activated protein kinase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; SUR1, sulphonylurea receptor 1; ABC, ATP-binding cassette; PNDM, permanent neonatal diabetes mellitus. * Corresponding author. Fax: +44 0 113 3434228. E-mail address: [email protected] (A. Sivaprasadarao). 0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.07.170
calcium channels, and influx of Ca2+ into the cell [2]. The resultant rise in intracellular Ca2+ promotes the fusion of insulin granules with the plasma membrane and release of insulin into the blood stream [3]. The importance of KATP channels in normal GSIS is underscored by the discovery of numerous genetic mutations of both Kir6.2 and SUR1 which lead to inherited disease states characterised by disrupted insulin secretion [4,5]. The pancreatic KATP channel is octameric, comprising four copies each of the two subunits, Kir6.2 and the sulphonylurea receptor1 (SUR1) [6]. Kir6.2 is a member of the inwardly rectifying potassium channel family and forms the K+ conducting pore of the channel [7,8]. SUR1 is a member of the ATP-binding cassette (ABC) transporter family [9], which confers nucleotide sensitivity on the channel through its nucleotide-binding folds, NBF1 and NBF2. In addition to these, a weak but a functionally important inhibitory binding site for ATP is located within the Kir6.2 tetramer [8,10]. SUR1 also contains binding
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sites for therapeutic drugs such as the sulphonylureas and diazoxide [11]. Sulphonylureas inhibit the KATP channel and are used to stimulate insulin secretion in diabetic patients [5]; diazoxide activates the KATP channel and is used to suppress insulin secretion in patients suffering from hyperinsulinaemia [4]. The net activity of KATP channels in a cell is a function of the number of channels present at the cell surface (N) and their open probability (Po). Whilst there have been extensive studies on how the Po of KATP channels is controlled by the blood glucose levels, there is little information on the regulation of channel density by glucose. Moritz et al. [12] reported that prolonged (72 h) exposure of pancreatic b-cells to high glucose concentrations reduces the level of KATP channel mRNA transcripts by 70%. Under physiological situations, however, changes in blood glucose levels can occur more acutely, for example, after a meal, and thus it is important to investigate if glucose can have acute effects on channel density. The importance of testing such a possibility is underscored by the fact that genetic mutations in KATP channels may cause either congenital hyperinsulinism by decreasing cell surface channel density [13,14] or permanent neonatal diabetes mellitus (PNDM) by increasing cell surface channel density [15]. Here we demonstrate that glucose can exert an acute effect on the density of KATP channels in the glucose responsive model b-cell line INS-1. In response to a sudden decline in glucose concentration, expression of both the KATP channel subunits increased acutely; conversely when the cells were exposed to high glucose levels the density of the channels decreased. The effects of lowering the glucose concentration could be mimicked by application of the 5 0 AMP-activated protein kinase (AMPK) activators 5aminoimidazole-4-carboxamide ribonucleotide (AICAR), and metformin [16,17]. The effects of both glucose and AMPK activation on KATP channel synthesis are absent in non-pancreatic cells and in b-cells (Rin-m), which have lost their ability to GSIS. Taken together our data suggest that glucose-mediated changes in KATP expression may have an important role in the physiology of insulin secretion. Experimental Materials. Antibodies to Kir6.2 and SUR1 (custom-made by SigmaGenosys) were raised in rabbits against peptides EDPAEPRYRARQR RARFVSKK and EEAAESEEDDNLSSVLHQRAK, respectively [13]. Donkey anti-rabbit fluorescein isothiocyanate (FITC)-conjugated antibody was supplied by Jackson ImmunoResearch Laboratories, Inc. RPMI 1640 cell culture media and heat-inactivated fetal calf serum were both obtained from Gibco-BRL. Diazoxide, tolbutamide, 5-aminoimidazole-4carboxamide ribonucleotide (AICAR) and metformin were obtained from Sigma, UK. Cell culture. INS-1 cells (passage 102–120) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 10 mM Hepes, 2 mM glutamine, 1 mM sodium pyruvate, 50 lM bmercaptoethanol, 100 U ml1 penicillin, and 100 lg ml1 streptomycin, at 37 C in a humidified atmosphere of air/5% CO2. RIN-m cells (ATCC No. CRL-2057) were grown as for INS-1 cells, but in a medium lacking Hepes
