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polymer conjugate to isolated rat islets
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Biomaterials 26 (2005) 3597–3606 www.elsevier.com/locate/biomaterials
Synthesis, bioactivity and specificity of glucagon-like peptide-1 (7–37)/polymer conjugate to isolated rat islets Sungwon Kima,b, Sung Wan Kima, You Han Baea, a
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 421 Wakara Way Suite 318, Salt Lake City, UT 84108, USA b Department of Materials Science and Engineering, Center for Biomaterials and Bioengineering, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea Received 26 April 2004; accepted 8 September 2004 Available online 10 November 2004
Abstract In order to increase the functionality of islets encapsulated in a biohybrid artificial pancreas (BAP), it was proposed that coencapsulation with insulinotropic agents would improve insulin secretion from islets. To prevent agents from leaking out, conjugation with high-molecular-weight polymers was inevitable. In this study, synthetic glucagon-like peptide-1 (GLP-1) (7–37) was conjugated to a water-soluble polymer, poly(N-vinyl-2-pyrroridone-co-acrylic acid) (5 mol% acrylic acid, Mw 445 kDa), via poly(ethylene glycol, Mw 3.4 kDa) spacer. The chemical conjugation was confirmed by reverse phase-HPLC and the GLP-1 content in the GLP-1/polymer conjugate (VAPG) was determined by UV spectrophotometry at 280 nm (ca. 29 wt/wt%). In a static insulin secretion test, the VAPG increased insulin secretion up to 200% over a control (no stimulation) at high glucose levels, although the insulinotropic activity of VAPG was slightly lower than that of native GLP-1. The bioactivity of VAPG was prolonged for at least 2 weeks, which was examined by co-encapsulation of the conjugate into islet microcapsules. Dose–response curve revealed that the half-maximal effective dose (ED50) of VAPG was about 55 nM (25 nM for native GLP-1). By N-terminal analysis using aminopeptidase and RP-HPLC, it was confirmed that the lowered bioactivity of VAPG stemmed from the polymer conjugation to N-terminal histidine moieties, which actively participate in binding to GLP-1 receptors, resulting in only 16% of N-terminal histidine remaining intact after the conjugation reaction. Finally, the specific interaction of the VAPG with isolated rat islets was investigated. Total cellular cyclic AMP levels were measured and confocal microscopy was conducted using GLP-1 and VAPG labeled with fluorescent probes. It was found that VAPG effectively increased the cAMP level in islet cells in a glucose concentration-dependent manner. Moreover, the confocal microscopy study showed that the binding of VAPG occurs at the same location where GLP-1 binds but with less affinity than that of native GLP-1. In summary, a GLP-1/polymer conjugate was synthesized for the first time, and its bioactivity was examined, which must result from its specific interaction with isolated islets. r 2004 Elsevier Ltd. All rights reserved. Keywords: Glucagon-like peptide-1 (GLP-1); Polymer; Insulin secretion; Islets of Langerhans; Biohybrid artificial pancreas
1. Introduction The biohybrid artificial pancreas (BAP) is an artificial organ designed to treat type 1 diabetes that results from irreversible destruction of insulin-secreting pancreatic bcells [1,2]. Even though numerous investigations have Corresponding author. Tel.: +1 801 585 1518; +1 801 585 3614. E-mail address: [email protected] (Y.H. Bae).
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0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.09.045
been accomplished to realize the application of a BAP in a clinical setting, significant obstacles still remain including the size of implant, the number of islets required for normoglycemia and the deteriorated insulin-secreting function of islets during isolation and encapsulation procedures [3]. To subjugate these problems, we have proposed that if insulinotropic agents are incorporated into the BAP, the number of islets needed to normalize blood glucose of a diabetic patient can be reduced by improving the insulin secretion ability
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of encapsulated islets, which has been reviewed by Hou and Bae [1]. To realize this hypothesis, our group has conducted several studies with sulfonylurea, a type of hypoglycemic drug, and glucagon-like peptide-1 (GLP1) [4–7]. When their functions are expired, the entire contents of a BAP can be removed and refreshed, allowing the restoration of normoglycemia. GLP-1 is one of the incretin hormones, which stimulates insulin secretion depending on the blood glucose concentration in the body, and responds effectively to higher glucose levels. The major portion of GLP-1 in plasma is produced by intestinal L-cells and processed from proglucagon by specific enzymatic cleavage [8]. While it has been known that the distribution of GLP-1 is limited to pancreatic a-cells, central nervous system and intestinal L-cells, GLP-1 receptors have a wide tissue distribution including pancreas, brain, hypothalamus, intestine, stomach, kidney, heart and lung [9]. The key role of GLP-1 in energy homeostasis is to regulate blood glucose levels by stimulating pancreatic b-cells after meal ingestion. Previously, we investigated the short-term bioactivity of GLP-1/Zn2+ crystal using islet macrocapsules [7]. In this study, GLP-1 was conjugated to a water-soluble polymer to examine the feasibility of creating a polymeric insulinotropic agent which would be incorporated into a BAP and expected to enhance the functionality of islets. The reason for conjugating a high-molecular-weight polymer to GLP-1 was to prevent the peptide from leaking out of a BAP device rather than to improve its biological activity as a therapeutic drug by elongating the circulation time. After confirming the chemical conjugation between GLP-1 and the polymer, the bioactivity and specificity of the conjugate were tested on isolated rat islets. We believe this is not only the first trial to conjugate GLP-1 to a macromolecule but we also will gain a better understanding of GLP-1 bioactivity and biochemistry.
2. Materials and methods 2.1. Materials Synthetic GLP-1 (7–37) was a kind gift from Dr. M. Baudysˇ (MacroMed Inc., Sandy, UT). A heterobifunctional o-amino-a-carboxyl poly(ethylene glycol) (Mw 3400; HCldNH2-PEG3400-COOH) was purchased from Shearwater Polymers (Huntsville, AL). N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile (AcN), dicyclohexylcarbodiimide (DCC), Nhydroxysuccinimide (NHS), triethylamine (TEA), L-histidine monohydrochloride monohydrate and anhydrous diethyl ether were purchased from Aldrich Chemical Co. (St. Louis, MO). Spectra/Pors6 dialysis tubing (MWCO 15,000) was purchased from Spectrum
Laboratories Inc., (Rancho Dominguez, CA). RPMI1640 medium, collagenase type V, Ficoll 400-DL, glucose, sodium bicarbonate (NaHCO3), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), glutaldehyde, aminopeptidase M and formaldehyde were obtained from SIGMA Chemicals Co. (St. Louis, MO). Hank’s balanced salt solution (HBSS), penicillin/ streptomycin antibiotics, fetal bovine serum (FBS) and sodium pyruvate were purchased from Invitrogen Corporation (GibcoBRL, Grand Island, NY). All chemical reagents and organic solvents used were at least ACS grade. 2.2. Synthesis PEG-grafted poly(N-vinyl-2-pyrrolidoneco-acrylic acid) (VAP) Fig. 1 illustrates the synthetic scheme of GLP-1/ polymer conjugate. The carboxyl groups (46.9 mmol) of 1.5 g poly(N-vinyl-2-pyrrolidone-co-acrylic acid) (VA) were activated by 14.4 mg of DCC (70.0 mmol), 5.75 mg of NHS (50.0 mmol) and dry 0.1 mL TEA in 100 mL anhydrous DMF. The VA was the same material as characterized in our previous report [4]; Mw 445 kDa by light scattering method, 312.5 mmol acrylic acid/g VA polymer. The reaction was carried out for 48 h under N2 atmosphere followed by precipitation twice against excess anhydrous diethyl ether (1.2 L). The product was dried and stored in vacuo with P2O5 for further reaction (Yield=1.4 g). The solution of 130 mg heterobifunctional PEG (HCldNH2-PEG3400-COOH, 38.3 mmol) pre-dissolved in 10 mL dry DMF with 0.1 mL TEA an hour before use was poured into the activated VA (100.1 mg, 29.4 mmol acid content) solution in 50 mL dried DMF together with 0.1 mL TEA. After 48 h reaction under N2 atmosphere, the product was collected by precipitation twice against excess diethyl ether. Then, the VA solution in 20 mL H2O was dialyzed against excess distilled water for 5 days (MWCO 15 kDa). Retrieved solution was lyophilized and stored in vacuo till further experiment (Yield=162.1 mg). By assuming that 100% coupling took place between PEG and VA, the acid content of VA (312.5 mmol) was adopted as the number of carboxyl ends of PEG in VAP without further characterization because the GLP-1 content was expected to be acquired by another method described later. 2.3. Synthesis of GLP-1/VAP conjugate (VAPG) First, the carboxylic end groups of PEG (9.2 mmol) of VAP (61.0 mg) were converted to active forms by reaction in 20 mL anhydrous DMSO with 1.2 mg NHS (10.0 mmol), 2.5 mg DCC (12.0 mmol), and 10 mL dry TEA. After 24 h reaction under N2 atmosphere, 40.0 mg GLP-1 (12.1 mmol) dissolved in 15 mL anhydrous DMSO with 0.1 mL TEA was added. Because GLP-1
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Shimadzu HPLC System, Shimadzu Scientific Instruments Inc., Columbia, MD) using an RP C4 protein column (4.6 250 mm2, 5 mm particle diameter, Vydac, Hisperia, CA). RP-HPLC conditions were modified from previously reported methods [10,11]. Before analysis, 0.5 mg of GLP-1, VAP and VAPG was dissolved in 1 mL AcN/H2O (50:50) containing 0.1 vol/ vol% TFA and filtered through a 0.45 mm syringe filter (Acrodiscs PVDF, GelmanSciences, Ann Arbor, MI). With a fixed flow rate (0.5 mL/min, Shimadzu LC-10AT Vp) and controlled oven temperature at 30 1C (Shimadzu CTO-10AC Vp), the RP-HPLC was carried out by binary gradient mode using eluent A (H2O/AcN/TFA, 5:95:0.1) and eluent B (H2O/AcN/TFA, 95:5:0.1). After 10 min equilibration by 30% eluent B, a linear gradient was applied from 30% eluent B up to 100% for another 40 min. Eluted peaks from 100 mL injection were detected at 280 nm by photodiode-array detector (Shimadzu SPD-M10A Vp). GLP-1 content in the VAPG was determined by UV spectrophotometry (Cary 3E UV–Visible Spectrophotometer, Varian Inc., Poway, CA, USA) because, while there was no aromatic moiety in VA and VAP, only GLP-1 contained one tryptophan residue that strongly absorbed 280 nm wavelength. A series of GLP-1 and VAPG solutions (1, 0.6, 0.2, 0.1, 0.075, 0.05, 0.025 and 0.01 mg/mL) in 0.01 N NaOH were used for calibration curves. By scanning from 190 to 400 nm (600 nm/min scan rate, 1 nm interval), it was confirmed that 280 nm was the most characteristic wavelength for both VAPG and GLP-1 because VAP did not absorb the wavelength. The optical density at 280 nm (OD280) for each sample was acquired as an averaged value from triplicate measurements. 2.5. Insulin secretion test
Fig. 1. Synthetic scheme of GLP-1/polymer conjugate.
