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Am J Physiol Cell Physiol 296: C346-C354, 2009. First published December 3, 2008; doi:10.1152/ajpcell.00444.2008
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RECEPTORS AND SIGNAL TRANSDUCTION

BLX-1002, a novel thiazolidinedione with no PPAR affinity, stimulates AMP-activated protein kinase activity, raises cytosolic Ca2+, and enhances glucose-stimulated insulin secretion in a PI3K-dependent manner

Fan Zhang,1 Deben Dey,2 Robert Bränström,3 Lars Forsberg,3 Ming Lu,3 Qimin Zhang,1 and Åke Sjöholm1

1Diabetes Research Center, Department of Internal Medicine, Karolinska Institutet, South Hospital, Stockholm, Sweden; 2Bexel Pharmaceuticals, Inc., Union City, California; and 3Department of Molecular Medicine and Surgery, Unit of Endocrine Surgery, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden

Submitted 27 August 2008 ; accepted in final form 25 November 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
BLX-1002 is a novel small thiazolidinedione with no apparent affinity to peroxisome proliferator-activated receptors (PPAR) that has been shown to reduce glycemia in type 2 diabetes without adipogenic effects. Its precise mechanisms of action, however, remain elusive, and no studies have been done with respect to possible effects of BLX-1002 on pancreatic β-cells. We have investigated the influence of the drug on β-cell function in mouse islets in vitro. BLX-1002 enhanced insulin secretion stimulated by high, but not low or intermediate, glucose concentrations. BLX-1002 also augmented cytoplasmic free Ca2+ concentration ([Ca2+]i) at high glucose, an effect that was abolished by pretreatment with the Ca2+-ATPase inhibitor thapsigargin. In contrast, BLX-1002 did not interfere with voltage-gated Ca2+ channel or ATP-sensitive K+ channel activities. In addition, cellular NAD(P)H stimulated by glucose was not affected by the drug. The stimulatory effect of BLX-1002 on insulin secretion at high glucose was completely abolished by treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY-294002. Stimulation of the β-cells with BLX-1002 also induced activation of AMP-activated protein kinase (AMPK) at high glucose. Our study suggests that BLX-1002 potentiates insulin secretion only at high glucose in β-cells in a PI3K-dependent manner. This effect of BLX-1002 is associated with an increased [Ca2+]i mediated through Ca2+ mobilization, and an enhanced activation of AMPK. The glucose-sensitive stimulatory impact of BLX-1002 on β-cell function may translate into substantial clinical benefits of the drug in the management of type 2 diabetes, by avoidance of hypoglycemia.

calcium; diabetes; islet; phosphatidylinositol 3-kinase; adenosine 5'-monophosphate

DIABETES MELLITUS IS CHARACTERIZED by lack of, or relative deficiency in, insulin production and insensitivity of target tissues to insulin. Impairment in glucose-sensitive insulin secretion of the pancreatic β-cell is an important pathogenetic feature (4), contributing to the development of the diabetic state (18). Although both insulin resistance and β-cell dysfunction contribute to the pathogenesis of type 2 diabetes, diabetes develops only when β-cells fail to compensate for increased demand (55). The changes in β-dysfunction in diabetes include decline or failure of the ability of the β-cell to respond to elevated glucose (54), disturbances in pulsatile insulin release (43), and impaired insulin synthesis (31). Improvement of β-cell function is a major goal in the clinical management of the disease.

Glucose is the major stimulator of insulin secretion under physiological conditions. The response of the β-cell to glucose is dependent on metabolism of the sugar (35). Elevation of extracellular glucose increases glucose metabolism, resulting in increased production of cytosolic ATP and an elevated ATP/ADP ratio. The latter serves as the principal regulator of ATP-sensitive K+ (KATP) channels present in the plasma membrane. An increase in the ATP/ADP ratio leads to closure of the KATP channel, followed by depolarization of the plasma membrane (3). The subsequent events include opening of voltage-gated Ca2+ channels and the attendant rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). Increase in [Ca2+]i triggers insulin secretion. The elevated β-cell metabolism also generates amplifying signals that augment the efficacy of Ca2+ on the exocytotic machinery (25), thereby contributing to sustained insulin secretion.