and b-mercaptoethanol. HEK293 cells stably expressing mouse Kir6.2 and hamster SUR1 were cultured as described previously [13]. Immunocytochemistry. INS-1 were cells grown on poly-L-lysine-coated glass coverslips and treated as required (see figure legends). The cells were rinsed in phosphate-buffered saline (PBS) (mM: NaCl, 137; KCl, 2.6; Na2HPO4, 1.0; and KH2PO4, 1.76; pH 7.4), fixed in 2% PFA for 15 min at room temperature, and then permeabilised in 50:50 mixture of acetone and methanol (pre-chilled to 20 C) for 15 min at 4 C. Cells were then blocked in 5% goat serum in PBS for 1 h, before extensive washing in PBS, followed by incubation in primary antibody for 2 h at room temperature. Primary antibodies used were rabbit anti-SUR1 (1:300 dilution) and rabbit anti-Kir6.2 (1:300 dilution). All antibodies were diluted in 5% goat serum in PBS. Cells were then washed extensively in PBS before incubation with FITC-conjugated donkey anti-rabbit IgG secondary antibody (1:100 dilution) for an hour at room temperature. Following extensive washing with PBS, cells were mounted onto microscope slides with VECTASHIELD (Vector Laboratories, CA) and air sealed with nail varnish. Cells were viewed on a Leica-NT laser scanning confocal microscope equipped with a 488 nm laser excitation filter for FITC. Fluorescent images were visualised under an oil-immersion 63· objective using the appropriate laser. Quantification of fluorescence intensity was performed using ImageJ software. Briefly, each individual cell was outlined by hand and the mean pixel intensity of the selected area was calculated. The mean pixel intensity of each cell was then normalised to the mean value of appropriate control cells (stated in the figure legends). The normalised data were then expressed as mean values ± SEM. Statistical significance was examined using Student’s t test. Northern analysis. Isolation of total RNA and Northern analysis were performed as described previously [18]. A 663 bp long SUR1 cDNA probe was amplified from total RNA isolated from INS-1 cells by reverse transcription PCR using 5 0 -AGAAGACACGCAGGAAGGAA-3 0 and 5 0 -CTGGAAGGCTGTTCCAGAAG-3 0 as primers. Actin probe was prepared as described before [18]. The hybridised RNA species were identified by phosphorimaging. The intensity of the bands was quantified using the Quantity 1 software (Bio-Rad). The density of SUR1 mRNA was normalised to that of actin bands. Electrophysiological measurement of whole cell currents. Whole cell currents from INS-1 cells, grown overnight in RPMI containing either 3 mM or 25 mM glucose, were recorded using the patch–clamp technique [19]. The pipette solution for whole cell recordings comprised (mM): 140 KCl, 0.6 MgCl2, 1.03 CaCl2, 10 EGTA, 10 Hepes, 0.1 MgATP, and 0.1 ADP (adjusted to pH 7.2). The bath solution comprised (mM): 122.5 NaCl, 5.0 KCl, 1.0 MgCl2, 1.0 CaCl2, and 10.0 Hepes (pH 7.4). This bath solution was supplemented with either 25 mM D-glucose or 3 mM D-glucose + 22 mM D-sorbitol as required. Currents were measured during a voltage ramp (100 mV to +100 mV) from a holding potential of 0 mV and normalised to the membrane capacitance. Diazoxide (200 lM) was perfused to activate the channel and tolbutamide (100 lM) to block the activated currents. Insulin assays. INS-1 cells grown in a 24-well plate were incubated in 200 ll KRB–Hepes buffer supplemented with either 3 mM or 25 mM glucose for 1 h. Aliquots of the supernatant were used to assay for the insulin content by radioimmunoassay using a kit from ICN Biomedicals Ltd, UK, according to the instructions of the manufacturer. Protein content of the detergent (0.5% Triton X-100 and 0.1% SDS) extracts of cells from each well was estimated by the bicinchoninic acid method. The insulin secretion data were normalised to the protein content of the cell.
