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Pioglitazone acutely influences glucose-sensitive insulin secretion in normal and diabetic human islets
BBRC Biochemical and Biophysical Research Communications 351 (2006) 750–755 www.elsevier.com/locate/ybbrc
Pioglitazone acutely influences glucose-sensitive insulin secretion in normal and diabetic human islets ˚ ke Sjo¨holm, Qimin Zhang Fan Zhang, A
*
Karolinska Institutet, Department of Internal Medicine, Stockholm South Hospital, SE-11883 Stockholm, Sweden Received 19 October 2006 Available online 30 October 2006
Abstract We have studied acute effects of the PPARc agonist pioglitazone in vitro on human islets from both non-diabetic and type 2 diabetic subjects. In 5 mM glucose, pioglitazone caused a transient increase in insulin secretion in non-diabetic, but not diabetic, islets. Continuous presence of the drug suppressed insulin release in both non-diabetic and diabetic islets. In islets from non-diabetic subjects, both high glucose and tolbutamide-stimulated insulin secretion was inhibited by pioglitazone. When islets were continuously perifused with 5 mM glucose, short-term pretreatment with pioglitazone caused approximately 2-fold increase in insulin secretion after drug withdrawal. Pioglitazone pretreatment of diabetic islets restored their glucose sensitivity. Examination of cytosolic free Ca2+ concentration ([Ca2+]i) in non-diabetic islets revealed slight Ca2+ transient by pioglitazone at 3 mM glucose with no significant changes at high glucose. Our data suggest that short-term pretreatment with pioglitazone primes both healthy and diabetic human islets for enhanced glucosesensitive insulin secretion. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Pioglitazone; Peroxisome proliferator-activated receptor (PPAR); Glucose sensitivity; Insulin secretion; Cytosolic free Ca2+ concentration ([Ca2+]i); Human islets; Diabetes
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, composed of three major isoforms (a, b/d, and c) that have been identified to date [1,2]. The receptor isoforms show tissue and cell specificity, and exert related but distinct functions upon activation [3–7]. Among the PPAR isoforms, PPARc is the best characterized and found to play an important role in the regulation of energy homeostasis [1,2,8–10]. Thiazolidinediones (TZDs), agonists of PPARc, reduce insulin resistance and thus influence free fatty acid flux and blood glucose levels. The glucose-lowering effect of TZDs is attributed to increased peripheral glucose disposal and decreased hepatic glucose output [8,11,12]. Synthetic ligands for PPARc are of particular interest for treating patients with type 2 diabetes because they restore the sensitivity of the tissues to insulin [8,9,11]. Pioglitazone is an orally administered insulin*
Corresponding author. Fax: +46 8 6163287. E-mail address: [email protected] (Q. Zhang).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.10.103
sensitizing TZD agent that activates PPARc, leading to the increased transcription of genes encoding various proteins regulating glucose and lipid metabolism [3,11]. In contrast to its action on insulin target tissues, little is known about the direct action of the PPARc agonist on pancreatic b-cell function and the b-cell sparing effects noted in clinical trials have generally been assumed to be secondary to the decreased insulin resistance. In the present study, we have studied the direct actions of the PPARc agonist pioglitazone on insulin secretion and Ca2+ handling in human islets from non-diabetic and diabetic subjects during short-term treatment in vitro. Materials and methods Pioglitazone was graciously donated by Takeda Pharmaceuticals North America, Inc. (Lincolnshire, IL). Fura-2/acetoxymethylester (Fura-2/AM) was from Sigma (St. Louis, MO). Bio-Gel P-4 (fine, 65 ± 20 lm, wet) was from Bio-Rad Laboratories (Hercules, CA). Insulin ELISA kits were from Mercodia (Uppsala, Sweden).
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Human islets Pancreatic islets from adult non-diabetic or diabetic donors were obtained from the Uppsala University Hospital facility for isolation of human islets from Scandinavian brain-dead donors. The 56-year-old diabetic donor was diagnosed with type 2 diabetes for 10 years and received insulin 90 U daily before death with BMI 33, HbA1c 9.5%, and C-peptide level of 0.3 nmol/l. The normal islets were from adult braindead non-diabetic patients. The islets were maintained in RPMI-1640 tissue culture medium in the presence of 5.5 mM glucose, supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 lg/ ml streptomycin, and were used for experiments 8 days after isolation. All experiments were approved by the Karolinska Ethics Committee. Insulin secretion Column perifusion. Dynamics of insulin secretion was studied by column perifusion as described [13,14]. About 100 human islets were carefully mixed with a small volume of pre-wetted Bio-Gel P-4, placed on top of each of the columns, and perifused with KRBH (Krebs–Ringer bicarbonate Hepes) buffer containing (in mM): 135 NaCl, 3.6 KCl, 5 NaHCO3, 0.5 NaH2PO4, 0.5 MgCl2, 1.5 CaCl2, and 10 Hepes, pH 7.4, with 0.1% BSA. Batch incubation. Islets were washed three times in KRBH buffer. Equal number of islets of comparable size was placed in each well of a 24-well plate, pre-added with 1 ml of the buffer in the presence of pioglitazone or DMSO at 3 mM glucose. After pre-incubation for 10 min at 37 °C, glucose or glucose with tolbutamide was added in the corresponding wells and incubated for 20 min. The medium was collected and centrifuged, the supernatant was for insulin assay. The islets were collected and lysed for protein assay (Bio-Rad). Measurement of [Ca2+]i. Fura-2 loaded cells were perifused with KRBH buffer in the presence of 3 mM glucose and 0.1% BSA, at 37 °C. Measurements of [Ca2+]i were performed on single cells prepared from islets as described [15,16] using a time-sharing spectrofluorometer (RM-5 System, PhotoMed, Denmark).