BLX-1002 is a novel thiazolidinedione, water-soluble antidiabetic compound (molecular weight < 500) with no structural resemblance to any existing compounds (41). Its structure is described in U.S. patent no. 6794401. It does not appear to affect peroxisome proliferator-activated receptors (PPAR) (13). There is evidence that BLX-1002 can improve hyperglycemia in diabetic animal models without body weight gain as observed in subjects treated with PPAR agonists (12, 14, 41), but the putative effects of the drug on β-cell function have not been studied. In the present study, we have investigated the effects of BLX-1002 on insulin secretion in mouse β-cells in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
BLX-1002 and BLX-1015 were graciously donated by Bexel Pharmaceuticals, Inc. (Union City, CA). LY-294002 was bought from Calbiochem (La Jolla, CA). Wortmannin, acetylcholine chloride, thapsigargin, fura-2 AM, and anti-β-actin were from Sigma-Aldrich (St. Louis, MO). Re-blot plus strong solution was from Chemicon (Temecula, CA). Anti-phospho-AMP-activated protein kinase (AMPK)-{alpha} (Thr172) was from Cell Signaling Upstate (Danvers, MA). Protease inhibitor cocktail and collagenase A was from Roche Diagnostics (Mannheim, Germany). Mouse insulin ELISA kits were from Mercodia (Uppsala, Sweden). RPMI-1640 culture medium and FBS were from Life Technologies Invitrogen (Paisley, UK).

Preparation of pancreatic islet cells. All animal use procedures were approved by the local animal ethics committee. Pancreatic islets, known to contain >90% β-cells, were isolated by collagenase and DNAse digestion as previously described (60) from 12-mo-old ob/ob mice bred at the KISÖS Stockholm colony. The Stockholm ob/ob colony was established at KISÖS in 2004 from breeding pairs kindly provided by Professor Janove Sehlin (Umeå University). Islets were dispersed into single cells by shaking in medium containing 1 mM EGTA (42). BKS.CG-M+/+ Leprdb (db/db) mice were purchased from Charles River Laboratories (Wilmington, MA). Pancreatic islets were isolated from 12-wk-old mice by collagenase digestion and handpicking of islets as previously described (15). Islets or cells were maintained in RPMI-1640 tissue culture medium supplemented with 10% (vol/vol) FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin overnight before experiments.

Insulin secretion from pancreatic islets or islet cells. Pancreatic islet cells from ob/ob mice or islets from db/db mice were washed three times in Krebs-Ringer bicarbonate HEPES (KRBH) 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, pH 7.4, in the presence of 3 mM glucose. Equal amounts of cells (2 x 104 cells) or islets (100 islets) were incubated in multiwell plates for 20 min at 37°C in the presence of 10 µM BLX-1002 or BLX-1015 or equal amount of vehicle (DMSO, at the final concentration of 0.05%) at different concentrations of glucose. To investigate the effects of kinase inhibitors on insulin secretion, cells, after being placed into 48-well plates, were preincubated in the presence of the inhibitors wortmannin (100 nM, 30 min, 37°C) or LY-294002 (10 µM, 15 min, 37°C) or equal amount of vehicle (DMSO, at the final concentration of 0.05%) at 3 mM glucose, followed by addition of BLX-1002 and glucose. Cells were further incubated for 20 min at 37°C. At the end of the incubation, cells or islets were spun down by centrifugation and the supernatants were collected. Insulin concentrations in the supernatants were measured using mouse insulin ELISA kits.

Measurement of [Ca2+]i. [Ca2+]i measurements were performed on single islet cells cultured overnight on plastic coverslips. Before the experiments, cells on coverslips were loaded with Fura-2 (1.5 µM) for 30 min at 37°C in KRBH buffer in the presence of 3 mM glucose and 0.1% BSA. The coverslips were subsequently rinsed once in the same buffer without the Ca2+ indicator and mounted at the bottom of a perifusion chamber on the stage of an inverted epifluorescence microscope (Olympus CK 40). The stage was thermostated to 37°C, and cells were superfused at a rate of 300 µl/min with KRBH buffer containing 3 mM glucose. Measurements of [Ca2+]i were performed as previously described (57) using a time-sharing spectrofluorometer (RM-5 system, PhotoMed) providing light flashes of 1-ms duration at 340 and 380 nm every 10 ms. Fluorescence was recorded at 510 nm from single cells.

