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Using a polymeric sulfonylurea (PSU) designed from glibenclamide, we examined the interactions of sulfonylurea with pancreatic islets rather than gene...
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Biomacromolecules 2003, 4, 1550-1557

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Visual Evidence and Quantification of Interaction of Polymeric Sulfonylurea with Pancreatic Islet Sungwon Kim†,‡,§ and You Han Bae*,† Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 421 Wakara Way, Suite 318, Salt Lake City, Utah 84108, and Center for Biomaterials and Bioengineering, Department of Materials Science and Engineering, Kwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea Received October 28, 2002; Revised Manuscript Received May 22, 2003

Using a polymeric sulfonylurea (PSU) designed from glibenclamide, we examined the interactions of sulfonylurea with pancreatic islets rather than genetically remodeled β-cell lines to clarify the biological roles of ATP-sensitive K+ (KATP) channels to which sulfonylurea binds. PSU enhanced insulin secretion from the islets with 10 nM (SU equivalent) treatment, especially at low glucose concentration, but its activity was inhibited by 100 µM diazoxide. Confocal microscopy visualized PSU interactions with the islet and revealed that the modulation of intracellular Ca2+ occurred in the same region of an islet where PSU was also bound. In quantification method of the confocal microscopic images, competition of PSU with glibenclamide on its binding sites and glucose inhibition against PSU binding were confirmed. In this study, it was concluded that the PSU was a comparable drug with glibenclamide and offered a new standard method to study intact islets. Introduction Pancreatic islets consist of R-, β-, δ-, and pancreatic polypeptide cells. Among these, β-cells occupy the largest volume portion of islets and tightly regulate the homeostasis of blood glucose by secreting insulin.1,2 However, the blood glucose level is not solely controlled by β-cells but is tempered by cross talking and antagonizing between different islet cells or by neuronal stimulation.3 Therefore, it seems essential to investigate the physiology of and drug action mechanisms (pharmacology) on islets as a whole. Nevertheless, numerous studies have been carried out using either cancer cell lines genetically remodeled from pancreatic β-cells4 or isolated single β-cells. This may stem from the fact that an islet is too intricate to colligate and schematize cellular events in a plain manner. Because there is no appropriate means available to delve into biological aspects of an islet in a molecular level, most experiments with whole islets have also limitation in only presenting consequential phenomena generated from the inputted conditions without any profound insight. One of the important drugs regulating biological functions of pancreatic islets is the sulfonylurea, which has been used for the treatment of type 2 diabetes mellitus because of its insulinotropic activity on pancreatic islets.5 In pancreatic β-cells, the drug binds to sulfonylurea receptor (SUR1), which forms an ATP-sensitive K+ (KATP) channel, and blocking KATP channels by sulfonylureas is influenced by * To whom correspondence should be addressed. Phone: (801) 5851518. Fax: (801) 585-3614. E-mail: [email protected]. † University of Utah. ‡ Kwangju Institute of Science and Technology. § E-mail: [email protected].

ATP, ADP, and divalent cations.6 This pharmacology of sulfonylureas has been investigated mostly by electrophysiological approaches. However, it is difficult to investigate the action mechanism of sulfonylureas on a whole islet by an electrophysiological method that inevitably requires a new method. In our previous studies, the insulinotropic activity of polymeric sulfonylureas (PSU) on isolated rat islets or MIN6 cells was examined.7-9 After firmly determining whether the PSU is a comparable drug to glibenclamide, visual evidences for the interaction of PSU with islets were provided by confocal microscopy using a fluorescent probe labeled PSU. In addition, a quantification method of confocal microscopic images was evaluated and used to standardize binding competition between PSU and glibenclamide, as well as glucose effect on PSU binding to islets. Materials and Methods Materials. Terephthalic acid, N-vinyl-2-pyrrolidinone, and poly(ethylene glycol) (PEG; molecular weight 1000 Da) were purchased from ACROS Organics (Geel, Belgium), and 4-(dimethylamino)pyridine (DMAP), N,N′-dicyclohexylcarbodiimide (DCC), Ficoll, collagenase type V, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), RPMI 1640, glibenclamide, diazoxide, rhodamine B isothiocyanate (RITC), and 37% formaldehyde were from SIGMA Chemicals Company (St. Louis, MO). 4-(2-Aminoethyl)benzene sulfonamide, acrylic acid, cyclohexylisocyanate, and Nhydroxysuccinimide (NHS) were obtained from Aldrich Chemical (St. Louis, MO). Antibiotics (streptomycin/penicillin), Hank’s balance salt solution (HBSS), phosphate buffer

10.1021/bm025713j CCC: $25.00 © 2003 American Chemical Society Published on Web 08/22/2003