Fig. 1. Bimodal effects of pioglitazone on insulin secretion dynamics in normal human islets. Human islets from non-diabetic donors were packed in a micro-column and perifused with KRBH buffer containing 0.1% BSA and 5 mM glucose. Pioglitazone (Pio; 10 lM) or vehicle (DMSO)containing buffer (as control) was introduced. Data show dynamic changes in insulin secretion from four separate experiments using islets from four different subjects. The average of the insulin content in the fractions collected before the drug addition (Control) was considered as 100%. Insulin content in each fraction was divided by the average. Results are expressed as percentage changes of insulin secretion. The insert figure shows the suppressed insulin secretion during perifusion with pioglitazone, and enhanced insulin secretion after withdrawal of the drug (peak b) in the four experiments. *P < 0.05, by ANOVA.
Results Bimodal effects of pioglitazone on glucose-sensitive insulin secretion in human islets When human islets from non-diabetic subjects were perifused with 5 mM glucose, administration of 10 lM pioglitazone elicited an approximately 20% transient increase in insulin secretion, followed by a suppression of insulin release from the islets (Fig. 1). The inhibitory effect of pioglitazone remained until withdrawal of the drug, resulting in up to 40% of decrease in insulin secretion, compared to those before the drug addition. Immediately after withdrawal of pioglitazone, insulin secretion was increased approximately threefold compared to the levels during pioglitazone administration, and twofold compared to the levels before addition of the drug. The same response pattern to pioglitazone was noted in all experiments performed using islets from different donors, but not in those with buffer changes alone.
Fig. 2. Effects of pioglitazone on glucose- and tolbutamide-stimulated insulin secretion in human islets. Human islets from non-diabetic donors were pre-incubated in the presence of pioglitazone (Pio) or vehicle (DMSO) for 10 min in KRBH buffer containing 0.1% BSA and 3 mM glucose at 37 °C, followed by addition of glucose (20 mM) or glucose with tolbutamide (Tol; 100 lM) in the corresponding wells. Insulin secretion from the islets in each well was normalized by protein content measured in the lysed islets. Results from four separate experiments using islets from three different subjects are shown and expressed as means ± SEM. *P < 0.05 by ANOVA.
Pioglitazone inhibits glucose-induced insulin secretion in normal human islets The effect of pioglitazone on insulin secretion was further investigated by acute exposure of human islets to the
drug in the presence of low and high glucose (Fig. 2). Compared to the inhibitory effect of pioglitazone on insulin secretion at 5 mM glucose, the drug did not result in
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significant changes in insulin secretion at a glucose concentration of 3 mM during 20-min incubation. In the presence of 20 mM glucose, however, pioglitazone caused approximately 50% decrease in the glucose-stimulated insulin release. In order to examine whether the pioglitazone also interfered with insulin secretion stimulated by other insulin secretagogues, islets were stimulated with the sulfonylurea tolbutamide in the presence of low and high glucose. Pioglitazone inhibited approximately 25% of tolbutamide-stimulated insulin secretion at an ambient glucose of 3 mM. At the glucose concentration of 20 mM, the effect of tolbutamide was inhibited to a similar extent (27%).