Electrophysiological recordings. K+ channel activity was recorded with the patch-clamp technique (21), using an HEKA EPC-10 patch-clamp amplifier (HEKA Elektronik). KATP channel current traces were displayed according to the prevailing convention, upward deflection denoting outward currents. All experiments were performed at room temperature (~22°C), and channel activity was measured at a membrane potential of 0 mV. The standard extracellular solution contained (in mM) 138 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, and 5 HEPES-NaOH at pH 7.4. For inside-out recordings, an intracellular-like solution (i.e., bath solution) consisted of (in mM) 125 KCl, 1 MgCl2, 10 EGTA, 25 KOH, and 5 HEPES-KOH at pH 7.15. ATP was added to the intracellular solution at the final concentration indicated in the text and figures. For Ca2+ channel recordings, pipette solution contained (in mM) 150 N-methyl-D-glucamine, 110 HCl, 1 MgCl2, 2 CaCl2, 10 EGTA, 3 MgATP, and 5 HEPES-NaOH (pH 7.15). N-methyl-D-glucamine was substituted for K+ in the pipette solution to prevent outwardly directed K+ currents. The standard extracellular solution (described above) was supplemented with 17.4 mM CaCl2, giving a total Ca2+ concentration of 20 mM. For analysis of whole cell currents, the recordings were filtered at 2.9 kHz and digitized at 10 kHz. Calculations were performed using TAC software (Bruxton, Seattle, WA).

Measurement of NAD(P)H. Temporal fluctuations in cellular NAD(P)H levels were measured as described previously (30) using the fluorescence system above. Pancreatic islet cells attached to coverslips were incubated in KRBH buffer in the presence of 0.1% BSA at 3 mM glucose for 30 min at 37°C. NAD(P)H fluorescence was monitored in single cells at an excitation wavelength of 366 nm, a dichroic mirror at 400 nm, and an emission band-pass filter at 450–470 nm (30). During the experiments, cells were perifused with KRBH buffer at 3 mM glucose. The effects of BLX-1002 on NAD(P)H fluorescence were evaluated by stimulation with 20 mM glucose in the presence or absence of 10 µM BLX-1002 or by stimulation with 20 mM glucose for 10 min, followed by addition of BLX-1002 for an additional 10 min.

Western blot analysis. Cells were washed three times in serum-free RPMI-1640 medium and incubated overnight in the medium in an incubator. Cells were washed twice in glucose-free RPMI-1640 with 0.1% BSA, resuspended in the same medium, and divided equally into tubes. Cells were incubated for 2 h at 37°C, followed by addition of BLX-1002 or glucose in corresponding tubes. The incubation was continued for the indicated time period. At the end of incubation, cells were collected by a brief centrifugation and lysed in a lysis buffer [25 mM Tris·HCl (pH 7.6) 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS (RIPA buffer) with 1 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor cocktail] for 30 min on ice. Cell debris was removed by centrifugation, and the resulting supernatants were collected. Equal amounts of protein (20 µg) were applied to SDS-PAGE. Proteins in the gel were subsequently electrotransferred onto nitrocellulose membranes. The membrane was blocked in 20 mM Tris base and 137 mM NaCl, pH 7.6 with 0.05% Tween 20 (TBS-T) with 5% nonfat dry milk, followed by an overnight incubation with anti-phospho-AMPK-{alpha} (Thr172) (1:1,000) in TBS-T-1% BSA at 4°C. After extensive washes in TBS-T, the membrane was incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:10,000) in TBS-T-1% BSA for 1 h at room temperature. The membrane was extensively washed, and the immunostained proteins were visualized by enhanced chemiluminescence. The blots were stripped in Re-blot plus strong solution and probed with anti-β-actin (1 µg/ml). The intensities of the bands were quantified by densitometry. The density of the phospho-AMPK was normalized to that of β-actin obtained from the same blot.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
BLX-1002 potentiates glucose-stimulated insulin secretion. To investigate the effects of BLX-1002 on insulin secretion, the compound was applied to ob/ob mouse islet cells at different glucose concentrations (Fig. 1A). BLX-1002 (10 µM) interfered with insulin secretion neither at 3 mM glucose nor at 8 mM of the sugar during a 20-min incubation. At 20 mM glucose, however, glucose-stimulated insulin secretion was significantly potentiated by BLX-1002, by ~40%. The sulfonylurea tolbutamide stimulated insulin secretion at both low and high glucose in the β-cells, although the stimulatory potency of tolbutamide at high glucose was greater (100 µM tolbutamide caused a double increase in insulin secretion from the cells under the same conditions; data not shown). In contrast, BLX-1015 (10 µM), the major metabolite of BLX-1002, did not significantly influence insulin secretion at any concentration of glucose applied.