was not likely to dissolve in DMSO, it was suspended in DMSO by adding TEA and slightly heated beforehand. The faintly turbid solution became clear with time. The reaction was performed under N2 atmosphere at RT for 24 h followed by dialysis (MWCO 15 kDa) against distilled water for 5 days. After lyophilization, the product was dissolved in 10 mL H2O and further purified by ultrafiltration (Centricons50, MWCO 50 kDa, Amicon Inc., Beverly, MA). Finally, the product was gained by lyophilization and stored at 20 1C for other experiments. Yield=42.0 mg. 2.4. Characterization of the VAPG The chemical conjugation between GLP-1 and VAP was conducted by reverse phase-HPLC (RP-HPLC,
Pancreatic islets were isolated from the pancreata of male Sprague–Dawley (SD) rats (200–250 g) by the conventional collagenase digestion method and by the Ficoll gradient method with minimal modification [12]. Isolated islets were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 5 mM NaHCO3, 20 mM HEPES and 11.2 mM D-glucose under humidified air containing 5% CO2 at 37 1C. After being cultured for 3–5 days, spherical islets that were obtained from five to six SD rats were hand-picked for further experiments. To examine the bioactivity of VAPG, islets were rinsed twice with cold HBSS and transferred into 24well culture plates (20 islets per well). Every well contained 1 mL HEPES-buffered Krebs (HK) solution (2.8 mM glucose, 130 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES, 0.2% BSA, pH 7.4). After 1 h, the solution was exchanged with 0.9 mL of fresh HK
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solution containing various glucose concentrations (2.8, 5.6, 11.1, 16.7 mM). Simultaneously, 0.1 mL of each stimulant (GLP-1 and VAPG) from stock solutions with different concentrations (100 nM and 10 mM; final concentrations became 10 nM and 1 mM) was added. For VAPG, the applied concentrations were equivalent to the GLP-1 content in the polymer. After 1 h incubation at 37 1C under 5% CO2 atmosphere, each solution (750 mL) was taken and centrifuged at 3500 r.p.m. for 3 min. The supernatant (400 mL) was kept at 20 1C till radioimmunoassay (RIA). The amount of insulin in each sample from six independent experiments was determined by the insulin 125I RIA kit (ICN Pharmaceuticals Inc., Costa Mesa, CA). In the dose–response experiment, 10 islets that had been starved with 2.8 mM glucose HK solution were placed in each well of a 96-well culture plate, which contained 180 mL of 5.6 mM glucose HK solution. At the same time, 20 mL VAPG stock solutions with different concentrations (10 and 1 mM, 100, 10, 1 nM) were added into each well. After 1 h incubation at 37 1C under 5% CO2 atmosphere, 180 mL of solution from every well was taken and centrifuged at 3500 r.p.m. for 3 min. RIA was carried out on every 100 mL supernatant obtained from five independent batches. Data were plotted as GLP-1 concentration in the VAPG versus the stimulation index (insulin secretion stimulated by VAPG/insulin secretion without VAPG). Microencapsulation of islets laminated by alginate and poly(L-lysine) was carried out as described elsewhere. For static culture, encapsulated islets with GLP-1 (1 mg/mL) or VAPG (1 mg GLP-1 equivalent/mL) or neither stimulant were placed in transwells (1.0 mm pore), which were inserted into 24-well plates. The medium was changed every single day and insulin concentration was measured by RIA. In semi-dynamic experiments, 300 G HK solution was perfused to encapsulated islets for 1 h after being stabilized in 50 G HK solution for 1 h, and were re-stabilized using 50 G HK solution for another 1 h as reported previously [13].
2.6. Cyclic AMP (cAMP) measurement After undergoing starvation with 2.8 mM glucose HK solution, 10 islets were hand-picked and transferred into a 96-well culture plate. Then, the volume of each well was adjusted to 160 mL with HK solution (no glucose). Immediately, 20 mL of 100 nM VAPG solution in HK solution with no glucose and 20 mL of different HK solutions with varying glucose concentration (2.8, 5.6, 11.1 and 16.7 mM) were added. After 1 h incubation at 37 1C under 5% CO2 atmosphere, the stimulation was stopped by heating for 5 min at 80 1C. Total cellular cAMP for each well was measured by following the user manual of cAMP enzymeimmunoassay kit (Biotrak
cellular communication assays, Amersham Pharmacia Biotech, Arlington Heights, IL). 2.7. Confocal microscopy To visualize the interactions between VAPG or GLP1 and isolated islets, fluorescent probes were attached to each molecule. The GLP-1 (1 mg, 303.2 nmol) and the VAPG (3 mg, 262.8 nmol) were reacted with RITC (16.3 mg, 30.3 nmol) and FITC (10.2 mg, 26.3 nmol) in 1.5 mL DMSO containing 10 mL TEA at room temperature for 24 h, respectively. Each reagent amount could be adjusted by dilution from high concentration stock solutions in DMSO. All reactions were stopped by adding excess H2O (10 mL), followed by ultrafiltration (MWCO 5 kDa for RITC-labeled GLP-1 and MWCO 10 kDa for FITC-labeled VAPG, Ultrafree-CL, Millipore Corporation, Bedford, MA). Each labeled molecule was obtained by lyophilization. Hand-picked islets were incubated with 100 nM of GLP-1, VAPG (GLP-1 equivalent) and VAP (acid content equivalent) for 1 h at 37 1C. After rinsing twice with PBS, islets were fixed with 2% glutaldehyde and 2% formaldehyde in PBS. Confocal microscopic images were gained by Olympus FluoviewTM 300 confocal microscope (Aurora, CO) with red helium neon (633 nm for RITC) and green helium neon (543 nm for FITC). The scan rate was 65,536 pixels/s and the magnitude was 200 by the same method as described previously [14]. Different fluorescent intensities and densities were normalized by calibration of RITC-labeled GLP-1 and FITC-labeled VAPG using microplate fluorescence reader. 2.8. N-terminal analysis by aminopeptidase Aminopeptidase (5 units) was incubated with 7.4 mg/ mL VAPG and 4 mg/mL GLP-1 in 0.5 mL PBS (pH 8.0) for 2.5 h at 37 1C. After filtration by 0.22 mm syringe filter, each sample and histidine were analyzed by RPHPLC (isocratic condition with 90% H2O and 10% AcN, Waters XTerraTM RP18 column, 5 mm pore size, 4.6 250 mm2). 2.9. Statistics For the static bioactivity and cAMP measurement tests, data were analyzed by Student’s t-test. Results from semi-dynamic and long-term culture experiments were analyzed by one-way ANOVA (Po0.05). Quantification of confocal microscopic images were followed by the same method as reported elsewhere [14]. All data from multiple experiments (n ¼ 5–6) are expressed by mean7standard error of means (SEM).
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3. Results and discussion Many groups have developed chemically modified polypeptides or proteins, which include PEG conjugation to monoclonal antibodies, insulin and growth factors. The conjugation of PEG to polypeptides or proteins, however, usually reduces the bioactivity by blocking the active sites and/or changing their original conformation. In this study, the GLP-1/polymer conjugate was originally designed as a component of a rechargeable BAP. Therefore, instead of relating the details of preparing well-defined and biologically active forms of the conjugate as a drug, this report will focus on examining the bioactivity of a particular synthesized conjugate and investigating its specific interaction with isolated rat islets to confirm whether the activity originates from the same action mechanism in the GLP-1 receptor signaling pathway. 3.1. Synthesis and characterization Synthesis of the GLP-1/polymer conjugate (VAPG) was performed as per scheme shown in Fig. 1, which was modified from a previously reported method [15]. To avoid cross-linking of the polymer, all carboxylic groups were activated before reactions and excess amounts of PEG and GLP-1 were added. Unreacted GLP-1 (about 3.3 kDa) was removed by dialysis (MWCO 15 kDa) as well as ultrafiltration (MWCO 50 kDa). The chemical conjugation between GLP-1 and VAP was confirmed by RP-HPLC. While GLP-1 gave a sharp single peak at 29.6 min (Fig. 2A), VAPG showed a dispersed peak from 29 to 36 min (Fig. 2B), due to the polydispersity of the polymer (VAP) and varying degrees of conjugation. Fig. 2C displays the chromatogram of the VAP which does not show any peaks throughout the elution time. Because the transition at 280 nm is a specific characteristic of the tryptophan [16,17], only GLP-1 and GLP-1containing polymer conjugates could absorb the wavelength. The GLP-1 content of VAPG could be also calculated from UV spectrophotometry. The UV spectra were scanned from 200 to 400 nm to confirm that the optical density at 280 nm (OD280) was sensitive enough to quantify the amount of GLP-1 in the VAPG. Calibration curves illustrated correlations between the concentrations of GLP-1 (a, y ¼ 0:0028+1.5018x, R2 ¼ 0:9999) or VAPG (b, y ¼ 0:0005+0.4361x, R2 ¼ 0:9999) and OD280. As a result, it was estimated that VAPG contained 28.9 wt% of GLP-1 (data not shown). 3.2. Bioactivity of VAPG On examining the insulin level of a control experiment without stimulation, in static insulin secretion test it was observed that both VAPG and GLP-1 enhanced the
Fig. 2. Representative RP-HPLC chromatograms of GLP-1 (A), VAPG (B) and VAP (C) at 280 nm. Because only GLP-1 and its polymer conjugate contain tryptophan moiety in their structure, there is no peak in VAP chromatogram.