human islet cells. After stimulation with high glucose, the cells were perifused with 3 mM glucose in the presence of pioglitazone for 10 min, followed by second pulse of glucose stimulation in the presence of the drug. Pioglitazone failed to alter glucose-induced rise in [Ca2+]i (Fig. 3B), while it inhibited insulin secretion stimulated by the sugar (Fig. 2). Pioglitazone restored glucose-sensitive insulin secretion in islets from type 2 diabetic patients In order to investigate whether the enhanced glucose sensitivity evoked by pioglitazone in non-diabetic islets would occur also under diabetic conditions, islets from a type 2 diabetic subject were placed in the micro-columns and perifused with a buffer containing 5 mM glucose (Fig. 4). Insulin secretion was suppressed during pioglitazone administration (Fig. 4, insert). Similar to the effect in non-diabetic islets (Fig. 1), pioglitazone evoked an immediate increase in insulin secretion from the diabetic islets after withdrawal of the drug, resulting in insulin secretion being increased approximately threefold compared to the levels during pioglitazone administration, and twofold compared to basal levels before addition of the drug. In contrast to the non-diabetic islets (Fig. 1), there was no pioglitazoneinduced transient increase in insulin secretion in the diabetic islets (Fig. 4, insert). The effect of pioglitazone on recovery of glucose sensitivity in diabetic islets was also investigated using two micro-columns, performed in parallel (Fig. 4). When the diabetic islets were perifused with 5 mM glucose, followed by stimulation with 20 mM glucose, the islets failed to respond to the glucose stimulation with increased insulin output. Exposure of islets to pioglitazone restored the
Pioglitazone does not significantly alter [Ca2+]i in human islets Since [Ca2+]i plays a crucial role in glucose-stimulated insulin secretion, we further investigated whether pioglitazone influences [Ca2+]i in human islet cells loaded with fura2 (Fig. 3). Islet cells were perifused with 3 mM or 5 mM glucose, during which a dose of 10 lM pioglitazone was applied. The cells were stimulated with glucose at the end of the experiments to verify the b-cell response. At 3 mM glucose, addition of pioglitazone elicited a Ca2+ transient that rapidly disappeared despite the continuous presence of the drug (Fig. 3A), while addition of same amount of vehicle (DMSO) was without effect. In contrast, application of pioglitazone at 5 mM glucose affected [Ca2+]i neither during drug administration nor after drug withdrawal (Fig. 3A, inset). In order to examine whether pioglitazone-induced inhibition on glucose-stimulated insulin secretion was associated with a change in [Ca2+]i, the effect of pioglitazone on glucose-stimulated rise in [Ca2+]i was investigated in A
B
Fig. 3. Effects of pioglitazone on [Ca2+]i in non-diabetic human islets. [Ca2+]i was measured in single cells prepared from non-diabetic human islets as described in Materials and methods. Cells were perifused with 3 mM glucose. Addition of pioglitazone (Pio; 10 lM) is indicated (A). Similar experiments were also performed when the cells were perifused with buffer containing 5 mM glucose. The changes in the Ca2+ transient amplitude elicited by pioglitazone at 3 or 5 mM glucose are shown in the insert figure, summarizing results derived from four separate experiments using islets from three subjects. Levels of fluorescence ratios before drug addition were considered as basal. Bars indicate means ± SEM and *P < 0.05 by ANOVA. The effects of pioglitazone on glucose-induced rises in [Ca2+]i are shown in (B). After the first stimulation with 20 mM glucose, cells were perifused with pioglitazone at 3 mM glucose for 10 min before and during a second glucose stimulation. A representative experiment out of four separate experiments using islets from three non-diabetic subjects is shown. The effects of pioglitazone on [Ca2+]i at 20 mM glucose are summarized in the insert figure. Bars represent mean percentage of the glucose-stimulated peak amplitude (±SEM) before pioglitazone addition (peak 1), compared to the peak stimulated by glucose in the presence of pioglitazone.
F. Zhang et al. / Biochemical and Biophysical Research Communications 351 (2006) 750–755
Fig. 4. Pioglitazone restores glucose-sensitive insulin secretion in human islets from a type 2 diabetic patient. Equal amounts of human islets from the diabetic patient were packed in two micro-columns and run in parallel. Islets were continuously perifused with KRBH buffer with 0.1% BSA and 5 mM glucose (G) in the presence of pioglitazone (10 lM Pio; empty circles) or vehicle (DMSO, filled circles) 10 min before and during the indicated period. Results from three experiments are shown and expressed as means ± SEM. *P < 0.05 by ANOVA. The insert figure shows a representative experiment performed on the human diabetic islets, perifused with 5 mM glucose as in Fig. 1. The average of insulin contents in the fractions collected before high glucose (Fig. 4) or pioglitazone (insert) additions was arbitrarily set at 100%.