Figure 1
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  		Fig. 1. BLX-1002 selectively potentiates insulin secretion at high glucose in β-cells. A: islet cells from ob/ob mice were incubated in different concentrations of glucose in the presence or absence of BLX-1002 or BLX-1015 (10 µM each) for 20 min and their insulin secretion was measured. Bars indicate means ± SE and are derived from three independent experiments, each performed in triplicate. B: islet cells from ob/ob mice were incubated at 20 mM glucose in the presence of indicated concentrations of BLX-1002 for 20 min. Bars indicate mean percentage of control ± SE and are derived from seven independent experiments, each performed in triplicate. C: effects of BLX-1002 or BLX-1015 (10 µM each) on glucose-sensitive insulin secretion over 20 min from pancreatic islets isolated from diabetic db/db mice. Bars indicate means ± SE and are derived from seven independent experiments, each performed in triplicate. *P < 0.05 for a chance difference vs. controls by ANOVA.

 
The dose dependency of the glucose-stimulated insulin secretion by BLX-1002 was further examined in the ob/ob mouse islet cells. Exposure to various concentrations of BLX-1002, ranging from 0.1 µM to 10 µM in the presence of 20 mM glucose, revealed a dose-dependent increase of insulin secretion by the drug (Fig. 1B). At the concentration of 0.1 µM, BLX-1002 significantly enhanced glucose-stimulated insulin secretion, and increasing the drug concentration to 1 or 10 µM caused further increase of insulin release.

The effect of BLX-1002 on insulin secretion was also evaluated in pancreatic islets isolated from diabetic db/db mice (Fig. 1C). Similar to the islet cells from ob/ob mice above, neither BLX-1002 nor BLX-1015 showed any effect on insulin secretion at 3 mM glucose. At 20 mM glucose, BLX-1002, but not its metabolite BLX-1015, significantly potentiated insulin secretion in the islets from db/db mice by some 70%.

BLX-1002 enhances glucose-induced rise in [Ca2+]i. Since a rise in [Ca2+]i is a crucial step in insulin secretion, we next studied whether the stimulatory effect of BLX-1002 on glucose-induced insulin secretion was associated with changes in [Ca2+]i measured in single ob/ob mouse islet cells after preloading the cells with the fluorescent Ca2+ probe Fura-2 (Fig. 2). Stimulation of the islet cells with 20 mM glucose induced a robust increase in [Ca2+]i (Fig. 2A). Addition of 10 µM BLX-1002 potentiated the glucose-stimulated rise in [Ca2+]i in all experiments performed(Fig. 2B). In contrast, administration of BLX-1002 at 3 mM glucose did not result in any change in [Ca2+]i (data not shown).


Figure 2
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  		Fig. 2. BLX-1002 enhances glucose-induced rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). Islet cells from ob/ob mice were perifused with a buffer containing 3 mM glucose, followed by a stimulation with 20 mM glucose (G) in the absence (n = 8; A) or presence (n = 12; B) of BLX-1002 (10 µM). C: cells were stimulated with 20 mM glucose after the Ca2+ channel antagonist nifedipine and BLX-1002 (10 µM each) were introduced as indicated. Restoration of the cells' responsiveness to BLX-1002 was verified after washout of the inhibitor (n = 5). D: similar experiments were performed, where cells were pretreated with the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (250 nM, 30 min, n = 9) before measurement. The filling conditions of the intracellular Ca2+ pools were evaluated by addition of acetylcholine (ACh, 1 µM). E and F: cells were pretreated with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 (10 µM, 15 min, n = 9; E) or the AMP-activated protein kinase (AMPK) inhibitor compound C (20 µM, 30 min, n = 5; F). The inhibitors were continuously present during the experiments.