Sulfonylurea-Islet Interactions

saline (PBS), and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT). Preparation of Polymeric Sulfonylurea (PSU). A carboxylated glibenclamide derivative (SU) and a PSU with a 1 KDa PEG tether were obtained by the method reported in the previous papers.7-9 Briefly, SU was synthesized by the reaction of 10 mmol of cyclohexylisocyanate with 9.3 mmol of SU precursor that was prepared from the reaction of 100 mmol of terephthalic acid and 100 mmol of 4-(2-aminoethyl)benzene sulfonamide catalyzed by 100 mmol of NHS and 130 mmol of DCC. Final product was purified by recrystallization in methanol. The carboxyl group of SU was conjugated to the end of PEG that was grafted to wellcharacterized poly(N-vinyl-2-pyrrolidone-co-acrylic acid). RITC-labeled PSU was synthesized and characterized as described elsewhere.9 Insulin Secretion Experiment. Islet isolation was carried out by the conventional collagenase digestion method and by Ficoll gradient method with minimal modification.10 Islets were isolated from pancreata of male Sprague-Dawley (SD) rats (200-250 g) and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% penicillin/ streptomycin, 5 mM sodium bicarbonate, 20 mM HEPES, and 11.2 mM D-glucose under humidified air containing 5% CO2 at 37 °C. After being cultured for 3-5 days, spherical islets pooled from five to six SD rat pancreata were handpicked for further experiments. Before the insulinotropic activity of PSU was tested, 20 islets in each well of 24-well culture plates were starved with 1 mL of HEPES-buffered Krebs solution (HK solution; 130 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.4) containing 2.8 mM glucose. After 1 h, the solution was replaced by 1 mL of fresh HK solution containing 2.8 or 11.1 mM glucose. At the same time, 5 µL of 2 µM glibenclamide or PSU solution (to make 10 nM SU equivalent in the solution) was added into all of the wells to stimulate islets. In a separate experimental setting, to investigate the antagonistic effect of the diazoxide against PSU, 5 µL of 20 mM diazoxide solution (final concentration of 100 µM) was added 20 min prior to applying PSU or glibenclamide. The amount of insulin released for 1 h incubation with five to six independent experiments was determined by radioimmunoassay (RIA) with 125I-labeled rat insulin kits (Rat Insulin RIA Kit from LINCO research, Inc., St. Charles, MO). Assay was conducted in duplicate. Confocal Microscopy Study. Competing Glibenclamide against PSU for Binding to Islets. After being incubated in 2.8 mM glucose HK solution for 1 h, islets were transferred to microcentrifuge tubes containing 1 mL of fresh HK solution with 2.8 mM glucose. At the same time, 5 µL of RITC-labeled PSU (2 µM SU equivalent; 10 nM final concentration in the solution) and 5 µL of glibenclamide solution (20, 2, or 0.2 µM; final concentrations of 100, 10, or 1 nM) were added to each tube. After 1 h at 37 °C without gas-phase supplement, the islets were collected and rinsed with PBS twice to remove unbound labeled PSU. The islets then were fixed with 4% formaldehyde solution in PBS. The confocal microscopic images of the islets were obtained with