glucose response, resulting in a 10-fold increase in insulin secretion compared to basal release (Fig. 4). Discussion Pioglitazone has been developed for the treatment of type 2 diabetes mellitus. The improved glycemic control by the drug is thought to be due to amplification of the post-receptor actions of insulin in target tissues such as skeletal muscle. By applying the drug in vitro to human islets from both non-diabetic and diabetic subjects, we show here that pioglitazone may also directly affect the function of the insulin-producing b-cell by influencing the glucose sensitivity of the b-cell. PPARc expression has been detected in islet cells [17–21] and in clonal b-cell lines [22]. The function of PPARc in the b-cell remains controversial [23–28]. The present study on human islets reveals that acute administration of pioglitazone rapidly inhibits glucose-induced insulin secretion at 5 mM or higher concentrations of glucose in islets from both diabetic and non-diabetic subjects. A slight transient increase in insulin secretion by the drug was observed in islets from all non-diabetic, but not in diabetic, subjects studied. The latter observation may indicate that the factor responsible for the transient effect of pioglitazone on insulin release is among the factors impaired during development of type 2 diabetes. The suppressive effect of pioglitazone on insulin secretion observed in the present studies is consistent with clinical observations where TZD treatment for several months results in suppressed levels of plasma insulin [29–31], and the report from rat islets
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using another PPARy agonist troglitazone [28]. This effect of pioglitazone should not be a result of cytotoxicity of the drug as incubation of human islets for 48 h at the same dosage as used here did not result in any significant changes in cell viability, evaluated by fluorescence microscopy after islets were vital stained with propidium iodide and bisbenzimide (unpublished observations). In addition, the pioglitazone-treated cells responded to glucose in terms of [Ca2+]i to a similar extent as control cells in the present study. In the clinical setting, the decrease in plasma insulin levels is believed to reflect decreased insulin resistance, and could be beneficial in arresting the natural unfolding of diabetes by sparing the b-cell and thereby impacting disease progression. Following the pioglitazone-induced suppression of insulin secretion at 5 mM glucose, insulin secretion was immediately increased upon withdrawal of the drug. This suggests that the lowering effect of pioglitazone on insulin secretion was not a toxic effect of the drug on hormone output at the concentration we employed, but rather that pretreatment of islets with the PPARc agonist enhanced or ‘‘primed’’ the sensitivity of the b-cell to glucose. The over-response of the cells to glucose after drug removal implies that the suppressed insulin secretion during pioglitazone exposure is reversible and is responsible for restoration of glucose sensitization or priming of insulin exocytosis. Importantly, and remarkably, pioglitazone restored a robust glucose-sensitive insulin secretion in islets from the type 2 diabetic patient, in which glucose sensitivity was completely lost. The mechanism by which pioglitazone acutely confers glucose competence to the diabetic b-cell is unknown, and whether PPARc is involved in mediating the acute effects of the drug remains unclear [32–35]. Most cellular responses contributing to the antidiabetic effect of TZDs occur over a long time (several weeks) and require altered gene transcription [36] and cannot explain the acute effect of pioglitazone noted here. Changes in islet morphology and gene expression after long-term treatment may lead to different results when evaluating the direct effect of the drug on insulin secretion [37]. In animal models, pioglitazone was shown to improve glucose-induced insulin secretory capacity by protecting the b-cell from oxidative stress [24,38], a feature of the drug that may also be functional in the human b-cells. However, unlike the animal models, the essential effect of pioglitazone on glucose-induced insulin secretion in human islets was suppressive and the enhanced glucose-stimulated secretory response was only observed after withdrawal of the drug. This action of pioglitazone in human islets may suggest a direct effect of the drug on the insulin exocytotic machinery. In agreement with this hypothesis, the postpioglitazone effect of glucose-stimulated insulin secretion was rapid and both glucose- and tolbutamide-induced insulin releases were suppressed by the drug. A direct action of pioglitazone on insulin exocytosis was also reflected by its effect on [Ca2+]i. Pioglitazone induced a slight increase in [Ca2+]i at 3 mM glucose without a
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significant change in insulin secretion. On the other hand, insulin secretion was suppressed by the drug at glucose concentrations of 5 and 20 mM without any changes in [Ca2+]i. This dissociation of glucose-induced insulin secretion and rise in [Ca2+]i does not support an involvement of activation of glucokinase through interaction of PPARc with the peroxisomal proliferator response element (PPRE) in the b-cell-specific glucokinase promoter [39] in the acute effect of pioglitazone on insulin secretion. It rather suggests a late action of the in drug the process of insulin secretion, distal to [Ca2+]i increase, namely the insulin exocytotic machinery. In comparison to human islet cells, pioglitazone stimulated insulin secretion in hamster insulinoma HIT-T15 cells, associated with an increased [Ca2+]i [26]. The different roles of pioglitazone on intracellular Ca2+ handling in different species may explain the discrepancies in acute effects of pioglitazone on insulin secretion. Taken together, the present results suggest that pioglitazone acutely induces a rapid, reversible, and Ca2+-independent inhibition of glucose-induced insulin secretion in human islets, but enhances or restores glucose sensitivity in healthy or diabetic human islets after immediate removal of the drug, therefore indicating a direct beneficial effect of pioglitazone on b-cell function.
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Acknowledgments Financial support was received from Takeda Pharmaceuticals North America, the Swedish Medical Research Council (# 72X-12550, 72X-14507, and 72P-14787), Berth von Kantzow’s Foundation, Petrus, and Augusta Hedlund’s Foundation, the Nutricia Research Foundation, the European Foundation for the Study of Diabetes, the Swedish Society of Medicine, the Sigurd, and Elsa Golje Memorial Foundation, Svenska Fo¨rsa¨kringsfo¨reningen, Svenska Diabetesstiftelsen, Magn. Bergvall Foundation, Barndiabe˚ ke Wiberg’s Foundation, Trygg-Hansa’s tesfonden, A Research Foundation, Torsten, and Ragnar So¨derberg’s Foundations, Harald Jeansson’s, and Harald and Greta Jeansson’s Foundations, Tore Nilson’s Foundation for Medical Research, Fredrik, and Inger Thuring’s Foundation, and Syskonen Svensson’s Fund.