 
To pin down the mechanisms involved in the stimulatory effect of BLX-1002 on glucose-induced rise in [Ca2+]i, we used the specific voltage-sensitive L-type Ca2+-channel blocker nifedipine in ob/ob mouse islet cells (Fig. 2C). Introduction of 10 µM nifedipine inhibited the glucose-stimulated rise in [Ca2+]i, as expected. Under these conditions, the effect of BLX-1002 on [Ca2+]i was also prevented, an effect that was readily reversed upon removal of the inhibitor.

The possible involvement of intracellular Ca2+ pools in the BLX-1002-induced rise in [Ca2+]i was also addressed. The effect of BLX-1002 on [Ca2+]i was abolished by pretreatment of the ob/ob mouse islet cells with thapsigargin (Fig. 2D), which inhibits Ca2+-ATPase in the endoplasmic reticulum (ER) and thereby depletes Ca2+ from intracellular stores. Under these conditions, addition of acetylcholine, which rapidly increases [Ca2+]i by mobilizing Ca2+ from intracellular pools, failed to induce a change in [Ca2+]i (Fig. 2, C and D), as expected.

The BLX-1002-induced rise in [Ca2+]i was further evaluated in the presence of the specific phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 (Fig. 2E). Treatment of the ob/ob mouse islet cells with 10 µM LY-294002 did not affect glucose-stimulated rise in [Ca2+]i, which is consistent with a previous report (16). In contrast, the [Ca2+]i response to BLX-1002 was attenuated by the inhibitor, with the amplitude being ~40% of that observed in controls.

To address the role of AMPK in BLX-1002-induced rise in [Ca2+]i, ob/ob mouse islet cells were treated with the AMPK-specific inhibitor compound C (Fig. 2F). At a concentration of 20 µM (32, 53), the glucose-stimulated rise in [Ca2+]i was suppressed ~75% by compound C, and the [Ca2+]i response to BLX-1002 was also significantly suppressed.

BLX-1002 does not significantly interfere with activities of plasma membrane ion channels. To further pin down the precise nature of the mechanism behind the rise in [Ca2+]i evoked by BLX-1002, we examined the effects of the drug on voltage-gated Ca2+ channels and the KATP channels in ob/ob mouse β-cells using patch-clamp technique (Fig. 3). The β-cell was voltage clamped at –80 mV, using the whole cell configuration (Fig. 3A). The cell was subsequently depolarized in steps of 20 mV to +80 mV. Addition of 10 µM BLX-1002 did not significantly change the voltage-gated Ca2+ current.


Figure 3
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  		Fig. 3. BLX-1002 does not interfere with ion channel activities. A: pancreatic β-cells from ob/ob mice were clamped using whole cell configuration at –80 mV and subsequently depolarized to 80 mV in steps of 20 mV. I, current; Vm, measuring voltage; bullet, control current; {circ}, current in the presence of 10 µM BLX-1002 (n = 6). B: representative recording of ATP-sensitive K+ (KATP) channel activity in an inside-out patch. C: KATP channel activity in percentage of control (means ± SE) in the presence of 10 µM BLX-1002 or 100 µM ATP (n = 9; NS, not significant; ***P < 0.001 for chance differences).

 
The impact of BLX-1002 on KATP channel activity was also examined. KATP channel activity was not affected by addition of 10 µM BLX-1002 on excised membrane patches (Fig. 3, B and C). Inclusion of 100 µM ATP (serving as a positive control) reversibly inhibited KATP channel activity, as expected (Fig. 3, B and C).

BLX-1002 does not alter NAD(P)H production. To address whether the BLX-1002-enhanced insulin secretion and [Ca2+]i were due to augmented glucose metabolism and thus secondary to ATP formation, we studied the impact of BLX-1002 on cellular NAD(P)H production, the key factor of ATP generation (Fig. 4). The effect of BLX-1002 on NAD(P)H production was examined both in ob/ob mouse islet cells stimulated with high glucose in the presence or absence of BLX-1002 (Fig. 4, A and B) and in cells first stimulated with high glucose, followed by addition of the drug (Fig. 4C). Addition of BLX-1002 (10 µM) did not influence glucose-induced NAD(P)H production in any setting, indicating that energy production was not influenced by the drug.