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Leica confocal laser microscope system (Leica TCS SP2 RS with Leica Confocal Software, Leica Microsystems, Wetzlar, Germany) at a magnification of ×100. Each image presented a representative islet among 150 islets from three independent experiments. Glucose-Dependent Binding of PSU. After starvation with 2.8 mM glucose HK solution, islets were incubated in 1 mL of fresh HK solutions with different glucose concentrations (2.8, 5.6, and 11.2 mM). Simultaneously, 5 µL of RITClabeled PSU (2 µM of SU concentration; final concentration of 10 nM in the solution) was added into each tube. Further experimental procedure was the same as described before. Calcium Imaging. Hand-picked islets (n ) 30) were incubated with 2 µM Fluo-3 AM (Molecular Probes, Inc., Eugene, OR) in HK solution for 1 h at 37 °C without gasphase supplement. After being washed with PBS twice, islets were subsequently incubated in 1 mL of 5.6 mM glucose HK solution and stimulated by 5 µL of 2 µM RITC-labeled PSU (10 nM final concentration) for 15 min at 37 °C. After the islets were quickly rinsed with PBS, they were fixed with 4% formaldehyde solution for a few minutes. Confocal microscopic images (×200) were obtained by continuously switching fluorescence filters for the labeled PSU (RITC; 570 nm excitation and 595 nm emission) and for the Fluo-3 AM (FITC; 490 nm excitation and 520 nm emission). Images for calcium mobilization and labeled PSU bound to the islet were displayed as a result of synchronous detection of RITC and FITC for the same islet. Quantification of Confocal Microscopic Images. For islets from the glibenclamide competition test against PSU and the glucose-dependent binding study, successive zsectional images were acquired by scanning each islet (scan rate was 65 536 pixels per second, 2 µm interval, ×200 magnitude) at the same laser power (725 V) (Figure 1A). Randomly selected islets (20-30) out of 150 islets of three independent experiments were analyzed in this process. Because the fluorescence intensity from the competition study using 10 nM PSU was too low to quantify the confocal microscopic images, all concentrations of RITC-labeled PSU and glibenclamide were doubled (20 nM labeled PSU and 2, 20, and 200 nM glibenclamide) without changing other experimental conditions. Glucose binding experiment was conducted with 10 nM labeled PSU. The fluorescence intensities in the circular area of each section from one islet (Figure 1B) were analyzed and quantified using NIH Image J software (version 1.27), which lead to a fluorescence intensity profile (x-axis) versus the number of pixels (y-axis) that corresponded to each intensity (Figure 1C). By the same method, the fluorescence intensities for each section were analyzed and plotted by a profile through the z-axis of an islet (Figure 1D). The averaged fluorescence intensity (the fluorescence intensity of islet per unit volume, FIU) was obtained from (area under the curve in Figure 1D)/(islet volume). Finally, FIUs were plotted versus islet diameter (ID; measured from an image that had the broadest area among z-sections of an islet). This process was calculated from the parameters defined as follows: In, the fluorescence intensity at the nth section; IT, the total fluorescence intensity of an islet (IT ) ∑n1In); r, the radius of an islet assuming a

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Figure 1. Quantification method using confocal microscopic images acquired from successive z-section. In panel A, an islet that has a hypothetical spherical shape (r ) radius) is illustrated in a diagram. Confocal sectioning was performed for every 2 µm throughout the islet at a rate of 65 536 pixels per second. In panel B, a representative confocal image from the nth section is displayed. RITC-labeled PSU in the islet emits red color in the figure. In panel C, the fluorescence intensity of the area closed by a circle (yellow circle in Figure 1B) was analyzed by NIH Image J software, which offered a plot showing the number of pixels (count) as a function of fluorescence intensity. For further calculation, the fluorescence intensity was summed up. In panel D, the summed fluorescence intensity for n sections is plotted along z-axis of the islet. Integrated value of this curve (area under the curve) was used as the total fluorescence intensity for an islet.

spherical shape; FIU, the fluorescence intensity of islet per unit volume (µm3), which is derived by FIU ) IT/V; V, the volume of an islet (V ) 4/3πr3). Data Analysis. Data from RIA experiments presented as mean ( standard error of the mean (SEM) were analyzed by Student’s t-test for unpaired data with P < 0.05 significance. Confocal microscopic images were illustrated as 12-step pseudocolor images to visualize the difference of fluorescence intensities. Quantitative data acquired from the confocal microscopic images were analyzed by a simple nonlinear regression model, y ) y0 + ax + bx2, where y represents FIU and x represents the ID. For the verification of this regression model, the normality and continuity of data was tested by Kolmogorov-Smirnov test (5% significance level), followed by identifying the correlation and the homogeneity of variance for residuals using Durbin-Watson statistics (5% significance level) and one-way analysis of variation (ANOVA, P < 0.01 considered significant), respectively. Results Characterization of SU and PSU. The characterization results of SU and PSU coincided with those reported in our previous study.9 Nonaqueous titration revealed that SU content in PSU was 0.256 mmol SU/gram PSU.

Kim and Bae

Figure 2. The insulinotropic activity of PSU to isolated rat islets (n ) 20) was compared with that of glibenclamide (Glib) in HK solution containing low (2.8 mM, A) and high (11.1 mM, B) glucose concentration. In panel A, at 2.8 mM glucose concentration, both glibenclamide (10 nM) and PSU (10 nM) enhanced the insulin secretion. This response was completely abolished by diazoxide treatment (100 µM). In panel B, neither glibenclamide nor PSU significantly increased the insulin secretion from islets at high glucose concentration compared with the control level, although the insulin secretion remarkably increased compared with that at low glucose. However, diazoxide eliminated the insulinotropic activity for all groups. Data from five to six different batches were expressed by mean ( SEM analyzed by unpaired t-test; / and // denote P < 0.05 and P < 0.1, respectively.