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Pioglitazone acutely influences glucose-sensitive insulin secretion in normal and diabetic human islets ˚ ke Sjo¨holm, Qimin Zhang Fan Zhang, A
*
Karolinska Institutet, Department of Internal Medicine, Stockholm South Hospital, SE-11883 Stockholm, Sweden Received 19 October 2006 Available online 30 October 2006
Abstract We have studied acute effects of the PPARc agonist pioglitazone in vitro on human islets from both non-diabetic and type 2 diabetic subjects. In 5 mM glucose, pioglitazone caused a transient increase in insulin secretion in non-diabetic, but not diabetic, islets. Continuous presence of the drug suppressed insulin release in both non-diabetic and diabetic islets. In islets from non-diabetic subjects, both high glucose and tolbutamide-stimulated insulin secretion was inhibited by pioglitazone. When islets were continuously perifused with 5 mM glucose, short-term pretreatment with pioglitazone caused approximately 2-fold increase in insulin secretion after drug withdrawal. Pioglitazone pretreatment of diabetic islets restored their glucose sensitivity. Examination of cytosolic free Ca2+ concentration ([Ca2+]i) in non-diabetic islets revealed slight Ca2+ transient by pioglitazone at 3 mM glucose with no significant changes at high glucose. Our data suggest that short-term pretreatment with pioglitazone primes both healthy and diabetic human islets for enhanced glucosesensitive insulin secretion. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Pioglitazone; Peroxisome proliferator-activated receptor (PPAR); Glucose sensitivity; Insulin secretion; Cytosolic free Ca2+ concentration ([Ca2+]i); Human islets; Diabetes
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, composed of three major isoforms (a, b/d, and c) that have been identified to date [1,2]. The receptor isoforms show tissue and cell specificity, and exert related but distinct functions upon activation [3–7]. Among the PPAR isoforms, PPARc is the best characterized and found to play an important role in the regulation of energy homeostasis [1,2,8–10]. Thiazolidinediones (TZDs), agonists of PPARc, reduce insulin resistance and thus influence free fatty acid flux and blood glucose levels. The glucose-lowering effect of TZDs is attributed to increased peripheral glucose disposal and decreased hepatic glucose output [8,11,12]. Synthetic ligands for PPARc are of particular interest for treating patients with type 2 diabetes because they restore the sensitivity of the tissues to insulin [8,9,11]. Pioglitazone is an orally administered insulin*
Corresponding author. Fax: +46 8 6163287. E-mail address: [email protected] (Q. Zhang).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.10.103
sensitizing TZD agent that activates PPARc, leading to the increased transcription of genes encoding various proteins regulating glucose and lipid metabolism [3,11]. In contrast to its action on insulin target tissues, little is known about the direct action of the PPARc agonist on pancreatic b-cell function and the b-cell sparing effects noted in clinical trials have generally been assumed to be secondary to the decreased insulin resistance. In the present study, we have studied the direct actions of the PPARc agonist pioglitazone on insulin secretion and Ca2+ handling in human islets from non-diabetic and diabetic subjects during short-term treatment in vitro. Materials and methods Pioglitazone was graciously donated by Takeda Pharmaceuticals North America, Inc. (Lincolnshire, IL). Fura-2/acetoxymethylester (Fura-2/AM) was from Sigma (St. Louis, MO). Bio-Gel P-4 (fine, 65 ± 20 lm, wet) was from Bio-Rad Laboratories (Hercules, CA). Insulin ELISA kits were from Mercodia (Uppsala, Sweden).
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Human islets Pancreatic islets from adult non-diabetic or diabetic donors were obtained from the Uppsala University Hospital facility for isolation of human islets from Scandinavian brain-dead donors. The 56-year-old diabetic donor was diagnosed with type 2 diabetes for 10 years and received insulin 90 U daily before death with BMI 33, HbA1c 9.5%, and C-peptide level of 0.3 nmol/l. The normal islets were from adult braindead non-diabetic patients. The islets were maintained in RPMI-1640 tissue culture medium in the presence of 5.5 mM glucose, supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 lg/ ml streptomycin, and were used for experiments 8 days after isolation. All experiments were approved by the Karolinska Ethics Committee. Insulin secretion Column perifusion. Dynamics of insulin secretion was studied by column perifusion as described [13,14]. About 100 human islets were carefully mixed with a small volume of pre-wetted Bio-Gel P-4, placed on top of each of the columns, and perifused with KRBH (Krebs–Ringer bicarbonate Hepes) buffer containing (in mM): 135 NaCl, 3.6 KCl, 5 NaHCO3, 0.5 NaH2PO4, 0.5 MgCl2, 1.5 CaCl2, and 10 Hepes, pH 7.4, with 0.1% BSA. Batch incubation. Islets were washed three times in KRBH buffer. Equal number of islets of comparable size was placed in each well of a 24-well plate, pre-added with 1 ml of the buffer in the presence of pioglitazone or DMSO at 3 mM glucose. After pre-incubation for 10 min at 37 °C, glucose or glucose with tolbutamide was added in the corresponding wells and incubated for 20 min. The medium was collected and centrifuged, the supernatant was for insulin assay. The islets were collected and lysed for protein assay (Bio-Rad). Measurement of [Ca2+]i. Fura-2 loaded cells were perifused with KRBH buffer in the presence of 3 mM glucose and 0.1% BSA, at 37 °C. Measurements of [Ca2+]i were performed on single cells prepared from islets as described [15,16] using a time-sharing spectrofluorometer (RM-5 System, PhotoMed, Denmark).