Figure 4
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  		Fig. 4. BLX-1002 does not influence NAD(P)H production. NAD(P)H fluorescence was monitored in single islet cells from ob/ob mice during perifusion with 3 mM and stimulation with 20 mM glucose in the presence or absence of 10 µM BLX-1002 (A and B) or in cells stimulated with high glucose, followed by addition of the drug (C). Fluorescence was normalized by setting basal fluorescence at 100%. Representative traces (A and C) out of six from each condition are shown [{circ}, control (Ctl); bullet, 10 µM BLX-1002]. Summary data of the increase in NAD(P)H fluorescence at 3 min or 6 min after addition of high glucose derived from A are shown in B. Bars indicate means ± SE; n = 6 for each condition.

 
BLX-1002-stimulated insulin secretion is abolished by inhibition of PI3K activity. The stimulatory effect of BLX-1002 on glucose-induced insulin secretion was further mechanistically addressed by application of specific inhibitors of PI3K (Fig. 5). Pretreatment of ob/ob mouse islet cells with wortmannin or LY-294002 enhanced glucose-stimulated insulin secretion, effects consistent with previous findings (16). In contrast, the enhanced insulin secretion by BLX-1002 at 20 mM glucose (bar 2 compared with bar 1) was completely abolished by either LY-294002 (bar 4 compared with bar 3) or wortmannin (bar 6 compared with bar 5) (Fig. 5).


Figure 5
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  		Fig. 5. BLX-1002-induced insulin secretion at high glucose requires PI3K activity. Experiments were performed as in Fig. 1. Islet cells from ob/ob mice were preincubated in the presence of the PI3K inhibitors wortmannin (Wort, 100 nM), LY-294002 (Ly, 10 µM), or DMSO for 15 min at 37°C followed by addition of BLX-1002 (10 µM) and glucose. Insulin content in the medium was measured after 20-min incubation at 37°C. Results from three separate experiments are shown in bars as means ± SE. *P < 0.05 for a chance difference vs. control (20 mM glucose alone) by ANOVA.

 
BLX-1002 activates AMPK in the β-cell. Since an important role has been assigned to AMPK in the β-cell stimulus-secretion coupling (34), we further investigated the effect of BLX-1002 on AMPK phosphorylation (a measure of its activation) by Western blotting using an antibody against phospho-AMPK-{alpha} (Thr172). Phosphorylation of this site (Thr172), located in the activation loop of the enzyme, is required for kinase activation (24). In this study, glucose and metformin, known to rapidly inhibit (48) and stimulate (10, 58) AMPK activity, respectively, were used as controls in ob/ob mouse islet cells. As shown in Fig. 6, AMPK activity was inhibited by high glucose and stimulated by metformin, as expected (Fig. 6, A, B, and D). Stimulation of the cells with BLX-1002 at 20 mM glucose caused a rapid activation of AMPK (Fig. 6, A, C, and D). Phosphorylation of AMPK increased after only 5 min of incubation with BLX-1002 and lasted for at least 1 h (Fig. 6, C and D).


Figure 6
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  		Fig. 6. BLX-1002 activates AMPK in pancreatic β-cells. Islet cells from ob/ob mice were incubated in the presence or absence of BLX-1002 (10 µM) during the time indicated in the presence of 20 mM glucose. Activated AMPK was detected by Western blot using anti-phospho-AMPK-{alpha} (Thr172). A: representative experiment out of eight is shown. Top: phospho (p)-AMPK. Bottom: bands after reblotting of the same membrane with anti-β-actin antibodies. B–D: comparisons of band densities of the phospho-AMPK normalized to β-actin (n = 8) in response to glucose (band density at 3 mM glucose as control; B), or to BLX-1002 after 5 or 15 min (C), or 60 min (D) of incubation at 20 mM glucose (high glucose alone as controls) are shown in bars as mean percentage of control ± SE. *P < 0.05 for chance difference vs. controls by ANOVA. Met, metformin (1 mM). E: phosphorylation of AMPK by the BLX-1002 metabolite BLX-1015 (10 µM) in ob/ob mouse islet cells. F: bars represent mean percentage of control (20 mM glucose alone) ± SE for three independent experiments of comparisons of phospho-AMPK band densities normalized to β-actin. *P < 0.05 for a chance difference vs. control, by Student's t-test. G: comparison between BLX-1002 and metformin on insulin secretion in islet cells from ob/ob mice after incubation for 20 min at 37°C in the presence or absence of BLX-1002 (10 µM) and/or metformin (1 mM) at 20 mM glucose. Results from six independent experiments, each performed in triplicate, are shown in bars as mean percentage of control (20 mM glucose alone) ± SE. *P < 0.05 for chance differences vs. control, by ANOVA.