PSU Mimics Glibenclamide in Insulin Secretion. Although the insulinotropic activity of PSU was preliminarily reported with an insulinoma cell line, MIN6, we tested whether PSU could impersonate glibenclamide against rat islets. Figure 2 presents the effect of PSU on insulin secretion from isolated rat islets. The amount of secreted insulin after treatment with PSU (10 nM SU equivalent) showed no significant difference from that after treatment with glibenclamide (10 nM) at both low (2.8 mM, Figure 2A) and high (11.1 mM, Figure 2B) glucose concentrations with 95% confidence level (/, P < 0.05). However, there existed statistical significance at low glucose with 90% level (n ) 5-6, //, P < 0.1). While neither insulinotropic agent gave any significant difference in insulin secretion at 11.1 mM glucose against control, they dragged up the insulin secretion over the control level at 2.8 mM glucose concentration. From the control level (1.42 ( 0.50 ng/mL/islet/h), glibenclamide increased the insulin secretion to 2.41 ( 0.47 (170%) and PSU to 3.80 ( 0.41 (268%) ng/mL/islet/h.

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Figure 3. Representative confocal microscopic images displaying the PSU-induced calcium mobilization in a pancreatic islet (×200): (A) the position of RITC-labeled PSU in an islet; (B) Ca2+ imaging obtained with Fluo-3 AM from the same islet; (C) overlay image of images A and B. In this experiment, labeled PSU was incubated with the islets for 15 min in HK solution (5.6 mM glucose) after 1 h of incubation with the Fluo-3 AM. These figures illustrate that red and green spots are mostly in periphery and that a gap exists between the position of the RITC-labeled PSU and Ca2+ mobilization. Each image is a representative one from 30 independent specimens. Panels D and E contain red- and greenfiltered images, respectively, of an islet without PSU treatment; panel F is the overlaid image.

The effect of diazoxide, a potassium channel opener, on insulin secretion is also presented in Figure 2. The insulin secretion, as expected, decreased regardless of the presence of insulinotropic agents at high glucose (/, P < 0.05, Figure 2B). While the diazoxide treatment did not show any significant variation in insulin secretion of either control or islets treated by glibenclamide at 2.8 mM glucose concentration, PSU significantly lost its activity to 2.08 ( 0.47 ng insulin/mL/islet/h at low glucose concentration. PSU Mobilizes the Intracellular Ca2+ Concentration ([Ca2+]i) of Isolated Islets. If PSU stimulates β-cells as glibenclamide does, [Ca2+]i of islet cells must increase after exposure to PSU. Figure 3 illustrates the localized RITClabeled PSU image (A, red), the mobilized calcium ions detected by hydrolysis of Fluo-3 AM (B, green), and the overlaid image (C). Figure 3D,E presents red- and greenfiltered images of an islet without treatment of PSU, respectively (Figure 3F, overlaid image). This observation in an islet describes the following: (1) PSU diffused inwardly from the periphery of the islet (Figure 3A). Because the islet was treated with PSU only for 15 min, the core of the islet did not emit fluorescence. (2) Evidently, PSU bound to somewhere so tightly that it, even in peripheral of the islet, was not subject to being washed out by rinsing twice with PBS. (3) Intracellular calcium ion was mobilized, and the majority of green light localized at periphery (Figure 3B). (4) Red and green fluorescence were not completely overlapped (Figure 3C). Collectively, FITC emission covered the region where the red color was strongly brightened, although the RITC-labeled PSU molecules diffused deeper into the islet.

Glibenclamide Competes against PSU for Binding to Islet Cells. The final attempt to match up the pharmacological property of PSU to that of a glibenclamide was conducted by competition study between both. Suppose that PSU works with a practically identical mechanism for insulin secretion to signal pathway of glibenclamide; PSU will vie with glibenclamide for binding to one site, expectedly the KATP channel. Figure 4 displays confocal microscopic images of each islet treated by 10 nM PSU, together with 1 nM (A), 10 nM (B), and 100 nM glibenclamide (C). All images are pointed up by 12-step pseudocolor transformed using Leica Confocal Software. As shown in Figure 4A, the white color presents the brightest spot, while the purple means almost basal level. It was evident that, when the higher glibenclamide concentration was applied, less fluorescence of RITClabeled PSU emitted. Especially, 100 nM glibenclamide so remarkably excluded 10 nM PSU that the islet was hardly distinguished from the basal noise. This result intuitively suggests that PSU properly competes with glibenclamide. For clearer explanation, we conducted a quantification method of confocal microscopic fluorescence images as described in the Materials and Methods section. Figure 5 presents the binding property of PSU influenced by glibenclamide concentration (PSU/glibenclamide ) 10:1 (A) or 1:1 (B)). For 200 nM glibenclamide treatment (20 nM PSU), the quantitative analysis was not possible because the confocal microscopic images were too dim. Within given significant levels, all statistical analyses informed that a nonlinear regression model (y ) y0 + ax + bx2) can fairly explain the data with the probability over 90%. The regression equations were y ) (- 2325.91x + 20.95x2 -