insulin level as shown in Fig. 3A. VAPG, however, gained statistical significance only at 1 mM (GLP-1 equivalent) while GLP-1 had significantly increased bioactivity at both 10 nM and 1 mM concentrations (Student’s t-test, Po0.1, Po0.05). Although 10 nM VAPG slightly increased insulin secretion over the control, it did not reach the required statistical level. One of the important features in Fig. 3A is the glucosedependent insulin secretion from isolated islets stimulated by GLP-1 or VAPG. The best insulinotropic activity was acquired at 200 G glucose concentration, and the increasing percentage of insulin secretion over the control at 300 G was similar to that at 200 G, although the amount of secreted insulin at 300 G was higher than that at 200 G, which agreed with a previously reported result [18]. Fig. 3B show the glucose-dependent manner of insulin secretion from a semi-dynamic test. All islet microcapsules strongly increased their insulin secretion when the glucose concentration was changed from 50 to 300 G, and returned to basal levels when the media were replaced with 50 G again. At 300 G condition, the VAPG gained outstanding bioactivity among the three groups (oneway ANOVA, Po0.05). In addition, Fig. 3C show that the microencapsulated islets containing VAPG strongly increased insulin secretion while those with GLP-1 did not. This result proves that the VAPG is successfully retained inside a BAP for a long time in contrast to GLP-1 leakage. In another experiment, insulinotropic activity of the VAPG inside the microcapsules lasted for 2 months and, moreover, VAPGcontaining capsules and control ones did not show
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islet stimulation by GLP-1 occurs only when the glucose level is high after a meal, not continuous [28], (4) the literature information tells us that transplanted encapsulated islets cannot survive for many years, leading to the design of a rechargeable system as previously suggested by us [1], and (5) GLP-1 may present islet proliferation capacity [9]. With all these reasons, the coencapsulation of GLP-1/polymer conjugate with islets may provide more advantages over metabolic load and the whole component including islets and VAPG can be refreshed when their functionality is exhausted. The physiological level of GLP-1 in the body is 5–15 pM, which increases up to 20–30 pM after meal ingestion [8]. For isolated islets, however, a higher dose of GLP-1 seems to be required to stimulate insulin secretion; the insulinotropic activity of GLP-1 against isolated rat pancreatic islets began at 2.5 nM and was saturated at 250 nM [18]. While the half-maximal effective dose (ED50) for native GLP-1 was reported as 25 nM, from the dose–response curve, the ED50 of VAPG was about 55 nM obtained by regression with a sigmoidal model equation (Fig. 4). 3.3. N-terminal analysis We hypothesized that the slightly lowered insulinotropic activity of GLP-1 moiety in VAPG was from random conjugation of VAP to GLP-1. Study of the structure–activity relationship informed us that Nterminal amino acids of GLP-1 were important for insulinotropic activity while C-terminal had relatively less influence on the bioactivity (Table 1) [21–25]. The histidine moiety at the end of N-terminus (His7) plays a central role in the GLP-1 activity for binding to GLP-1 receptors [21,22] and for producing cAMP [23–27]. Liberation of two amino acids (His7-Ala8) from the N-
Fig. 3. (A) Insulinotropic activity of GLP-1 and VAPG to isolated rat islets. Results are presented as mean7SEM from six independent experiments. Po0.01, Po0.5 from the Student’s t-test. (B) Semidynamic insulin secretion test by microencapsulation of islets with VAPG or GLP-1. Po0.05 by one-way ANOVA. (C) Insulin secretion profile of islet microcapsules from 2-week static culture. Po0.05 by one-way ANOVA.
different islets viabilities during the period (data not shown). The islet stimulation has been justified by noting that (1) the insulin secretion capacity of islets after isolation and encapsulation is approximately 10–15% of residing islets in a pancreas due to poor oxygen supply and damage during isolation and encapsulation, giving us enough room in metabolic load [19], (2) the oxygen supply issue can be addressed by introducing macromolecular oxygen carriers as published by us [20], (3)
Fig. 4. Dose–response curve of the VAPG. Each point was acquired from five independent experiments and presented as mean7SEM a stimulation index ¼(insulin secretion with VAPG)/(insulin secretion without VAPG).
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Table 1 Summary of amino acids that play a key role in the bioactivity of GLP-1 (7–36) or GLP-1 (7–37) 7
10
15
20
25
30
35
References
HAE HAE HAE HAE HAE
GTFTS GTFTS GTFTS GTFTS GTFTS
DVSSY DVSSY DVSSY DVSSY DVSSY
LEGQA LEGQA LEGQA LEGQA LEGQA
AKEFI AKEFI AKEFI AKEFI AKEFI
AWLVK AWLVK AWLVK AWLVK AWLVK
GR GR GR GRG GR
Adelhorst et al. [21] Gallwitz et al. [22] Watanabe et al. [23] Gefel et al. [24] Suzuki et al. [25]
Each letter abbreviates corresponding amino acid. The position of amino acid in the sequence of GLP-1 (1–37) is indicated at the upper row. Based on the results from each report, the boxed amino acids are important to maintain the bioactivity of GLP-1, while the underlined amino acids do not significantly influence on the GLP-1 activity.
terminus of GLP-1 by dipeptidyl peptidase IV not only diminished the bioactivity of GLP-1 but also antagonized the activity of GLP-1 [10,11,28,29]. In addition, it was reported that modifications of His7 to generate dipeptidyl peptidase IV-resistant forms of GLP-1 significantly lowered both the bioactivity and the cAMP producing ability of GLP-1 [11,27]. Because protein/polymer conjugates are not a good substrate for an automatic amino acid sequencer and several reports exist to analyze protein terminus using peptidases, we attempted to analyze the N-terminal using aminopeptidase and RP-HPLC for monitoring the chemical modification [30–32]. Fig. 5A illustrates the chromatograms of histidine itself or His7 released through enzymatic degradation by an aminopeptidase that can liberate every single amino acid from the end of N-terminus. Fig. 5B shows that much less histidine (relative amount of 15.6% by integration from 2.30 to 2.65 min) was released by the degradation of VAPG than from the native GLP-1. This means that most of His7 at the end of GLP-1 was employed for conjugation to carboxylic groups of VAP. Because two lysine moieties (Lys26 and Lys34) hardly influence the activity of GLP-1 when substituted with other amino acids (Table 1) or when coupled to other chemicals [33], the bioactivity of VAPG was attributed to the conjugate population containing intact His7. Although the method employed here, aminopeptidase degradation–HPLC coupling, is not a common one, recent analytical methods using peptidase to analyze peptides and proteins have been employed in developing new substrates and drugs, assaying enzyme–substrate reaction, and examining structural biology become important tools [34,35]. Such method was suggested as an alternative way to determine peptide sequence in a highmolecular-weight polymer/peptide conjugation system. 3.4. Specific interactions of VAPG with isolated rat islets Figs. 3A and B already suggested evidence of specific interactions because the insulin secretion stimulated by VAPG showed glucose dependence similar to GLP-1
Fig. 5. (A) Representative RP-HPLC chromatograms of VAPG and GLP-1 (after treatment of aminopeptidase), and histidine. (B) The relative amount of free N-terminal histidine moieties in the VAPG calculated from RP-HPLC (n ¼ 3).
[18,36,37]. Fig. 6 displays glucose-dependent cAMP production stimulated by VAPG, which is a marker of the GLP-1 action mechanism. Upon comparison with the control levels, the cAMP level significantly increased at 200 and 300 G glucose concentrations in the presence of VAPG, while it was negligible at low glucose
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Fig. 6. Total cellular cAMP level with or without treating 100 nM VAPG. Data were analyzed by Student’s t-test (Po0.01, n ¼ 3) and presented as mean7SEM.
concentrations (Po0.01 by Student’s t-test). To obtain more direct evidence for the specific interaction of VAPG with isolated rat islets, confocal microscopy was used to visualize fluorescently labeled VAPG or GLP-1. Fig. 7A presents the confocal microscopic images of islets treated with FITC-labeled VAPG (a, green) and RITC-labeled GLP-1 (b, red). Both GLP-1 and VAPG seem to be incorporated inside the pancreatic cells, which stain the cytoplasmic compartment without reaching nuclei. The relationship between islet size (islet diameter, mm) and fluorescence intensity per unit volume (mm3) was quantified (Fig. 7B) by a method described elsewhere [13]. The plot indicates that the binding affinity of VAPG is less than that of GLP-1. This result implies that VAPG could interact with rat islets by means of GLP-1 receptors, although its binding affinity was lower than that of GLP-1 due to polymer conjugation to His7.