Fig. 1. Bimodal effects of pioglitazone on insulin secretion dynamics in normal human islets. Human islets from non-diabetic donors were packed in a micro-column and perifused with KRBH buffer containing 0.1% BSA and 5 mM glucose. Pioglitazone (Pio; 10 lM) or vehicle (DMSO)containing buffer (as control) was introduced. Data show dynamic changes in insulin secretion from four separate experiments using islets from four different subjects. The average of the insulin content in the fractions collected before the drug addition (Control) was considered as 100%. Insulin content in each fraction was divided by the average. Results are expressed as percentage changes of insulin secretion. The insert figure shows the suppressed insulin secretion during perifusion with pioglitazone, and enhanced insulin secretion after withdrawal of the drug (peak b) in the four experiments. *P < 0.05, by ANOVA.
Results Bimodal effects of pioglitazone on glucose-sensitive insulin secretion in human islets When human islets from non-diabetic subjects were perifused with 5 mM glucose, administration of 10 lM pioglitazone elicited an approximately 20% transient increase in insulin secretion, followed by a suppression of insulin release from the islets (Fig. 1). The inhibitory effect of pioglitazone remained until withdrawal of the drug, resulting in up to 40% of decrease in insulin secretion, compared to those before the drug addition. Immediately after withdrawal of pioglitazone, insulin secretion was increased approximately threefold compared to the levels during pioglitazone administration, and twofold compared to the levels before addition of the drug. The same response pattern to pioglitazone was noted in all experiments performed using islets from different donors, but not in those with buffer changes alone.
Fig. 2. Effects of pioglitazone on glucose- and tolbutamide-stimulated insulin secretion in human islets. Human islets from non-diabetic donors were pre-incubated in the presence of pioglitazone (Pio) or vehicle (DMSO) for 10 min in KRBH buffer containing 0.1% BSA and 3 mM glucose at 37 °C, followed by addition of glucose (20 mM) or glucose with tolbutamide (Tol; 100 lM) in the corresponding wells. Insulin secretion from the islets in each well was normalized by protein content measured in the lysed islets. Results from four separate experiments using islets from three different subjects are shown and expressed as means ± SEM. *P < 0.05 by ANOVA.
Pioglitazone inhibits glucose-induced insulin secretion in normal human islets The effect of pioglitazone on insulin secretion was further investigated by acute exposure of human islets to the
drug in the presence of low and high glucose (Fig. 2). Compared to the inhibitory effect of pioglitazone on insulin secretion at 5 mM glucose, the drug did not result in
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significant changes in insulin secretion at a glucose concentration of 3 mM during 20-min incubation. In the presence of 20 mM glucose, however, pioglitazone caused approximately 50% decrease in the glucose-stimulated insulin release. In order to examine whether the pioglitazone also interfered with insulin secretion stimulated by other insulin secretagogues, islets were stimulated with the sulfonylurea tolbutamide in the presence of low and high glucose. Pioglitazone inhibited approximately 25% of tolbutamide-stimulated insulin secretion at an ambient glucose of 3 mM. At the glucose concentration of 20 mM, the effect of tolbutamide was inhibited to a similar extent (27%).