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Figure 4. Confocal microscopic images representing the competition between RITC-labeled PSU and glibenclamide (×100). Each islet was incubated in HK solution containing 2.8 mM glucose for 1 h with labeled PSU and unlabeled glibenclamide followed by fixation using 4% formaldehyde in PBS. The applied concentrations were varied by RITC-labeled PSU 10 nM: glibenclamide 1 nM (A), 10 nM (B), and 100 nM (C). As shown, as higher concentration of glibenclamide was applied, less fluorescence of PSU was emitted. Each image that was transformed into 12-step pseudocolor images displays a representative islet among 150 islets from three independent experiments.

0.9735, n ) 30) for 2.8 mM glucose (Figure 7A), y ) (77.89x + 7.42x2 + 204.27) × 104 (R2 ) 0.9276, n ) 20) for 5.6 mM glucose (Figure 7B), and y ) (-1488.91x + 6.41x2 86 492.22) × 104 (R2 ) 0.8802, n ) 19) for 11.1 mM glucose (Figure 7C). This analysis apparently indicates that the binding of labeled PSU is inhibited by increased glucose concentration. Discussion

Figure 5. Quantitative comparison of confocal microscopic images obtained from the competition study. In this experiment, the concentration of glibenclamide and RITC-labeled PSU was doubled with fixed labeled PSU concentration of (b) 20 nM labeled PSU/2 nM glibenclamide (y ) (-2325.91x + 20.95x2 - 64 556.77) × 104, R2 ) 0.9391, n ) 29) and (0) 20 nM labeled PSU/20 nM glibenclamide (y ) (-505.11x + 5.21x2 - 12 242.62) × 104, R2 ) 0.9209, n ) 29) because the confocal microscopic images acquired in Figure 4 were too dim to be quantified. It was not possible to analyze islets from 200 nM glibenclamide treatment because of too low intensity. At a given islet diameter (ID), it presented that the fluorescence intensity per unit islet volume (FIU) decreased by treatment of more glibenclamide. Images for this experiment were randomly chosen from 150 islets of three different batches.

64 556.77) × 104 (R2 ) 0.9391, n ) 29) for 2 nM glibenclamide treatment (Figure 5A) and y ) (-505.11x + 5.21x2 - 12 242.62) × 104 (R2 ) 0.9209, n ) 29) for 20 nM glibenclamide (Figure 5B), respectively. This result implies that raising the glibenclamide concentration lowered the amount of PSU bound at a given islet size and that FIU increased in proportion to the increment of islet size. High Glucose Concentration Interferes with PSU (Sulfonylurea) Binding to Islets. By the same quantification method, we last endeavored to find out a relationship between glucose concentration and PSU binding. Figure 6 exhibits the representative images of each islet treated with 10 nM PSU at 2.8 mM (A), 5.6 mM (B), and 11.1 mM glucose (C). It is clearly noted that the decline of bound fluorescence, that is, the amount of PSU, relied on increasing glucose concentration regardless of islet size. This glucose-dependent binding property was quantified with the same method as described above and plotted in Figure 7. Acquired equations were y ) (-1779.49x + 19.37x2 - 40 871.14) × 104 (R2 )

Although the action mechanism of sulfonylureas for triggering insulin secretion has been intensively studied, limitation still exists because of the use of insulinoma model cell lines or primary single cells enzymatically digested from intact islets. To address this issue and to establish a new methodology that allows investigating the binding behavior of sulfonylureas with intact islets, we utilized a polymeric sulfonylurea (PSU). In this study, it was aimed (1) to compare the bioactivity and the mechanism of action between PSU and glibenclamide and (2) to evaluate a new methodology for identifying the action mechanism of glibenclamide using the PSU. One important finding in this study is that bioactivity and signaling mechanism of PSU were similar to those of glibenclamide. In pancreatic β-cells, glibenclamide binds to sulfonylurea receptors (SUR1s) that form ATP-sensitive potassium (KATP) channels with inwardly rectifying potassium channels (Kir6.2s).11 Binding of sulfonylurea to the receptors induces closure of KATP channel, resulting in depolarization of the cells. This electrical signal opens voltage-gated Ca2+ channels (VGCC), and in turn, the increased intracellular Ca2+ concentration ([Ca2+]i) triggers the insulin secretion.12 Although the bioactivity of PSU is possibly higher than that of glibenclamide because of a multivalent effect of the polymer, at the concentration of 10 nM PSU (SU equivalent), which is a submaximal concentration of glibenclamide,13 insulin secretion level (Figure 2) and Ca2+ mobilization image (Figure 3) suggest that the insulinotropic activity of PSU follows the same sequence as glibenclamide induces. Moreover, the competition study confirmed that PSU, as evidenced by the interference of glibenclamide, bound to KATP channel (Figures 4 and 5). Because there was a report on the uptake of tritiated