Fig. 7. (A) Confocal microscopic images of islets treated with RITC-labeled GLP-1 (a) and with FITC-labeled VAPG (b). (B) Quantification results of confocal microscopic images that show the different binding affinity between labeled GLP-1 (n ¼ 23) and VAPG (n ¼ 23).
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4. Conclusion A GLP-1/polymer conjugate (VAPG) was synthesized and conjugation was characterized by RP-HPLC. Using the specific transition of a tryptophan moiety in GLP-1 structure at 280 nm, UV spectrophotometry revealed that there existed 29.9 wt/wt% of GLP-1 in VAPG. Static insulin secretion tests showed that VAPG could stimulate isolated rat islets up to 200% (1 mM GLP-1 equivalent), although bioactivity of the polymer was slightly less than that of native GLP-1. By using islet microcapsulation, it was shown that bioactivity was maintained at least 2 weeks. The experiment also showed that insulinotropic activity of VAPG increased as the glucose concentration became higher, which is a physiological property of native GLP-1. Dose–response curve of VAPG showed that ED50 was about 55 nM. This reduced bioactivity resulted from the conjugation of polymer to the amine groups of GLP-1, especially, Nterminal histidine. Aminopeptidase treatment combined with RP-HPLC proved that only 15.6% of histidine was free without polymer conjugation. However, the VAPG still had binding affinity to GLP-1 receptors on isolated islets, which was confirmed by confocal microscopy. Quantitative analysis of confocal microscopic images revealed that the binding affinity of VAPG was less than that of GLP-1. The specific interaction was also confirmed by cAMP measurement. The cAMP level increased in a glucose-dependent manner with significant distinction from the control level at high glucose concentrations. The long-term insulinotropic activity of VAPG in conventional alginate and poly L-lysine islet microcapsules is under investigation.
[7]
[8]
[9] [10]
[11]
[12] [13]
[14]
[15]
[16]
[17]
[18]
[19]
Acknowledgement This research was supported by funding from NIH DK 56884 in USA and by BK 21 program in South Korea.
[20]
[21]
[22]
References [1] Hou QP, Bae YH. Biohybrid artificial pancreas based on macrocapsule device. Adv Drug Deliv Rev 1999;35:271–87. [2] Lanza RP, Sullivan SJ, Chick WL. Islet transplantation with immunoisolation. Diabetes 1992;41:1503–10. [3] Suzuki K, Bonner-Weir S, Hollister-Lock J, Colton CK, Weir GC. Number and volume of islets transplanted in immunobarrier devices. Cell Transplant 1998;7:47–52. [4] Park K-H, Kim S, Bae YH. Interaction of sulfonylureaconjugated polymer with insulinoma cell line of MIN6 and its effect on insulin secretion. J Biomed Mater Res 2001;55:72–8. [5] Kikuchi A, Bae YH, Kim SW. Stimulatory effect of a sulfonylurea analogue and its polymer conjugate on insulin secretion from rat islets. Biotechnol Prog 1994;43:673–7. [6] Hwang JS, Chae SY, Lee MK, Bae YH. Synthesis of sulfonylurea conjugated copolymer via PEO spacer and its in vitro short-term
[23]
[24]
[25]
3605
bioactivity in insulin secretion from islets of Langerhans. Biomaterials 1998;19:1189–95. Gappa H, Baudysˇ M, Koh JJ, Kim SW, Bae YH. The effect of zinc-crystallized glucagon-like peptide-1 on insulin secretion of macroencapsulated pancreatic islets. Tissue Eng 2001;7:35–44. Ahre´n B. Glucagon-like peptide-1 (GLP-1): a gut hormone of potential interest in the treatment of diabetes. Bioassays 1998;20:642–51. Kieffer TJ, Habener F. The glucagon-like peptides. Endocr Rev 1999;20:876–913. Ritzel U, Leonhardt U, Ottleben M, Ru¨hmann A, Eckart K, Spiess J, Ramadori G. A synthetic glucagon-like peptide-1 analog with improved plasma stability. J Endocrinol 1998;158:93–102. Siegel EG, Scharf G, Gallwitz B, Mentlein R, Morys-Wortmann M, Fo¨lsch UR, Schmidt WE. Comparison of the effect of native glucagon-like peptide I and dipeptidyl peptidase IV-resistant analogues on insulin release from rat pancreatic islets. Eur J Clin Invest 1999;29:610–4. Lacy PE, Kostianovsky M. Method for the isolation of intact islets of Langerhans from rat pancreas. Diabetes 1967;16:35–9. Kim S, Chae SY, Na K, Kim SW, Bae YH. Insulinotropic activity of sulfonylurea/pullulan conjugate in rat islet microcapsule. Biomaterials 2003;24:4843–51. Kim S, Bae YH. Visual evidence and quantification of interaction of polymeric sulfonylurea with pancreatic islet. Biomacromolecules 2003;4:1550–7. Liu F, Song SC, Mix D, Baudysˇ M, Kim SW. Glucose-induced release of glycosylpoly(ethylene glycol) insulin bound to a soluble conjugate of concanavalin A. Bioconjug Chem 1997;8:664–72. Kazmin D, Edwards RA, Turner RJ, Larson E, Starkey J. Visualization of proteins in acrylamide gels using ultraviolet illumination. Anal Biochem 2002;301:91–6. He Q-Y, Mason AB, Lyons BA, Tam BM, Nguyen V, Macgillivray RTA, Woodworth RC. Spectral and metal-binding properties of three single-point tryptophan mutants of the human transferrin N-lobe. Biochem J 2001;354:423–39. Schmidt WE, Siegel EG, Creutzfeldt W. Glucagon-like peptide-1 but not glucagon-like peptide-2 stimulates insulin release from isolated rat pancreatic islets. Diabetologia 1985;28:704–7. Arita S, Une S, Ohtsuka S, Kawahara T, Kasraie A, Smith CV, Mullen Y. Increased islet viability by addition of beraprost sodium to collagenase solution. Pancreas 2001;23:62–7. Chae SY, Kim SW, Bae YH. The effect of crosslinked hemoglobin on functionality and viability of microencapsulated pancreatic islets. Tissue Eng 2002;8:379–94. Adelhorst K, Hedegaard BB, Knudsen LB, Kirk O. Structure– activity studies of glucagon-like peptide-1. J Biol Chem 1994;269:6275–8. Gallwitz B, Witt M, Paetzold G, Morys-wortmann C, Zimmermann B, Eckart K, Fo¨lsch UR, Schmidt WE. Structure/activity characterization of glucagon-like peptide-1. Eur J Biochem 1994;225:1151–6. Watanabe Y, Kawai K, Ohashi S, Yokota C, Suzuki S, Yamashita K. Structure–activity relationships of glucagon-like peptide-1 (7–36) amide: insulinotropic activities in perfused rat pancreases, and receptor binding and cyclic AMP production in RINm5F cells. J Endocrinol 1994;140:45–52. Gefel D, Hendrick GK, Mojsov S, Habener J, Weir GC. Glucagon-like peptide-I analogs, effects on insulin secretion and adenosine 30 ,50 -monophosphate formation. Endocrinology 1990;126:2164–8. Suzuki S, Kawai K, Ohashi S, Mukai H, Yamashita K. Comparison of the effects of various C-terminal and N-terminal fragment peptides of glucagon-like peptide-1 on insulin and glucagon release from the isolated perfused rat pancreas. Endocrinology 1989;125:3109–14.
ARTICLE IN PRESS 3606
S. Kim et al. / Biomaterials 26 (2005) 3597–3606
[26] Drucker DJ, Philippe J, Mojsov S, Chick WL, Habener JF. Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line. Proc Natl Acad Sci USA 1987;84:3434–8. [27] Xiao Q, Giuere J, Parisien M, Jeng W, St-Pierre SA, Brubaker PL, Wheeler MB. Biological activities of glucagon-like peptide-1 analogues in vitro and in vivo. Biochemistry 2001;40:2860–9. [28] Kieffer TJ, McIntosh CHS, Pederson RA. Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV. Endocrinology 1995;136:3585–96. [29] Deacon CF, Hughes TE, Holst JJ. Dipeptidyl peptidase IV inhibition potentiates the insulinotropic effect of glucagon-like peptide 1 in the anesthetized pig. Diabetes 1998;47:764–9. [30] Sechi S, Chait BT. A method to define the carboxyl terminal of proteins. Anal Chem 2000;72:3374–8. [31] Breci LA, Tabb DL, Yates III JR, Wysocki VH. Cleavage Nterminal to proline, analysis of a database of peptide tandem mass spectra. Anal Chem 2003;75:1963–71. [32] Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C. Identifying proteins from two-dimensional gels by
[33]
[34]
[35]
[36]
[37]
molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci USA 1993;90:5011–5. Chou JZ, Place GD, Waters DG, Kirkwood JA, Bowsher RR. A radioimmunoassay for LY315902, an analog of glucagon-like insulinotropic peptide, and its application in the study of canine pharmacokinetics. J Pharm Sci 1997;86:768–73. Ribo M, Bosch M, Torrent G, Benito A, Beaumelle B, Vilanova M. Quantitative analysis, using MALDI-TOF mass spectrometry, of the N-terminal hydrolysis and cyclization reactions of the activation process of onconase. Eur J Biochem 2004;271:1163–71. Grant SK, Sklar JG, Cummings RT. Development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates. J Biomol Screen 2002;7:531–40. Go¨ke R, Wagner B, Fehmann H-C, Go¨ke B. Glucose-dependency of the insulin stimulatory effect of glucagon-like peptide-1 (7–36) amide on the rat pancreas. Res Exp Med 1993;193: 97–103. Hargrove DM, Nardone NA, Persson LM, Parker JC, Stevenson RW. Glucose-dependent action of glucagon-like peptide-1 (7–37) in vivo during short- or long-term administration. Metabolism 1995;44:1231–7.