 
The ability of the BLX-1002 metabolite, BLX-1015, to stimulate phosphorylation of AMPK was also investigated in ob/ob mouse islet cells in the presence of 20 mM glucose (Fig. 6, E and F). AMPK phosphorylation was again, as expected (7), suppressed by high glucose (Fig. 6E). Incubation of the cells with 10 µM BLX-1015 significantly enhanced AMPK phosphorylation, to an extent similar to that of BLX-1002.

Since BLX-1002 functioned similar to metformin in terms of AMPK activation, we compared BLX-1002 with metformin with regard to their action on insulin secretion. Unlike BLX-1002, incubation of ob/ob mouse islet cells with metformin did not induce any significant change in glucose-stimulated insulin secretion (Fig. 6G). Combination of metformin with BLX-1002 also did not influence insulin secretion in response to BLX-1002.

BLX-1002 potentiates pioglitazone-, but not fenofibrate-induced insulin secretion. Since BLX-1002 belongs to the thiazolidinedione drug family, we further investigated whether BLX-1002 interfered with insulin secretion induced by PPAR agonists (Fig. 7). Incubation of ob/ob mouse islet cells with either the PPAR-{alpha} agonist fenofibrate or the PPAR-{gamma} agonist pioglitazone alone potentiated glucose-stimulated insulin secretion. Combining BLX-1002 and pioglitazone resulted in a further increase of insulin secretion above the effect of either pioglitazone or BLX-1002 alone. In contrast, BLX-1002 did not significantly influence insulin secretion induced by fenofibrate.


Figure 7
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  		Fig. 7. Interaction of BLX-1002 with peroxisome proliferator-activated receptor (PPAR) agonists on insulin secretion in the β-cell. Islet cells from ob/ob mice were incubated for 20 min at 37°C in the presence or absence of BLX-1002 and/or the PPAR-{gamma} agonist pioglitazone (Pio) or the PPAR-{alpha} agonist fenofibrate (Fen) (10 µM each) at 20 mM glucose. Results from six independent experiments, each performed in triplicate, are shown in bars as mean percentage of control (20 mM glucose alone) ± SE. *P < 0.05 for chance differences vs. controls by ANOVA; #P < 0.05 for chance differences vs. the indicated conditions by ANOVA.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In human type 2 diabetes, loss of glucose-sensitive insulin secretion is an important pathogenetic event that is also of significance in type 1 diabetes and in the context of islet transplantation (61). The change in β-cell phenotype involves a selective loss of glucose-stimulated insulin secretion even in the very earliest stages of disease progression. The molecular basis for this functional defect remains elusive but also constitutes an inviting target for attempts to intervene against β-cell failure and outbreak of type 2 diabetes. Hence, improvement of β-cell function is a major goal not only in diabetes research but also in the clinical management of the disease. Contemporary diabetes treatment is hampered by several problems in terms of efficacy, durability, and side effects of available drugs. The sulfonylurea class of drugs has been used for >50 yr and stimulates insulin secretion from the β-cell (36). However, sulfonylurea drugs do this irrespective of whether they are needed; thus, since they promote insulin secretion even when normal or low glucose levels prevail, hypoglycemia and weight gain are common and dreaded side effects of these drugs (23). In the present study, we show that the novel low-molecular-weight non-PPAR thiazolidinedione BLX-1002 stimulates insulin secretion from isolated pancreatic islets or dispersed β-cells only at high glucose, an effect associated with AMPK activation and seemingly dependent on PI3K activation. While BLX-1002 potentiated insulin secretion at high glucose, it did not influence insulin secretion at either 3 mM or 8 mM glucose, suggesting a low potential for hypoglycemic side effects by this drug (as opposed to the sulfonylureas). Such glucose-dependent action manner of the drug shares similarities with incretin-based drugs (6, 26). Notably, the stimulatory effect of BLX-1002 on glucose-induced insulin secretion was not mimicked by its major metabolite, BLX-1015. This suggests that BLX-1002 directly stimulates β-cell function only when it has not yet metabolized and that the direct action of BLX-1002 on the β-cell may participate in the antidiabetic effect of BLX-1002. Again, this feature appears advantageous in a clinical setting, since it may translate into lack of sustained hypoglycemic effects by drug metabolites, a substantial problem with many sulfonylurea drugs (e.g., glibenclamide) (23).