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Figure 6. Confocal microscopic images for attenuated binding affinity of the labeled PSU to isolated islets by glucose (×100). RITC-labeled PSU (10 nM) was treated for 1 h in HK solution containing different glucose concentrations: (A) 2.8 mM; (B) 5.6 mM; (C) 11.1 mM. The binding of labeled PSU was inhibited by high glucose concentration regardless of islet size. The representative images among 150 islets from independent three experiments are exhibited.

Figure 7. Quantitative comparison of confocal microscopic images from the RITC-labeled PSU (10 nM) binding depending on different glucose concentrations: (A) 2.8 mM; (B) 5.6 mM; (C) 11.1 mM. Nonlinear regression curves express the difference of FIU at a given ID well: (b) y ) (-1779.49x + 19.37x2 - 40 871.14) × 104, R2 ) 0.9735, n ) 30; (0) y ) (77.89x + 7.42x2 + 204.27) × 104, R2 ) 0.9276, n ) 20; (4) y ) (- 1488.91x + 6.41x2 - 86 492.22) × 104, R2 ) 0.8802, n ) 19. The binding of labeled PSU was inhibited by high glucose concentration. Images for this experiment were randomly chosen from 150 islets of three different batches.

glibenclamide,14 in our separate study, internalization of a different kind of FITC-labeled PSU into isolated rat islets also indicated the share of the same binding sites with glibenclamide (data not shown). Recently, several reports suggested that KATP channels were expressed not only on β-cells but also on R-, δ-, and PP cells of rodent islets.15,16 On the basis of our confocal microscopic images, it is probable that SUR may exist in whole pancreatic cells. Especially, Figure 3 shows that RITClabeled PSU is present in the periphery even after washing. Given that R-cells are peripherally located, the existence of KATP channels on R-cells is highly suspected. In addition, the mobilization of [Ca2+]i occurred around the circumference of the islet. Because it was reported that glucose did not affect the ratio of ATP/ADP in R-cells of rodent and human islets,17 we can deduce that the change in Ca2+ concentration detected in Figure 3B is possibly due to binding of PSU to KATP channels of R-cells, as well as β-cells. In contrast, there was a report by Quesada et al. demonstrating that tolbutamide (a first generation sulfonylurea) treatment was not able to induce [Ca2+]i oscillation in immunofluorescence-identifying R-cells.18 Moreover, this

Figure 8. Structures of four sulfonylurea derivatives: (A) glibenclamide; (B) tolbutamide; (C) PSU; (D) glibenclamide-BODIPY. As seen, tolbutamide lacks the benzamido group of a glibenclamide. While PSU has a flexible and hydrophilic side chain at the benzamido group, glibenclamide-BODIPY contains a bulky and hydrophobic group.

report addressed that binding of glibenclamide-BODIPY (40 nM, Figure 8D) was not detected in R-cells but only in β-cells. However, it is known that the binding affinity of tolbutamide (Figure 8B) is too low to rival glibenclamide (Figure 8A) for KATP channels, which results from the structure-activity relationship. Gribble et al. described that the half-maximal inhibition concentrations of tolbutamide were 5 µM for high-affinity sites (Ki1) and 2 mM for lowaffinity sites (Ki2) of Kir6.2/SUR1 channels, while glibenclamide presented only Ki1 (4.2 nM). For Kir6.2/SUR2A, tolbutamide had 1.7 mM Ki and glibenclamide had 27 nM Ki1 and 110 µM Ki2.19 Together with other evidences,6 it is reasonable that two kinds of binding sites, low- and highaffinity, of sulfonylureas exist depending on their structure and tissue types. In the experiment by Quesada et al.,18 the applied concentration of tolbutamide was 40 µM, which might be effective only on high-affinity Kir6.2/SUR1. If, in R-cells, there exist other types of KATP channels rather than Kir6.2/SUR1, it is possible that the applied tolbutamide concentration was not high enough to initiate calcium mobilization. Besides, the structure of glibenclamide-