Biomaterials 26 (2005) 3597–3606 www.elsevier.com/locate/biomaterials
Synthesis, bioactivity and specificity of glucagon-like peptide-1 (7–37)/polymer conjugate to isolated rat islets Sungwon Kima,b, Sung Wan Kima, You Han Baea, a
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 421 Wakara Way Suite 318, Salt Lake City, UT 84108, USA b Department of Materials Science and Engineering, Center for Biomaterials and Bioengineering, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea Received 26 April 2004; accepted 8 September 2004 Available online 10 November 2004
Abstract In order to increase the functionality of islets encapsulated in a biohybrid artificial pancreas (BAP), it was proposed that coencapsulation with insulinotropic agents would improve insulin secretion from islets. To prevent agents from leaking out, conjugation with high-molecular-weight polymers was inevitable. In this study, synthetic glucagon-like peptide-1 (GLP-1) (7–37) was conjugated to a water-soluble polymer, poly(N-vinyl-2-pyrroridone-co-acrylic acid) (5 mol% acrylic acid, Mw 445 kDa), via poly(ethylene glycol, Mw 3.4 kDa) spacer. The chemical conjugation was confirmed by reverse phase-HPLC and the GLP-1 content in the GLP-1/polymer conjugate (VAPG) was determined by UV spectrophotometry at 280 nm (ca. 29 wt/wt%). In a static insulin secretion test, the VAPG increased insulin secretion up to 200% over a control (no stimulation) at high glucose levels, although the insulinotropic activity of VAPG was slightly lower than that of native GLP-1. The bioactivity of VAPG was prolonged for at least 2 weeks, which was examined by co-encapsulation of the conjugate into islet microcapsules. Dose–response curve revealed that the half-maximal effective dose (ED50) of VAPG was about 55 nM (25 nM for native GLP-1). By N-terminal analysis using aminopeptidase and RP-HPLC, it was confirmed that the lowered bioactivity of VAPG stemmed from the polymer conjugation to N-terminal histidine moieties, which actively participate in binding to GLP-1 receptors, resulting in only 16% of N-terminal histidine remaining intact after the conjugation reaction. Finally, the specific interaction of the VAPG with isolated rat islets was investigated. Total cellular cyclic AMP levels were measured and confocal microscopy was conducted using GLP-1 and VAPG labeled with fluorescent probes. It was found that VAPG effectively increased the cAMP level in islet cells in a glucose concentration-dependent manner. Moreover, the confocal microscopy study showed that the binding of VAPG occurs at the same location where GLP-1 binds but with less affinity than that of native GLP-1. In summary, a GLP-1/polymer conjugate was synthesized for the first time, and its bioactivity was examined, which must result from its specific interaction with isolated islets. r 2004 Elsevier Ltd. All rights reserved. Keywords: Glucagon-like peptide-1 (GLP-1); Polymer; Insulin secretion; Islets of Langerhans; Biohybrid artificial pancreas
1. Introduction The biohybrid artificial pancreas (BAP) is an artificial organ designed to treat type 1 diabetes that results from irreversible destruction of insulin-secreting pancreatic bcells [1,2]. Even though numerous investigations have Corresponding author. Tel.: +1 801 585 1518; +1 801 585 3614. E-mail address: [email protected] (Y.H. Bae).
fax:
0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.09.045
been accomplished to realize the application of a BAP in a clinical setting, significant obstacles still remain including the size of implant, the number of islets required for normoglycemia and the deteriorated insulin-secreting function of islets during isolation and encapsulation procedures [3]. To subjugate these problems, we have proposed that if insulinotropic agents are incorporated into the BAP, the number of islets needed to normalize blood glucose of a diabetic patient can be reduced by improving the insulin secretion ability
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of encapsulated islets, which has been reviewed by Hou and Bae [1]. To realize this hypothesis, our group has conducted several studies with sulfonylurea, a type of hypoglycemic drug, and glucagon-like peptide-1 (GLP1) [4–7]. When their functions are expired, the entire contents of a BAP can be removed and refreshed, allowing the restoration of normoglycemia. GLP-1 is one of the incretin hormones, which stimulates insulin secretion depending on the blood glucose concentration in the body, and responds effectively to higher glucose levels. The major portion of GLP-1 in plasma is produced by intestinal L-cells and processed from proglucagon by specific enzymatic cleavage [8]. While it has been known that the distribution of GLP-1 is limited to pancreatic a-cells, central nervous system and intestinal L-cells, GLP-1 receptors have a wide tissue distribution including pancreas, brain, hypothalamus, intestine, stomach, kidney, heart and lung [9]. The key role of GLP-1 in energy homeostasis is to regulate blood glucose levels by stimulating pancreatic b-cells after meal ingestion. Previously, we investigated the short-term bioactivity of GLP-1/Zn2+ crystal using islet macrocapsules [7]. In this study, GLP-1 was conjugated to a water-soluble polymer to examine the feasibility of creating a polymeric insulinotropic agent which would be incorporated into a BAP and expected to enhance the functionality of islets. The reason for conjugating a high-molecular-weight polymer to GLP-1 was to prevent the peptide from leaking out of a BAP device rather than to improve its biological activity as a therapeutic drug by elongating the circulation time. After confirming the chemical conjugation between GLP-1 and the polymer, the bioactivity and specificity of the conjugate were tested on isolated rat islets. We believe this is not only the first trial to conjugate GLP-1 to a macromolecule but we also will gain a better understanding of GLP-1 bioactivity and biochemistry.
2. Materials and methods 2.1. Materials Synthetic GLP-1 (7–37) was a kind gift from Dr. M. Baudysˇ (MacroMed Inc., Sandy, UT). A heterobifunctional o-amino-a-carboxyl poly(ethylene glycol) (Mw 3400; HCldNH2-PEG3400-COOH) was purchased from Shearwater Polymers (Huntsville, AL). N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile (AcN), dicyclohexylcarbodiimide (DCC), Nhydroxysuccinimide (NHS), triethylamine (TEA), L-histidine monohydrochloride monohydrate and anhydrous diethyl ether were purchased from Aldrich Chemical Co. (St. Louis, MO). Spectra/Pors6 dialysis tubing (MWCO 15,000) was purchased from Spectrum
Laboratories Inc., (Rancho Dominguez, CA). RPMI1640 medium, collagenase type V, Ficoll 400-DL, glucose, sodium bicarbonate (NaHCO3), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), glutaldehyde, aminopeptidase M and formaldehyde were obtained from SIGMA Chemicals Co. (St. Louis, MO). Hank’s balanced salt solution (HBSS), penicillin/ streptomycin antibiotics, fetal bovine serum (FBS) and sodium pyruvate were purchased from Invitrogen Corporation (GibcoBRL, Grand Island, NY). All chemical reagents and organic solvents used were at least ACS grade. 2.2. Synthesis PEG-grafted poly(N-vinyl-2-pyrrolidoneco-acrylic acid) (VAP) Fig. 1 illustrates the synthetic scheme of GLP-1/ polymer conjugate. The carboxyl groups (46.9 mmol) of 1.5 g poly(N-vinyl-2-pyrrolidone-co-acrylic acid) (VA) were activated by 14.4 mg of DCC (70.0 mmol), 5.75 mg of NHS (50.0 mmol) and dry 0.1 mL TEA in 100 mL anhydrous DMF. The VA was the same material as characterized in our previous report [4]; Mw 445 kDa by light scattering method, 312.5 mmol acrylic acid/g VA polymer. The reaction was carried out for 48 h under N2 atmosphere followed by precipitation twice against excess anhydrous diethyl ether (1.2 L). The product was dried and stored in vacuo with P2O5 for further reaction (Yield=1.4 g). The solution of 130 mg heterobifunctional PEG (HCldNH2-PEG3400-COOH, 38.3 mmol) pre-dissolved in 10 mL dry DMF with 0.1 mL TEA an hour before use was poured into the activated VA (100.1 mg, 29.4 mmol acid content) solution in 50 mL dried DMF together with 0.1 mL TEA. After 48 h reaction under N2 atmosphere, the product was collected by precipitation twice against excess diethyl ether. Then, the VA solution in 20 mL H2O was dialyzed against excess distilled water for 5 days (MWCO 15 kDa). Retrieved solution was lyophilized and stored in vacuo till further experiment (Yield=162.1 mg). By assuming that 100% coupling took place between PEG and VA, the acid content of VA (312.5 mmol) was adopted as the number of carboxyl ends of PEG in VAP without further characterization because the GLP-1 content was expected to be acquired by another method described later. 2.3. Synthesis of GLP-1/VAP conjugate (VAPG) First, the carboxylic end groups of PEG (9.2 mmol) of VAP (61.0 mg) were converted to active forms by reaction in 20 mL anhydrous DMSO with 1.2 mg NHS (10.0 mmol), 2.5 mg DCC (12.0 mmol), and 10 mL dry TEA. After 24 h reaction under N2 atmosphere, 40.0 mg GLP-1 (12.1 mmol) dissolved in 15 mL anhydrous DMSO with 0.1 mL TEA was added. Because GLP-1
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Shimadzu HPLC System, Shimadzu Scientific Instruments Inc., Columbia, MD) using an RP C4 protein column (4.6 250 mm2, 5 mm particle diameter, Vydac, Hisperia, CA). RP-HPLC conditions were modified from previously reported methods [10,11]. Before analysis, 0.5 mg of GLP-1, VAP and VAPG was dissolved in 1 mL AcN/H2O (50:50) containing 0.1 vol/ vol% TFA and filtered through a 0.45 mm syringe filter (Acrodiscs PVDF, GelmanSciences, Ann Arbor, MI). With a fixed flow rate (0.5 mL/min, Shimadzu LC-10AT Vp) and controlled oven temperature at 30 1C (Shimadzu CTO-10AC Vp), the RP-HPLC was carried out by binary gradient mode using eluent A (H2O/AcN/TFA, 5:95:0.1) and eluent B (H2O/AcN/TFA, 95:5:0.1). After 10 min equilibration by 30% eluent B, a linear gradient was applied from 30% eluent B up to 100% for another 40 min. Eluted peaks from 100 mL injection were detected at 280 nm by photodiode-array detector (Shimadzu SPD-M10A Vp). GLP-1 content in the VAPG was determined by UV spectrophotometry (Cary 3E UV–Visible Spectrophotometer, Varian Inc., Poway, CA, USA) because, while there was no aromatic moiety in VA and VAP, only GLP-1 contained one tryptophan residue that strongly absorbed 280 nm wavelength. A series of GLP-1 and VAPG solutions (1, 0.6, 0.2, 0.1, 0.075, 0.05, 0.025 and 0.01 mg/mL) in 0.01 N NaOH were used for calibration curves. By scanning from 190 to 400 nm (600 nm/min scan rate, 1 nm interval), it was confirmed that 280 nm was the most characteristic wavelength for both VAPG and GLP-1 because VAP did not absorb the wavelength. The optical density at 280 nm (OD280) for each sample was acquired as an averaged value from triplicate measurements. 2.5. Insulin secretion test
Fig. 1. Synthetic scheme of GLP-1/polymer conjugate.