Results The density of KATP channels in INS-1 cells is dependent on the glucose concentration of the medium To investigate if glucose regulates the density of KATP channels, we have incubated the pancreatic b-cell line INS-1 in media supplemented with varying concentrations
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of glucose for 2 h. The cells were then immunolabelled with anti-Kir6.2 or anti-SUR1 antibodies and examined by confocal microscopy. The specificity of each antibody for its target antigen has previously been demonstrated [13]. Fig. 1A shows that the expression of both the KATP channel subunits is high at and below 5 mM glucose, but declines as the ambient glucose concentration is raised to 11 mM and beyond. A similar level of expression was observed for both subunits at all glucose concentrations, suggesting that the changes had occurred in a stoichiometric fashion allowing the formation of fully assembled channels. Measurement of the average pixel intensity of labelled cells revealed that fluorescence in cells incubated in 0.5 mM glucose was 2.5-fold higher compared to those cells incubated in 25 mM glucose (Fig 1B). Substitution of mannitol, a non-metabolisable glucose derivative, failed to show such an effect (Fig. 1C), indicating that the effect is not due to changes in the osmolarity of the medium. Reducing the glucose concentration causes an acute increase in the density of KATP channels We next asked whether the observed dependence of KATP channel density on glucose concentration is due to an
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increase in the synthesis of channel subunits when exposed to low glucose or due to an increase in channel degradation in high glucose. For this INS-1 cells were incubated in high (25 mM) or low (3 mM) glucose medium overnight to allow the channel density to reach steady-state before changing to media containing low (3 mM) and high (25 mM) glucose concentrations, respectively. At different time intervals the cells were fixed and stained for SUR1 to follow the resulting changes in channel density. The results show that the density of SUR1 increases acutely when the cells were shifted from a high to low glucose (Fig. 2A, top panels; Fig. 2B), with a noticeable increase within 15 min. This increase in fluorescence appeared uniformly throughout the cytoplasm. In contrast, the fluorescence decreases comparatively more slowly when the cells were switched from low to high glucose (Fig. 2A, bottom panels; Fig. 2C), the decrease being more prominent from the perinuclear region of the cell. Quantitative analysis of the fluorescence intensity revealed that significant increase (1.4-fold; p < 0.05) in channel expression occurs as early as 15 min following the shift from high to low glucose; after 10 h the fluorescence intensity was 2.7-fold greater compared to the zero time point (Fig. 2B). In contrast, it takes 2 h for a significant decrease in fluorescence to be observed following the shift from low to high glu-
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B
C
Fig. 1. The density of KATP channels in INS-1 cells decreases when the glucose concentration of the bathing medium exceeds the physiological range. (A) Effect of medium glucose concentration on the expression of KATP channel subunits. INS-1 cells were incubated for 2 h in RPMI medium supplemented with indicated concentrations of glucose. The cells were then fixed, permeabilised, and immunostained for Kir6.2 (upper panel) or SUR1 (lower panels) as described in Experimental. Representative images taken from one dataset of three using identical settings are shown. (B) The average pixel intensity of the fluorescence of cells incubated for 2 h in either 0.5 mM glucose (light grey) or 25 mM glucose (dark grey) was calculated. The data represent means ± SEM normalised to the mean of 0.5 mM glucose values. *p < 0.01 (Student’s two-tailed t test). n = number of individual cells examined for fluorescence intensity from three separate experiments. (C) The decline in the channel density is not due to increased osmolarity. INS-1 cells exposed to either 3 mM glucose or 3 mM glucose + 22 mM mannitol for 2 h were stained for SUR1. Representative images taken using identical settings are shown. Scale bars = 10 lm.
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A
B
C
Fig. 2. Density of KATP channels in INS-1 cells increases rapidly during glucose deprivation. (A) INS-1 cells were incubated overnight (16 h) in media supplemented with 25 mM glucose (top panel) or 3 mM glucose (bottom panel) before switching to 3 mM or 25 mM glucose, respectively. Following incubation for the indicated times in the new media, the cells were fixed and immunolabelled with anti-SUR1 antibodies. Representative confocal images taken using identical settings are shown. (B, C) The average pixel intensity of the fluorescence of cells switched from 25 mM to 3 mM glucose (B) or 3 mM to 25 mM glucose (C) were calculated. The data represent means ± SEM normalized to the mean of 0 min values. *p < 0.01 (Student’s two-tailed t test). Scale bars = 10 lm.