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BODIPY contains a very bulky and hydrophobic side group attached to the benzamido group of glibenclamide. Because the benzamido group endows glibenclamide with the highaffinity binding property,19 it is doubtful that glibenclamideBODIPY acts exactly as glibenclamide. Because of the structure of PSU (Figure 8C) carrying relatively small and flexible chains at benzamido group, however, it seemed that PSU bioactivity was comparable to the glibenclamide although the binding affinity of PSU was possibly lower than that of glibenclamide. Despite requiring further investigation for more explicit understanding, our observation combined with the results from other reports can support the possibility that pancreatic R-cells possess either low-affinity Kir6.2/ SUR1 or other types of KATP channels, like Kir6.2/SUR2A. As a second achievement in this study, the feasibility of a new method to quantify confocal microscopic images, which identified the inhibitory effect of glucose against binding of PSU in a concentration-dependent manner, was confirmed. About whole islets, several attempts have been carried out to visualize the viability of thawed islets after cryopreservation using fluorescence probes and pancreatic β-cells by radioactive monosaccharide or to visualize the cytoplasmic Ca2+ concentration dynamics.20-22 But, our present study was the first challenge to visualize the binding property of a sulfonylurea with intact islets. Figures 5 and 7 give a tool to compare differences of the fluorescence intensities per unit islet volume (FIUs) varied by treatment conditions such as different glibenclamide and glucose concentrations. In agreement with the confocal microscopic images (Figures 4 and 6), the FIU at a given islet diameter (ID) decreased with increase in glibenclamide or glucose concentration. Since it was reported that glucose induced closing of KATP channels of β-cells,23 extensive studies have been accomplished to identify the effect of glucose or its metabolites (e.g., ATP and ADP) on KATP channels,24 the pharmacological action of KATP channel modulators,6 and the structures of the channels.25,26 It was reported that both MgATP and MgADP inhibited binding of tritiated glibenclamide to HIT T15 cells, a hamster pancreatic β-cell line27 and that glibenclamide was also a frequenter to the nucleotide-binding folds (NBFs) to which ATP and ADP bound.28 On the basis of these observations, our result entails that the diminished binding affinity of sulfonylurea may be due to the metabolites of glucose, which brings out the lowered insulinotropic activity of sulfonylurea with increasing glucose.29 However, the entwinement of glucose and its metabolites, ions, and KATP channel modulators makes it more complicated and unclear to understand exact mechanisms. Glucose metabolism leading to increased ATP/ADP ratio cannot solely explicate the regulation of sulfonylurea binding because many scrutinies reporting the inhibitory and stimulatory effects of ATP and ADP on KATP channel activity combined with Mg2+ exist.30-33 By the quantification method, we were able to normalize the amount of PSU bound to intact islets as presented in Figure 7. From this result, we conclude the following: (1) Along with the result of Figure 6, the RITC-labeled PSU binds more favorably to islets at low than at high glucose

Kim and Bae

concentrations. (2) As the islet size increases, the FIU value is amplified. This can be explained by cellular uptake of labeled PSU. It was observed in one of our separate studies that a similar type of PSU was swallowed by pancreatic cells (data not shown). Within 1 h, labeled PSU was located throughout the entire islet and most polymers were spotted on and inside the plasma membrane. (3) This method gives us a comparative tool to overcome the obstacle that originates from the heterogeneously populated islet in size. With excluding the variation of islet size and at a given ID, calculated equations and plots make it possible to directly compare the fluorescence intensities depending on different experimental conditions. In other words, without taking the islet size into consideration, any experiment using islets may run a risk due to the uncertainty of islet size effect. However, the relationship between FIU and ID can be influenced by other factors, possibly diffusion of PSU, residual PSU in the interstitial space of islet, cell-cell communication influencing PSU uptake, etc. To accomplish more understanding toward the biological feature of a whole islet, it is essential to reduce or normalize these parameters in experiments. In summary, pharmacological properties of the PSU to intact isolated islets were actually identical to those of a glibenclamide. It increased insulin secretion at low glucose concentration rather than at high one, as well as modulated [Ca2+]i. In addition, the PSU competed with glibenclamide for binding to islets. These observations support that the PSU binds to a common site, KATP channel, where a glibenclamide binds. Also, confocal microscopy illustrated that KATP channels possibly existed in pancreatic R-cells, as well as in β-cells. Using a new method to quantify confocal microscopic images, we showed that the PSU weakened its binding affinity to islets as the glucose concentration increased, which might be explained by inhibition from glucose metabolites. Consequently, the PSU can be a useful tool for biomedical and pharmacological investigations about the physiology of intact islets. Acknowledgment. This research was partially supported by NIH Grant DK 56884 and by BK 21 program in Korea. References and Notes (1) Prentki, M.; Tornheim, K.; Corkey, B. E. Diabetologia 1997, 40, S32-S41. (2) Matschinsky, F. M.; Glaser, B.; Magnuson, M. A. Diabetes 1998, 47, 307-315. (3) Schuit, F. C.; Huypens, P.; Heimberg, H.; Pipeleers, D. G. Diabetes 2001, 50, 1-11. (4) Ulrich, A. B.; Schmied, B. M.; Standop, J.; Schneider, M. B.; Pour, P. M. Pancreas 2002, 24, 111-120. (5) Groop, L. C. Diabetes Care 1992, 15, 737-754. (6) Ashcroft, S. J. H. J. Membr. Biol. 2000, 176, 187-206. (7) Kikuchi, A.; Bae, Y. H.; Kim, S. W. Biotechnol. Prog. 1994, 10, 630-635. (8) Hwang, J. S.; Chae, S. Y.; Lee, M. K.; Bae, Y. H. Biomaterials 1998, 19, 1189-1195. (9) Park, K.-H.; Kim, S.; Bae, Y. H. J. Biomed. Mater. Res. 2001, 55, 72-78. (10) Lacy, P. E.; Kostianovsky, M. Diabetes 1967, 16, 35-39. (11) Yokoshiki, H.; Sunagawa, M.; Seki, T.; Sperelakis, N. Am. J. Physiol. 1998, 274, C25-C37. (12) Ashcroft, F. M.; Rorsman, P. Prog. Biophys. Mol. Biol. 1989, 54, 87-143. (13) Panten, U.; Burgfeld, J.; Goerke, F.; Rennicke, M.; Schwanstecher, M.; Wallasch, A.; Zu¨nkler, B. J.; Lenzen, S. Biochem. Pharmacol. 1989, 38, 1217-1229.