was not likely to dissolve in DMSO, it was suspended in DMSO by adding TEA and slightly heated beforehand. The faintly turbid solution became clear with time. The reaction was performed under N2 atmosphere at RT for 24 h followed by dialysis (MWCO 15 kDa) against distilled water for 5 days. After lyophilization, the product was dissolved in 10 mL H2O and further purified by ultrafiltration (Centricons50, MWCO 50 kDa, Amicon Inc., Beverly, MA). Finally, the product was gained by lyophilization and stored at 20 1C for other experiments. Yield=42.0 mg. 2.4. Characterization of the VAPG The chemical conjugation between GLP-1 and VAP was conducted by reverse phase-HPLC (RP-HPLC,
Pancreatic islets were isolated from the pancreata of male Sprague–Dawley (SD) rats (200–250 g) by the conventional collagenase digestion method and by the Ficoll gradient method with minimal modification [12]. Isolated islets were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 5 mM NaHCO3, 20 mM HEPES and 11.2 mM D-glucose under humidified air containing 5% CO2 at 37 1C. After being cultured for 3–5 days, spherical islets that were obtained from five to six SD rats were hand-picked for further experiments. To examine the bioactivity of VAPG, islets were rinsed twice with cold HBSS and transferred into 24well culture plates (20 islets per well). Every well contained 1 mL HEPES-buffered Krebs (HK) solution (2.8 mM glucose, 130 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES, 0.2% BSA, pH 7.4). After 1 h, the solution was exchanged with 0.9 mL of fresh HK
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solution containing various glucose concentrations (2.8, 5.6, 11.1, 16.7 mM). Simultaneously, 0.1 mL of each stimulant (GLP-1 and VAPG) from stock solutions with different concentrations (100 nM and 10 mM; final concentrations became 10 nM and 1 mM) was added. For VAPG, the applied concentrations were equivalent to the GLP-1 content in the polymer. After 1 h incubation at 37 1C under 5% CO2 atmosphere, each solution (750 mL) was taken and centrifuged at 3500 r.p.m. for 3 min. The supernatant (400 mL) was kept at 20 1C till radioimmunoassay (RIA). The amount of insulin in each sample from six independent experiments was determined by the insulin 125I RIA kit (ICN Pharmaceuticals Inc., Costa Mesa, CA). In the dose–response experiment, 10 islets that had been starved with 2.8 mM glucose HK solution were placed in each well of a 96-well culture plate, which contained 180 mL of 5.6 mM glucose HK solution. At the same time, 20 mL VAPG stock solutions with different concentrations (10 and 1 mM, 100, 10, 1 nM) were added into each well. After 1 h incubation at 37 1C under 5% CO2 atmosphere, 180 mL of solution from every well was taken and centrifuged at 3500 r.p.m. for 3 min. RIA was carried out on every 100 mL supernatant obtained from five independent batches. Data were plotted as GLP-1 concentration in the VAPG versus the stimulation index (insulin secretion stimulated by VAPG/insulin secretion without VAPG). Microencapsulation of islets laminated by alginate and poly(L-lysine) was carried out as described elsewhere. For static culture, encapsulated islets with GLP-1 (1 mg/mL) or VAPG (1 mg GLP-1 equivalent/mL) or neither stimulant were placed in transwells (1.0 mm pore), which were inserted into 24-well plates. The medium was changed every single day and insulin concentration was measured by RIA. In semi-dynamic experiments, 300 G HK solution was perfused to encapsulated islets for 1 h after being stabilized in 50 G HK solution for 1 h, and were re-stabilized using 50 G HK solution for another 1 h as reported previously [13].
2.6. Cyclic AMP (cAMP) measurement After undergoing starvation with 2.8 mM glucose HK solution, 10 islets were hand-picked and transferred into a 96-well culture plate. Then, the volume of each well was adjusted to 160 mL with HK solution (no glucose). Immediately, 20 mL of 100 nM VAPG solution in HK solution with no glucose and 20 mL of different HK solutions with varying glucose concentration (2.8, 5.6, 11.1 and 16.7 mM) were added. After 1 h incubation at 37 1C under 5% CO2 atmosphere, the stimulation was stopped by heating for 5 min at 80 1C. Total cellular cAMP for each well was measured by following the user manual of cAMP enzymeimmunoassay kit (Biotrak
cellular communication assays, Amersham Pharmacia Biotech, Arlington Heights, IL). 2.7. Confocal microscopy To visualize the interactions between VAPG or GLP1 and isolated islets, fluorescent probes were attached to each molecule. The GLP-1 (1 mg, 303.2 nmol) and the VAPG (3 mg, 262.8 nmol) were reacted with RITC (16.3 mg, 30.3 nmol) and FITC (10.2 mg, 26.3 nmol) in 1.5 mL DMSO containing 10 mL TEA at room temperature for 24 h, respectively. Each reagent amount could be adjusted by dilution from high concentration stock solutions in DMSO. All reactions were stopped by adding excess H2O (10 mL), followed by ultrafiltration (MWCO 5 kDa for RITC-labeled GLP-1 and MWCO 10 kDa for FITC-labeled VAPG, Ultrafree-CL, Millipore Corporation, Bedford, MA). Each labeled molecule was obtained by lyophilization. Hand-picked islets were incubated with 100 nM of GLP-1, VAPG (GLP-1 equivalent) and VAP (acid content equivalent) for 1 h at 37 1C. After rinsing twice with PBS, islets were fixed with 2% glutaldehyde and 2% formaldehyde in PBS. Confocal microscopic images were gained by Olympus FluoviewTM 300 confocal microscope (Aurora, CO) with red helium neon (633 nm for RITC) and green helium neon (543 nm for FITC). The scan rate was 65,536 pixels/s and the magnitude was 200 by the same method as described previously [14]. Different fluorescent intensities and densities were normalized by calibration of RITC-labeled GLP-1 and FITC-labeled VAPG using microplate fluorescence reader. 2.8. N-terminal analysis by aminopeptidase Aminopeptidase (5 units) was incubated with 7.4 mg/ mL VAPG and 4 mg/mL GLP-1 in 0.5 mL PBS (pH 8.0) for 2.5 h at 37 1C. After filtration by 0.22 mm syringe filter, each sample and histidine were analyzed by RPHPLC (isocratic condition with 90% H2O and 10% AcN, Waters XTerraTM RP18 column, 5 mm pore size, 4.6 250 mm2). 2.9. Statistics For the static bioactivity and cAMP measurement tests, data were analyzed by Student’s t-test. Results from semi-dynamic and long-term culture experiments were analyzed by one-way ANOVA (Po0.05). Quantification of confocal microscopic images were followed by the same method as reported elsewhere [14]. All data from multiple experiments (n ¼ 5–6) are expressed by mean7standard error of means (SEM).
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3. Results and discussion Many groups have developed chemically modified polypeptides or proteins, which include PEG conjugation to monoclonal antibodies, insulin and growth factors. The conjugation of PEG to polypeptides or proteins, however, usually reduces the bioactivity by blocking the active sites and/or changing their original conformation. In this study, the GLP-1/polymer conjugate was originally designed as a component of a rechargeable BAP. Therefore, instead of relating the details of preparing well-defined and biologically active forms of the conjugate as a drug, this report will focus on examining the bioactivity of a particular synthesized conjugate and investigating its specific interaction with isolated rat islets to confirm whether the activity originates from the same action mechanism in the GLP-1 receptor signaling pathway. 3.1. Synthesis and characterization Synthesis of the GLP-1/polymer conjugate (VAPG) was performed as per scheme shown in Fig. 1, which was modified from a previously reported method [15]. To avoid cross-linking of the polymer, all carboxylic groups were activated before reactions and excess amounts of PEG and GLP-1 were added. Unreacted GLP-1 (about 3.3 kDa) was removed by dialysis (MWCO 15 kDa) as well as ultrafiltration (MWCO 50 kDa). The chemical conjugation between GLP-1 and VAP was confirmed by RP-HPLC. While GLP-1 gave a sharp single peak at 29.6 min (Fig. 2A), VAPG showed a dispersed peak from 29 to 36 min (Fig. 2B), due to the polydispersity of the polymer (VAP) and varying degrees of conjugation. Fig. 2C displays the chromatogram of the VAP which does not show any peaks throughout the elution time. Because the transition at 280 nm is a specific characteristic of the tryptophan [16,17], only GLP-1 and GLP-1containing polymer conjugates could absorb the wavelength. The GLP-1 content of VAPG could be also calculated from UV spectrophotometry. The UV spectra were scanned from 200 to 400 nm to confirm that the optical density at 280 nm (OD280) was sensitive enough to quantify the amount of GLP-1 in the VAPG. Calibration curves illustrated correlations between the concentrations of GLP-1 (a, y ¼ 0:0028+1.5018x, R2 ¼ 0:9999) or VAPG (b, y ¼ 0:0005+0.4361x, R2 ¼ 0:9999) and OD280. As a result, it was estimated that VAPG contained 28.9 wt% of GLP-1 (data not shown). 3.2. Bioactivity of VAPG On examining the insulin level of a control experiment without stimulation, in static insulin secretion test it was observed that both VAPG and GLP-1 enhanced the
Fig. 2. Representative RP-HPLC chromatograms of GLP-1 (A), VAPG (B) and VAP (C) at 280 nm. Because only GLP-1 and its polymer conjugate contain tryptophan moiety in their structure, there is no peak in VAP chromatogram.