cose with an eventual decrease of 60% after 10 h (Fig. 2C). By this time the intensity of the cells was similar to that of cells incubated in the continuous presence of high glucose. A similar pattern was observed for the Kir6.2 subunit (data not shown). Stimulation of KATP channel expression during glucose deficiency occurs via increased translation Various pharmacological agents were used to investigate whether the increase in protein synthesis was due to an increase in the rate of transcription or translation. Fig. 3A shows that inhibitors of transcription, a-amanitin and actinomycin D, both failed to prevent the increase in the expression of SUR1 during a shift to lower glucose concentrations. By contrast, cycloheximide, an inhibitor of translation, prevented the increase in the expression of SUR1. These data suggest that glucose deficiency has little effect on the transcription of the SUR1 (ABCC8) gene but appears to increase the translation of SUR1 mRNA. We performed Northern analysis on RNA isolated from cells exposed to 3 mM glucose and 25 mM glucose for 2 h (Figs. 3B and C) and the data (Fig. 3C) show no significant difference (p > 0.05) in the SUR1 mRNA content (expressed as means ± SEM of SUR1/actin ratio) of cells treated with 3 mM glucose (3.00 ± 0.27) from that of 25 mM glucose
(3.14 ± 0.4). The lack of glucose-dependent changes in mRNA levels during short-term exposure is consistent with other reports [20,21]. Electrophysiological measurements reveal no significant differences in the cell surface channel density As mentioned above the increased fluorescence appeared uniformly throughout the cytoplasm. As a result it is not clear if the increase in channel synthesis is accompanied by a corresponding increase in the channel density at the cell surface. Both antibodies are directed to cytoplasmic domains of their respective subunits making them unsuitable for selective labelling of cell surface channels. For this reason, we have determined the cell surface channel density by measuring whole cell currents by the patch– clamp technique. KATP channel currents were maximally activated by application of diazoxide and inhibited by application of tolbutamide (Figs. 4A and B). The diazoxide-stimulated, tolbutamide-sensitive currents were normalised to the capacitance of the cell (which gives a measure of cell surface area) to obtain a measure of the channel density at the cell surface. Fig. 4C shows that the density of functional channels in cells incubated in 3 mM glucose (636 ± 125 pA/pF; n = 7) is approximately 30% higher than that of cells in 25 mM glucose (428 ± 70
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A
B
C
Fig. 3. The induction of KATP channel synthesis during glucose deprivation is due to an increase in translation. (A) Inhibitors of translation, but not transcription, prevent the induction of KATPchannel synthesis. INS-1 cells were pre-incubated for 4 h in 25 mM glucose (in RPMI) supplemented with either a-amanitin (5 lg/ml), actinomycin-D (Act-D, 1 lg/ml) or cycloheximide (CHX, 25 lg/ml) followed by a further 2 h incubation in medium containing 3 mM glucose, supplemented with the relevant pharmacological agent. Cells were then fixed, permeabilised and immunostained for SUR1. For comparison, cells incubated in 25 mM glucose alone were also stained in a parallel experiment. Representative confocal images taken using identical settings are shown. (B, C) Lack of effect of glucose concentration on SUR1 mRNA. Northern blots of total RNA isolated from INS-1 cells incubated in 3 mM (lanes 1 and 2) or 25 mM (lanes 3 and 4) glucose, showing the SUR1 and actin bands; data generated using RNA isolated from two separate flasks of cells are shown for each glucose concentration. (C) Means ± SEM (n = 4) of SUR1 mRNA, normalised to actin mRNA band intensity; p > 0.05 (Student’s two-tailed t-test). Scale bars = 10 lm.
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C
B
Fig. 4. The density of KATP channels at the cell surface does not appear to be affected by glucose concentration. (A, B) KATP channel currents measured from INS-1 cells cultured in 3 mM glucose. Currents were measured in the whole cell patch–clamp configuration. Data points represent currents measured at 0 mV during perfusion of bath solution alone or bath solution supplemented with diazoxide (D) or tolbutamide (T), as indicated by horizontal bars. (B) Current–voltage relationships measured during the application of diazoxide or tolbutamide, corresponding to time points (gray filled circles) labelled 1 and 2 in (A). (C) Histogram of means ± SEM of currents from cells cultured overnight in 3 and 25 mM glucose as indicated.
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pA/pF; n = 8); however, the difference is not significant (p = 0.16).