human islet cells. After stimulation with high glucose, the cells were perifused with 3 mM glucose in the presence of pioglitazone for 10 min, followed by second pulse of glucose stimulation in the presence of the drug. Pioglitazone failed to alter glucose-induced rise in [Ca2+]i (Fig. 3B), while it inhibited insulin secretion stimulated by the sugar (Fig. 2). Pioglitazone restored glucose-sensitive insulin secretion in islets from type 2 diabetic patients In order to investigate whether the enhanced glucose sensitivity evoked by pioglitazone in non-diabetic islets would occur also under diabetic conditions, islets from a type 2 diabetic subject were placed in the micro-columns and perifused with a buffer containing 5 mM glucose (Fig. 4). Insulin secretion was suppressed during pioglitazone administration (Fig. 4, insert). Similar to the effect in non-diabetic islets (Fig. 1), pioglitazone evoked an immediate increase in insulin secretion from the diabetic islets after withdrawal of the drug, resulting in insulin secretion being increased approximately threefold compared to the levels during pioglitazone administration, and twofold compared to basal levels before addition of the drug. In contrast to the non-diabetic islets (Fig. 1), there was no pioglitazoneinduced transient increase in insulin secretion in the diabetic islets (Fig. 4, insert). The effect of pioglitazone on recovery of glucose sensitivity in diabetic islets was also investigated using two micro-columns, performed in parallel (Fig. 4). When the diabetic islets were perifused with 5 mM glucose, followed by stimulation with 20 mM glucose, the islets failed to respond to the glucose stimulation with increased insulin output. Exposure of islets to pioglitazone restored the
Pioglitazone does not significantly alter [Ca2+]i in human islets Since [Ca2+]i plays a crucial role in glucose-stimulated insulin secretion, we further investigated whether pioglitazone influences [Ca2+]i in human islet cells loaded with fura2 (Fig. 3). Islet cells were perifused with 3 mM or 5 mM glucose, during which a dose of 10 lM pioglitazone was applied. The cells were stimulated with glucose at the end of the experiments to verify the b-cell response. At 3 mM glucose, addition of pioglitazone elicited a Ca2+ transient that rapidly disappeared despite the continuous presence of the drug (Fig. 3A), while addition of same amount of vehicle (DMSO) was without effect. In contrast, application of pioglitazone at 5 mM glucose affected [Ca2+]i neither during drug administration nor after drug withdrawal (Fig. 3A, inset). In order to examine whether pioglitazone-induced inhibition on glucose-stimulated insulin secretion was associated with a change in [Ca2+]i, the effect of pioglitazone on glucose-stimulated rise in [Ca2+]i was investigated in A
B
Fig. 3. Effects of pioglitazone on [Ca2+]i in non-diabetic human islets. [Ca2+]i was measured in single cells prepared from non-diabetic human islets as described in Materials and methods. Cells were perifused with 3 mM glucose. Addition of pioglitazone (Pio; 10 lM) is indicated (A). Similar experiments were also performed when the cells were perifused with buffer containing 5 mM glucose. The changes in the Ca2+ transient amplitude elicited by pioglitazone at 3 or 5 mM glucose are shown in the insert figure, summarizing results derived from four separate experiments using islets from three subjects. Levels of fluorescence ratios before drug addition were considered as basal. Bars indicate means ± SEM and *P < 0.05 by ANOVA. The effects of pioglitazone on glucose-induced rises in [Ca2+]i are shown in (B). After the first stimulation with 20 mM glucose, cells were perifused with pioglitazone at 3 mM glucose for 10 min before and during a second glucose stimulation. A representative experiment out of four separate experiments using islets from three non-diabetic subjects is shown. The effects of pioglitazone on [Ca2+]i at 20 mM glucose are summarized in the insert figure. Bars represent mean percentage of the glucose-stimulated peak amplitude (±SEM) before pioglitazone addition (peak 1), compared to the peak stimulated by glucose in the presence of pioglitazone.
F. Zhang et al. / Biochemical and Biophysical Research Communications 351 (2006) 750–755
Fig. 4. Pioglitazone restores glucose-sensitive insulin secretion in human islets from a type 2 diabetic patient. Equal amounts of human islets from the diabetic patient were packed in two micro-columns and run in parallel. Islets were continuously perifused with KRBH buffer with 0.1% BSA and 5 mM glucose (G) in the presence of pioglitazone (10 lM Pio; empty circles) or vehicle (DMSO, filled circles) 10 min before and during the indicated period. Results from three experiments are shown and expressed as means ± SEM. *P < 0.05 by ANOVA. The insert figure shows a representative experiment performed on the human diabetic islets, perifused with 5 mM glucose as in Fig. 1. The average of insulin contents in the fractions collected before high glucose (Fig. 4) or pioglitazone (insert) additions was arbitrarily set at 100%.