insulin level as shown in Fig. 3A. VAPG, however, gained statistical significance only at 1 mM (GLP-1 equivalent) while GLP-1 had significantly increased bioactivity at both 10 nM and 1 mM concentrations (Student’s t-test, Po0.1, Po0.05). Although 10 nM VAPG slightly increased insulin secretion over the control, it did not reach the required statistical level. One of the important features in Fig. 3A is the glucosedependent insulin secretion from isolated islets stimulated by GLP-1 or VAPG. The best insulinotropic activity was acquired at 200 G glucose concentration, and the increasing percentage of insulin secretion over the control at 300 G was similar to that at 200 G, although the amount of secreted insulin at 300 G was higher than that at 200 G, which agreed with a previously reported result [18]. Fig. 3B show the glucose-dependent manner of insulin secretion from a semi-dynamic test. All islet microcapsules strongly increased their insulin secretion when the glucose concentration was changed from 50 to 300 G, and returned to basal levels when the media were replaced with 50 G again. At 300 G condition, the VAPG gained outstanding bioactivity among the three groups (oneway ANOVA, Po0.05). In addition, Fig. 3C show that the microencapsulated islets containing VAPG strongly increased insulin secretion while those with GLP-1 did not. This result proves that the VAPG is successfully retained inside a BAP for a long time in contrast to GLP-1 leakage. In another experiment, insulinotropic activity of the VAPG inside the microcapsules lasted for 2 months and, moreover, VAPGcontaining capsules and control ones did not show
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islet stimulation by GLP-1 occurs only when the glucose level is high after a meal, not continuous [28], (4) the literature information tells us that transplanted encapsulated islets cannot survive for many years, leading to the design of a rechargeable system as previously suggested by us [1], and (5) GLP-1 may present islet proliferation capacity [9]. With all these reasons, the coencapsulation of GLP-1/polymer conjugate with islets may provide more advantages over metabolic load and the whole component including islets and VAPG can be refreshed when their functionality is exhausted. The physiological level of GLP-1 in the body is 5–15 pM, which increases up to 20–30 pM after meal ingestion [8]. For isolated islets, however, a higher dose of GLP-1 seems to be required to stimulate insulin secretion; the insulinotropic activity of GLP-1 against isolated rat pancreatic islets began at 2.5 nM and was saturated at 250 nM [18]. While the half-maximal effective dose (ED50) for native GLP-1 was reported as 25 nM, from the dose–response curve, the ED50 of VAPG was about 55 nM obtained by regression with a sigmoidal model equation (Fig. 4). 3.3. N-terminal analysis We hypothesized that the slightly lowered insulinotropic activity of GLP-1 moiety in VAPG was from random conjugation of VAP to GLP-1. Study of the structure–activity relationship informed us that Nterminal amino acids of GLP-1 were important for insulinotropic activity while C-terminal had relatively less influence on the bioactivity (Table 1) [21–25]. The histidine moiety at the end of N-terminus (His7) plays a central role in the GLP-1 activity for binding to GLP-1 receptors [21,22] and for producing cAMP [23–27]. Liberation of two amino acids (His7-Ala8) from the N-
Fig. 3. (A) Insulinotropic activity of GLP-1 and VAPG to isolated rat islets. Results are presented as mean7SEM from six independent experiments. Po0.01, Po0.5 from the Student’s t-test. (B) Semidynamic insulin secretion test by microencapsulation of islets with VAPG or GLP-1. Po0.05 by one-way ANOVA. (C) Insulin secretion profile of islet microcapsules from 2-week static culture. Po0.05 by one-way ANOVA.
different islets viabilities during the period (data not shown). The islet stimulation has been justified by noting that (1) the insulin secretion capacity of islets after isolation and encapsulation is approximately 10–15% of residing islets in a pancreas due to poor oxygen supply and damage during isolation and encapsulation, giving us enough room in metabolic load [19], (2) the oxygen supply issue can be addressed by introducing macromolecular oxygen carriers as published by us [20], (3)
Fig. 4. Dose–response curve of the VAPG. Each point was acquired from five independent experiments and presented as mean7SEM a stimulation index ¼(insulin secretion with VAPG)/(insulin secretion without VAPG).
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Table 1 Summary of amino acids that play a key role in the bioactivity of GLP-1 (7–36) or GLP-1 (7–37) 7
10
15
20
25
30
35
References
HAE HAE HAE HAE HAE
GTFTS GTFTS GTFTS GTFTS GTFTS
DVSSY DVSSY DVSSY DVSSY DVSSY
LEGQA LEGQA LEGQA LEGQA LEGQA
AKEFI AKEFI AKEFI AKEFI AKEFI
AWLVK AWLVK AWLVK AWLVK AWLVK
GR GR GR GRG GR
Adelhorst et al. [21] Gallwitz et al. [22] Watanabe et al. [23] Gefel et al. [24] Suzuki et al. [25]
Each letter abbreviates corresponding amino acid. The position of amino acid in the sequence of GLP-1 (1–37) is indicated at the upper row. Based on the results from each report, the boxed amino acids are important to maintain the bioactivity of GLP-1, while the underlined amino acids do not significantly influence on the GLP-1 activity.
terminus of GLP-1 by dipeptidyl peptidase IV not only diminished the bioactivity of GLP-1 but also antagonized the activity of GLP-1 [10,11,28,29]. In addition, it was reported that modifications of His7 to generate dipeptidyl peptidase IV-resistant forms of GLP-1 significantly lowered both the bioactivity and the cAMP producing ability of GLP-1 [11,27]. Because protein/polymer conjugates are not a good substrate for an automatic amino acid sequencer and several reports exist to analyze protein terminus using peptidases, we attempted to analyze the N-terminal using aminopeptidase and RP-HPLC for monitoring the chemical modification [30–32]. Fig. 5A illustrates the chromatograms of histidine itself or His7 released through enzymatic degradation by an aminopeptidase that can liberate every single amino acid from the end of N-terminus. Fig. 5B shows that much less histidine (relative amount of 15.6% by integration from 2.30 to 2.65 min) was released by the degradation of VAPG than from the native GLP-1. This means that most of His7 at the end of GLP-1 was employed for conjugation to carboxylic groups of VAP. Because two lysine moieties (Lys26 and Lys34) hardly influence the activity of GLP-1 when substituted with other amino acids (Table 1) or when coupled to other chemicals [33], the bioactivity of VAPG was attributed to the conjugate population containing intact His7. Although the method employed here, aminopeptidase degradation–HPLC coupling, is not a common one, recent analytical methods using peptidase to analyze peptides and proteins have been employed in developing new substrates and drugs, assaying enzyme–substrate reaction, and examining structural biology become important tools [34,35]. Such method was suggested as an alternative way to determine peptide sequence in a highmolecular-weight polymer/peptide conjugation system. 3.4. Specific interactions of VAPG with isolated rat islets Figs. 3A and B already suggested evidence of specific interactions because the insulin secretion stimulated by VAPG showed glucose dependence similar to GLP-1
Fig. 5. (A) Representative RP-HPLC chromatograms of VAPG and GLP-1 (after treatment of aminopeptidase), and histidine. (B) The relative amount of free N-terminal histidine moieties in the VAPG calculated from RP-HPLC (n ¼ 3).
[18,36,37]. Fig. 6 displays glucose-dependent cAMP production stimulated by VAPG, which is a marker of the GLP-1 action mechanism. Upon comparison with the control levels, the cAMP level significantly increased at 200 and 300 G glucose concentrations in the presence of VAPG, while it was negligible at low glucose
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Fig. 6. Total cellular cAMP level with or without treating 100 nM VAPG. Data were analyzed by Student’s t-test (Po0.01, n ¼ 3) and presented as mean7SEM.
concentrations (Po0.01 by Student’s t-test). To obtain more direct evidence for the specific interaction of VAPG with isolated rat islets, confocal microscopy was used to visualize fluorescently labeled VAPG or GLP-1. Fig. 7A presents the confocal microscopic images of islets treated with FITC-labeled VAPG (a, green) and RITC-labeled GLP-1 (b, red). Both GLP-1 and VAPG seem to be incorporated inside the pancreatic cells, which stain the cytoplasmic compartment without reaching nuclei. The relationship between islet size (islet diameter, mm) and fluorescence intensity per unit volume (mm3) was quantified (Fig. 7B) by a method described elsewhere [13]. The plot indicates that the binding affinity of VAPG is less than that of GLP-1. This result implies that VAPG could interact with rat islets by means of GLP-1 receptors, although its binding affinity was lower than that of GLP-1 due to polymer conjugation to His7.