Glucose mediated changes in the KATP channel density are dependent on the ability of the cells for GSIS
The increase in KATP channel expression in low glucose may involve the activation of AMPK
We next asked if the glucose-regulation of channel expression is specific to pancreatic b-cells. We tested the effect of glucose concentration on HEK cells heterologously expressing Kir6.2 and SUR1, and found that glucose has no effect on channel density (Fig. 6A). We then investigated whether glucose-regulated channel expression is dependent on the ability of the pancreatic b-cell for GSIS. For this we utilised Rin-m cells, another model pancreatic b-cell line which have lost their ability for GSIS and tested them in parallel with INS-1 cells. Whilst INS-1 cells exposed to 25 mM glucose secrete 10-fold more insulin (p < 0.01; Student’s t test) than cells incubated in 3 mM glucose, the late passage Rin-m cells failed to elicit GSIS (Fig. 6B, C). When examined for glucose effects on KATP channel density, INS-1 cells showed increased channel density in low glucose relative to high glucose as has been shown in Fig. 1, but Rin-m cells did not show such differences (Fig. 6A). These data suggest that the effect of glucose on the KATP channel density is not only b-cell specific but more importantly, depends on the ability of the cells for normal GSIS.
One consequence of glucose starvation is decreased metabolism and an increase in the intracellular levels of ADP and AMP potentially leading to the activation of AMPK [22,23]. Indeed, it has been shown that in pancreatic b-cells the activity of AMPK increases with the decrease in ambient glucose concentration [24,25]. To address a possible role for AMPK activation in mediating glucose-regulated KATP channel expression, we tested the ability of two activators of AMPK, AICAR [16] and metformin [17,26], to mediate changes in channel expression. Both compounds activate AMPK regardless of ambient glucose concentration and would thus mimic the effects of glucose deprivation leading to increased KATP channel expression even at high ambient glucose concentrations. Both AICAR and metformin enhanced the expression of KATP channels in cells incubated in high (25 mM) glucose to the same level as in cells subjected to glucose deprivation (3 mM) (Fig. 5). By contrast, AICAR and metformin caused no apparent increase in channel expression in cells incubated in 3 mM glucose, which is consistent with the expectation that in low glucose AMPK would be already fully activated. Thus glucose starvation appears to induce expression of KATP channels via activation of AMPK.
Discussion We have investigated the effect of ambient glucose concentration on the density of KATP channels in the rat pancreatic INS-1 b-cell line. We found that in response to a decrease in ambient glucose concentration, synthesis
Fig. 5. Pharmacological activation of AMPK mimics the effect of low glucose on the expression of KATP channels incubated in high glucose. INS-1 cells were incubated for 2 h in media containing either 3 mM or 25 mM glucose supplemented with AICAR (0.5 mM) or metformin (0.5 mM) as required. The cells were then fixed and immunolabelled using anti-SUR1 antibodies. Representative confocal images taken from one dataset of three using identical settings are shown. Scale bars = 10 lm.
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Fig. 6. Glucose mediated regulation of the density of KATP channels is dependent on the ability of b-cells for GSIS. (A) Cell specific effect of glucose. HEK cells stably co-transfected with SUR1 and Kir6.2, Rin-m cells and INS-1 cells were incubated in low (0.5 mM) and high (25 mM) glucose for 2 h before immunostaining for SUR1. Representative confocal images taken from one dataset of three are shown. The inset panel for high glucose INS-1 cells (bottom right panel) shows the same field of view as the main picture but with increased brightness and contrast to show that cells are present in the field of view. (B, C) Glucose induced insulin secretion from INS-1 (B) and Rin-m (C) cells. Cells were incubated in 3 mM or 25 mM glucose for 2 h and the amount of insulin secreted into the medium was assayed. The data represent means ± SEM of four independent experiments. Scale bars = 10 lm.