glucose response, resulting in a 10-fold increase in insulin secretion compared to basal release (Fig. 4). Discussion Pioglitazone has been developed for the treatment of type 2 diabetes mellitus. The improved glycemic control by the drug is thought to be due to amplification of the post-receptor actions of insulin in target tissues such as skeletal muscle. By applying the drug in vitro to human islets from both non-diabetic and diabetic subjects, we show here that pioglitazone may also directly affect the function of the insulin-producing b-cell by influencing the glucose sensitivity of the b-cell. PPARc expression has been detected in islet cells [17–21] and in clonal b-cell lines [22]. The function of PPARc in the b-cell remains controversial [23–28]. The present study on human islets reveals that acute administration of pioglitazone rapidly inhibits glucose-induced insulin secretion at 5 mM or higher concentrations of glucose in islets from both diabetic and non-diabetic subjects. A slight transient increase in insulin secretion by the drug was observed in islets from all non-diabetic, but not in diabetic, subjects studied. The latter observation may indicate that the factor responsible for the transient effect of pioglitazone on insulin release is among the factors impaired during development of type 2 diabetes. The suppressive effect of pioglitazone on insulin secretion observed in the present studies is consistent with clinical observations where TZD treatment for several months results in suppressed levels of plasma insulin [29–31], and the report from rat islets
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using another PPARy agonist troglitazone [28]. This effect of pioglitazone should not be a result of cytotoxicity of the drug as incubation of human islets for 48 h at the same dosage as used here did not result in any significant changes in cell viability, evaluated by fluorescence microscopy after islets were vital stained with propidium iodide and bisbenzimide (unpublished observations). In addition, the pioglitazone-treated cells responded to glucose in terms of [Ca2+]i to a similar extent as control cells in the present study. In the clinical setting, the decrease in plasma insulin levels is believed to reflect decreased insulin resistance, and could be beneficial in arresting the natural unfolding of diabetes by sparing the b-cell and thereby impacting disease progression. Following the pioglitazone-induced suppression of insulin secretion at 5 mM glucose, insulin secretion was immediately increased upon withdrawal of the drug. This suggests that the lowering effect of pioglitazone on insulin secretion was not a toxic effect of the drug on hormone output at the concentration we employed, but rather that pretreatment of islets with the PPARc agonist enhanced or ‘‘primed’’ the sensitivity of the b-cell to glucose. The over-response of the cells to glucose after drug removal implies that the suppressed insulin secretion during pioglitazone exposure is reversible and is responsible for restoration of glucose sensitization or priming of insulin exocytosis. Importantly, and remarkably, pioglitazone restored a robust glucose-sensitive insulin secretion in islets from the type 2 diabetic patient, in which glucose sensitivity was completely lost. The mechanism by which pioglitazone acutely confers glucose competence to the diabetic b-cell is unknown, and whether PPARc is involved in mediating the acute effects of the drug remains unclear [32–35]. Most cellular responses contributing to the antidiabetic effect of TZDs occur over a long time (several weeks) and require altered gene transcription [36] and cannot explain the acute effect of pioglitazone noted here. Changes in islet morphology and gene expression after long-term treatment may lead to different results when evaluating the direct effect of the drug on insulin secretion [37]. In animal models, pioglitazone was shown to improve glucose-induced insulin secretory capacity by protecting the b-cell from oxidative stress [24,38], a feature of the drug that may also be functional in the human b-cells. However, unlike the animal models, the essential effect of pioglitazone on glucose-induced insulin secretion in human islets was suppressive and the enhanced glucose-stimulated secretory response was only observed after withdrawal of the drug. This action of pioglitazone in human islets may suggest a direct effect of the drug on the insulin exocytotic machinery. In agreement with this hypothesis, the postpioglitazone effect of glucose-stimulated insulin secretion was rapid and both glucose- and tolbutamide-induced insulin releases were suppressed by the drug. A direct action of pioglitazone on insulin exocytosis was also reflected by its effect on [Ca2+]i. Pioglitazone induced a slight increase in [Ca2+]i at 3 mM glucose without a
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significant change in insulin secretion. On the other hand, insulin secretion was suppressed by the drug at glucose concentrations of 5 and 20 mM without any changes in [Ca2+]i. This dissociation of glucose-induced insulin secretion and rise in [Ca2+]i does not support an involvement of activation of glucokinase through interaction of PPARc with the peroxisomal proliferator response element (PPRE) in the b-cell-specific glucokinase promoter [39] in the acute effect of pioglitazone on insulin secretion. It rather suggests a late action of the in drug the process of insulin secretion, distal to [Ca2+]i increase, namely the insulin exocytotic machinery. In comparison to human islet cells, pioglitazone stimulated insulin secretion in hamster insulinoma HIT-T15 cells, associated with an increased [Ca2+]i [26]. The different roles of pioglitazone on intracellular Ca2+ handling in different species may explain the discrepancies in acute effects of pioglitazone on insulin secretion. Taken together, the present results suggest that pioglitazone acutely induces a rapid, reversible, and Ca2+-independent inhibition of glucose-induced insulin secretion in human islets, but enhances or restores glucose sensitivity in healthy or diabetic human islets after immediate removal of the drug, therefore indicating a direct beneficial effect of pioglitazone on b-cell function.
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Acknowledgments Financial support was received from Takeda Pharmaceuticals North America, the Swedish Medical Research Council (# 72X-12550, 72X-14507, and 72P-14787), Berth von Kantzow’s Foundation, Petrus, and Augusta Hedlund’s Foundation, the Nutricia Research Foundation, the European Foundation for the Study of Diabetes, the Swedish Society of Medicine, the Sigurd, and Elsa Golje Memorial Foundation, Svenska Fo¨rsa¨kringsfo¨reningen, Svenska Diabetesstiftelsen, Magn. Bergvall Foundation, Barndiabe˚ ke Wiberg’s Foundation, Trygg-Hansa’s tesfonden, A Research Foundation, Torsten, and Ragnar So¨derberg’s Foundations, Harald Jeansson’s, and Harald and Greta Jeansson’s Foundations, Tore Nilson’s Foundation for Medical Research, Fredrik, and Inger Thuring’s Foundation, and Syskonen Svensson’s Fund.
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