Fig. 7. (A) Confocal microscopic images of islets treated with RITC-labeled GLP-1 (a) and with FITC-labeled VAPG (b). (B) Quantification results of confocal microscopic images that show the different binding affinity between labeled GLP-1 (n ¼ 23) and VAPG (n ¼ 23).
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4. Conclusion A GLP-1/polymer conjugate (VAPG) was synthesized and conjugation was characterized by RP-HPLC. Using the specific transition of a tryptophan moiety in GLP-1 structure at 280 nm, UV spectrophotometry revealed that there existed 29.9 wt/wt% of GLP-1 in VAPG. Static insulin secretion tests showed that VAPG could stimulate isolated rat islets up to 200% (1 mM GLP-1 equivalent), although bioactivity of the polymer was slightly less than that of native GLP-1. By using islet microcapsulation, it was shown that bioactivity was maintained at least 2 weeks. The experiment also showed that insulinotropic activity of VAPG increased as the glucose concentration became higher, which is a physiological property of native GLP-1. Dose–response curve of VAPG showed that ED50 was about 55 nM. This reduced bioactivity resulted from the conjugation of polymer to the amine groups of GLP-1, especially, Nterminal histidine. Aminopeptidase treatment combined with RP-HPLC proved that only 15.6% of histidine was free without polymer conjugation. However, the VAPG still had binding affinity to GLP-1 receptors on isolated islets, which was confirmed by confocal microscopy. Quantitative analysis of confocal microscopic images revealed that the binding affinity of VAPG was less than that of GLP-1. The specific interaction was also confirmed by cAMP measurement. The cAMP level increased in a glucose-dependent manner with significant distinction from the control level at high glucose concentrations. The long-term insulinotropic activity of VAPG in conventional alginate and poly L-lysine islet microcapsules is under investigation.
[7]
[8]
[9] [10]
[11]
[12] [13]
[14]
[15]
[16]
[17]
[18]
[19]
Acknowledgement This research was supported by funding from NIH DK 56884 in USA and by BK 21 program in South Korea.
[20]
[21]
[22]
References [1] Hou QP, Bae YH. Biohybrid artificial pancreas based on macrocapsule device. Adv Drug Deliv Rev 1999;35:271–87. [2] Lanza RP, Sullivan SJ, Chick WL. Islet transplantation with immunoisolation. Diabetes 1992;41:1503–10. [3] Suzuki K, Bonner-Weir S, Hollister-Lock J, Colton CK, Weir GC. Number and volume of islets transplanted in immunobarrier devices. Cell Transplant 1998;7:47–52. [4] Park K-H, Kim S, Bae YH. Interaction of sulfonylureaconjugated polymer with insulinoma cell line of MIN6 and its effect on insulin secretion. J Biomed Mater Res 2001;55:72–8. [5] Kikuchi A, Bae YH, Kim SW. Stimulatory effect of a sulfonylurea analogue and its polymer conjugate on insulin secretion from rat islets. Biotechnol Prog 1994;43:673–7. [6] Hwang JS, Chae SY, Lee MK, Bae YH. Synthesis of sulfonylurea conjugated copolymer via PEO spacer and its in vitro short-term
[23]
[24]
[25]
3605
bioactivity in insulin secretion from islets of Langerhans. Biomaterials 1998;19:1189–95. Gappa H, Baudysˇ M, Koh JJ, Kim SW, Bae YH. The effect of zinc-crystallized glucagon-like peptide-1 on insulin secretion of macroencapsulated pancreatic islets. Tissue Eng 2001;7:35–44. Ahre´n B. Glucagon-like peptide-1 (GLP-1): a gut hormone of potential interest in the treatment of diabetes. Bioassays 1998;20:642–51. Kieffer TJ, Habener F. The glucagon-like peptides. Endocr Rev 1999;20:876–913. Ritzel U, Leonhardt U, Ottleben M, Ru¨hmann A, Eckart K, Spiess J, Ramadori G. A synthetic glucagon-like peptide-1 analog with improved plasma stability. J Endocrinol 1998;158:93–102. Siegel EG, Scharf G, Gallwitz B, Mentlein R, Morys-Wortmann M, Fo¨lsch UR, Schmidt WE. Comparison of the effect of native glucagon-like peptide I and dipeptidyl peptidase IV-resistant analogues on insulin release from rat pancreatic islets. Eur J Clin Invest 1999;29:610–4. Lacy PE, Kostianovsky M. Method for the isolation of intact islets of Langerhans from rat pancreas. Diabetes 1967;16:35–9. Kim S, Chae SY, Na K, Kim SW, Bae YH. Insulinotropic activity of sulfonylurea/pullulan conjugate in rat islet microcapsule. Biomaterials 2003;24:4843–51. Kim S, Bae YH. Visual evidence and quantification of interaction of polymeric sulfonylurea with pancreatic islet. Biomacromolecules 2003;4:1550–7. Liu F, Song SC, Mix D, Baudysˇ M, Kim SW. Glucose-induced release of glycosylpoly(ethylene glycol) insulin bound to a soluble conjugate of concanavalin A. Bioconjug Chem 1997;8:664–72. Kazmin D, Edwards RA, Turner RJ, Larson E, Starkey J. Visualization of proteins in acrylamide gels using ultraviolet illumination. Anal Biochem 2002;301:91–6. He Q-Y, Mason AB, Lyons BA, Tam BM, Nguyen V, Macgillivray RTA, Woodworth RC. Spectral and metal-binding properties of three single-point tryptophan mutants of the human transferrin N-lobe. Biochem J 2001;354:423–39. Schmidt WE, Siegel EG, Creutzfeldt W. Glucagon-like peptide-1 but not glucagon-like peptide-2 stimulates insulin release from isolated rat pancreatic islets. Diabetologia 1985;28:704–7. Arita S, Une S, Ohtsuka S, Kawahara T, Kasraie A, Smith CV, Mullen Y. Increased islet viability by addition of beraprost sodium to collagenase solution. Pancreas 2001;23:62–7. Chae SY, Kim SW, Bae YH. The effect of crosslinked hemoglobin on functionality and viability of microencapsulated pancreatic islets. Tissue Eng 2002;8:379–94. Adelhorst K, Hedegaard BB, Knudsen LB, Kirk O. Structure– activity studies of glucagon-like peptide-1. J Biol Chem 1994;269:6275–8. Gallwitz B, Witt M, Paetzold G, Morys-wortmann C, Zimmermann B, Eckart K, Fo¨lsch UR, Schmidt WE. Structure/activity characterization of glucagon-like peptide-1. Eur J Biochem 1994;225:1151–6. Watanabe Y, Kawai K, Ohashi S, Yokota C, Suzuki S, Yamashita K. Structure–activity relationships of glucagon-like peptide-1 (7–36) amide: insulinotropic activities in perfused rat pancreases, and receptor binding and cyclic AMP production in RINm5F cells. J Endocrinol 1994;140:45–52. Gefel D, Hendrick GK, Mojsov S, Habener J, Weir GC. Glucagon-like peptide-I analogs, effects on insulin secretion and adenosine 30 ,50 -monophosphate formation. Endocrinology 1990;126:2164–8. Suzuki S, Kawai K, Ohashi S, Mukai H, Yamashita K. Comparison of the effects of various C-terminal and N-terminal fragment peptides of glucagon-like peptide-1 on insulin and glucagon release from the isolated perfused rat pancreas. Endocrinology 1989;125:3109–14.
ARTICLE IN PRESS 3606
S. Kim et al. / Biomaterials 26 (2005) 3597–3606
[26] Drucker DJ, Philippe J, Mojsov S, Chick WL, Habener JF. Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line. Proc Natl Acad Sci USA 1987;84:3434–8. [27] Xiao Q, Giuere J, Parisien M, Jeng W, St-Pierre SA, Brubaker PL, Wheeler MB. Biological activities of glucagon-like peptide-1 analogues in vitro and in vivo. Biochemistry 2001;40:2860–9. [28] Kieffer TJ, McIntosh CHS, Pederson RA. Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV. Endocrinology 1995;136:3585–96. [29] Deacon CF, Hughes TE, Holst JJ. Dipeptidyl peptidase IV inhibition potentiates the insulinotropic effect of glucagon-like peptide 1 in the anesthetized pig. Diabetes 1998;47:764–9. [30] Sechi S, Chait BT. A method to define the carboxyl terminal of proteins. Anal Chem 2000;72:3374–8. [31] Breci LA, Tabb DL, Yates III JR, Wysocki VH. Cleavage Nterminal to proline, analysis of a database of peptide tandem mass spectra. Anal Chem 2003;75:1963–71. [32] Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C. Identifying proteins from two-dimensional gels by
[33]
[34]
[35]
[36]
[37]
molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci USA 1993;90:5011–5. Chou JZ, Place GD, Waters DG, Kirkwood JA, Bowsher RR. A radioimmunoassay for LY315902, an analog of glucagon-like insulinotropic peptide, and its application in the study of canine pharmacokinetics. J Pharm Sci 1997;86:768–73. Ribo M, Bosch M, Torrent G, Benito A, Beaumelle B, Vilanova M. Quantitative analysis, using MALDI-TOF mass spectrometry, of the N-terminal hydrolysis and cyclization reactions of the activation process of onconase. Eur J Biochem 2004;271:1163–71. Grant SK, Sklar JG, Cummings RT. Development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates. J Biomol Screen 2002;7:531–40. Go¨ke R, Wagner B, Fehmann H-C, Go¨ke B. Glucose-dependency of the insulin stimulatory effect of glucagon-like peptide-1 (7–36) amide on the rat pancreas. Res Exp Med 1993;193: 97–103. Hargrove DM, Nardone NA, Persson LM, Parker JC, Stevenson RW. Glucose-dependent action of glucagon-like peptide-1 (7–37) in vivo during short- or long-term administration. Metabolism 1995;44:1231–7.