of KATP channels is acutely stimulated and that this increase in channel synthesis occurs via increased translation. The effect of glucose deprivation can be mimicked by the pharmacological stimulation of AMPK by AICAR and metformin. More importantly, the glucose-mediated change in channel expression is dependent on the ability of b-cells for GSIS, indicating that glucose induced changes in channel density have potential (patho-)physiological significance. Immunolabelling experiments revealed that the density of KATP channel subunits, Kir6.2 and SUR1, is inversely dependent on ambient glucose concentration (Fig. 1). These changes appear to result from a stimulation of channel expression in low glucose and suppression in high glucose media. The induction of channel expression following the sudden switch from a high to low glucose medium was quite rapid with a significant (1.4-fold; p < 0.05) increase occurring within only 15 min (Fig. 2). This increase was due to increased translation of pre-existing mRNA and not due to an increase in gene transcription (Fig. 3). In this regard it resembles proinsulin whose expression is stimulated by glucose [27] or the iron homeostatic proteins transferrin and ferritin whose expression is determined by iron availability [28,29]. Since translation alone can produce a much faster response compared to transcription-coupled translation, the finding that glucose-mediated changes in
channel expression is regulated at the translational level seems consistent with the speed with which the b-cells responded to glucose depravation (Fig. 2). Several studies have investigated the effect of either acute or chronic exposure to elevated glucose levels on KATP channel mRNA expression [12,20,21]. It appears that short-term exposure to high glucose (<24 h) has no measurable effect on the level of either Kir6.2 or SUR1 mRNA [20,21]. By contrast, more prolonged exposure to high glucose has been shown to significantly reduce mRNA levels in both INS1 cells [12] and in b-cells isolated from mice with induced diabetes [30]. Taken together with the published data, it seems that the channel density is regulated at the translational level to produce a rapid response and at the transcriptional level to maintain a sustained response. In contrast to the marked differences in the total density of the channels between low and high glucose treated cells, patch–clamp-data (Fig. 4) showed only a small (30%) difference in the density of functional channels at the plasma membrane. This lack of correlation between functional data and the immunofluorescence data suggests that there may be other mechanisms that control the number of functional channels at the cell surface. Emerging evidence suggests that the cell surface density of KATP channels is subjected to downregulation by endocytic mechanisms [15,31]. Consistent with this idea in cells exposed to high
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glucose the fluorescence appears to be more prominent at the periphery of cells in the form of puncta (Figs. 1–3) which may represent endocytic/exocytic vesicles. We have demonstrated that the effect of glucose deprivation could be mimicked by the pharmacological activators of AMPK, AICAR, and metformin (Fig. 5). These two compounds activate AMPK by different mechanisms [16,17,26], yet produce the same stimulatory effect on channel expression. Since both compounds are able to maximally stimulate KATP channel expression it is likely that the response is mediated via AMPK activation and not simply because of another drug-mediated non-specific mechanism. The finding that AMPK activation mimics the effect of glucose deprivation on KATP channel synthesis is interesting because previous studies have established a clear relationship between the activity of AMPK in pancreatic b-cells and ambient glucose concentration [24,25] as well as between AMPK activity and insulin secretion [24,32]. With the decrease in glucose concentration, the activity of AMPK increases and insulin secretion is suppressed [24,32]. Although these studies suggest that glucose deprivation inhibits insulin secretion by activating AMPK, the mechanism by which AMPK activation suppresses insulin secretion remained unclear. Our present data suggest that the suppression of insulin secretion by AMPK may be mediated in part by the upregulation of KATP channel expression by AMPK; greater channel numbers might be expected to have an increased capacity for maintaining a sustained membrane hyperpolarisation thus more effectively suppressing insulin secretion. Another important finding is that cells which have lost the ability for GSIS have also lost their ability to induce KATP channel expression during glucose starvation. Salt et al. [24] reported that the late passage b-cell lines which have lost their ability for GSIS also fail to show glucosemediated changes in AMPK activity. Taken together these findings suggest that glucose-mediated regulation of KATP channel expression (via AMPK) has an essential role in GSIS. Since loss of GSIS is the hallmark of type2 diabetes, loss of glucose mediated changes in AMPK activity, and hence of KATP channel expression, may be another contributing factor in type-2 diabetes mellitus. In this context, it is also interesting to note that metformin has been shown to restore a normal secretory pattern in rat pancreatic islets whose function has been impaired by chronic exposure to high glucose concentrations [33,34]; this useful property may be related to the ability of this anti-diabetic drug to induce KATP channel expression via AMPK activation. Acknowledgments The work was supported by the Medical Research Council, UK. A.S. is supported by Emma and Leslie Reid Scholarship. We thank Dr. C.B. Wollheim and Dr. P. Maechler for kindly providing INS-1 cells.
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