Sulfonylurea-Islet Interactions (14) Ladriere, L.; Malaisse-Lagae, F.; Malaisse, W. J. Endocrine 2000, 12, 133-136. (15) Suzuki, M.; Fujikura, K.; Inagaki, N.; Seino, S.; Takata, K. Diabetes 1997, 46, 1440-1444. (16) Suzuki, M.; Fujikura, K.; Kotake, K.; Inagaki, N.; Seino, S.; Takata, K. Diabetologia 1999, 42, 1204-1211. (17) Detimary, P.; Dejonghe, S.; Ling, Z.; Pipeleers, D.; Schuit, F.; Henquin, J.-C. J. Biol. Chem. 1998, 273, 33905-33908. (18) Quesada, I.; Nadal, A.; Soria, B. Diabetes 1999, 48, 2390-2397. (19) Gribble, F. M.; Tucker, S. J.; Seino, S.; Ashcroft, F. M. Diabetes 1998, 47, 1412-1418. (20) Merchant, F. A.; Diller, K. R.; Aggarwal, S. J.; Bovik, A. C. Cryobiology 1996, 33, 236-252. (21) Malaisse, W. J. Diabetologia 2001, 44, 393-406. (22) Asada, N.; Shibuya, I.; Iwanaga, T.; Niwa, K.; Kanno, T. Diabetes 1998, 47, 751-757. (23) Ashcroft, F. M.; Harrison, D. E.; Ashcroft, S. J. H. Nature 1984, 312, 446-448. (24) Henquin, J.-C. Diabetes 2000, 49, 1751-1760. (25) Aguilar-Bryan, L.; Clement, J. P., IV; Gonzalez, G.; Kunjilwar, K.; Babenko, A.; Bryan, J. Physiol. ReV. 1998, 78, 227-245.

Biomacromolecules, Vol. 4, No. 6, 2003 1557 (26) Babenko, A. P.; Aguilar-Bryan, L.; Bryan, J. Annu. ReV. Physiol. 1998, 60, 667-687. (27) Niki, I.; Nicks, J. H.; Ashcroft, S. J. H. Biochem. J. 1990, 277, 619624. (28) Ueda, K.; Komine, J.; Matsuo, M.; Seino, S.; Amachi, T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1268-1272. (29) Anello, M.; Gilon, P.; Henquin, J.-C. Br. J. Pharmacol. 1999, 127, 1883-1891. (30) Gribble, F. M.; Tucker, S. J.; Haug, T.; Ashcroft, F. M. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 7185-7190. (31) Mukai, E.; Ishida, H.; Kato, S.; Tsuura, Y.; Fujimoto, S.; IshidaTakahashi, A.; Horie, M.; Tsuda, K.; Seino, Y. Am. J. Physiol. 1998, 274, E38-E44. (32) Proks, P.; Gribble, F. M.; Adhikari, R.; Tucker, S.; Ashcroft, F. M. J. Physiol. 1999, 514, 19-25. (33) Tucker, S. J.; Gribble, F. M.; Proks, P.; Trapp, S.; Ryder, T. J.; Haug, T.; Reimann, F.; Ashcroft, F. M. EMBO J. 1998, 17, 3290-3296.

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