Interestingly, BLX-1002 potentiated the insulinotropic effect of the thiazolidinedione PPAR-{gamma} agonist pioglitazone, whereas it did not affect the secretory response to the PPAR-{alpha} agonist fenofibrate. These results suggest that BLX-1002-induced insulin secretion might be mediated through a pathway not shared by pioglitazone (56), consistent with the view that BLX-1002 is a non-PPAR thiazolidinedione.

A rise in [Ca2+]i is an important event regulating early-phase insulin secretion (5). Entirely consistent with such a role, insulin secretion potentiated by BLX-1002 at high glucose was associated with an enhanced [Ca2+]i in the β-cell. The BLX-1002-induced rise in [Ca2+]i was abolished by blocking the voltage-sensitive L-type Ca2+ channel, implying an involvement of Ca2+ influx in this event. However, the drug affected neither voltage-gated Ca2+ channels nor KATP channels in the β-cell as demonstrated by the patch-clamp technique. These findings do not support the view that the [Ca2+]i increase elicited by BLX-1002 is mediated through Ca2+ influx from the extracellular medium. Additionally, the stimulatory effect of BLX-1002 on [Ca2+]i was completely abolished after depletion of intracellular Ca2+ pools by pretreatment with thapsigargin (an inhibitor of ER Ca2+-ATPase). These results suggest that an elevated [Ca2+]i through the L-type Ca2+ channels is required for the BLX-1002 action. In agreement with this notion, BLX-1002 did not raise [Ca2+]i at low glucose. From this point of view, BLX-1002 may elevate [Ca2+]i by facilitating Ca2+-induced Ca2+ release in the β-cell (59). The inability of BLX-1002 to influence KATP channel activity also suggests that the drug may not directly affect β-cell glucose metabolism, through which KATP channel activity is modified by an altered ATP/ADP ratio. In agreement with this notion, BLX-1002 did not evoke any changes in cellular NAD(P)H production in response to high glucose, suggesting that energy production is not impacted by the drug.

The molecular mechanism behind the Ca2+ mobilization induced by BLX-1002 is not clear. Release of Ca2+ from intracellular stores is a common event in cellular signaling on activation of membrane receptors (20, 27). Since the precise nature of the mechanisms by which BLX-1002 activates the β-cell remain to be identified, further studies are warranted to explore the pathways involved.

Interestingly, insulin secretion potentiated by BLX-1002 at high glucose was completely abolished by specific inhibitors of PI3K, a key element in transducing insulin signaling (52) and controlling vesicle trafficking and secretory function in many cell types (11, 44), suggesting an essential role of PI3K activity in the BLX-1002 insulinotropic effects. The β-cell contains PI3K activity (2), and the kinase has been shown to be important in controlling insulin gene transcription (9). In addition, an important role of the enzyme in β-cell secretory function has been suggested (16). Studies on islets from class I PI3K p85{alpha} regulatory subunit-deficient (p85–/–) mice revealed that PI3K plays a suppressive role in the regulation of glucose-stimulated insulin secretion (16). Another class I PI3K, the single class IB isoform PI3K-{gamma}, is also expressed in pancreatic islets (39) and in the β-cell (37). Unlike class IA, which is linked to tyrosine kinase signaling, PI3K-{gamma} is activated by the β{gamma}-subunits of the GTP-binding protein Gβ{gamma} (49). Knockout of PI3K-{gamma} in mice, or transfection of small interfering RNA against PI3K-{gamma} in β-cells, significantly inhibits glucose-stimulated insulin secretion (37). The partial inhibition in the [Ca2+]i response and complete inhibition in BLX-1002-induced insulin secretion by the specific PI3K inhibitor suggest that this kinase may be involved both in the regulation of [Ca2+]i (19, 40) and insulin exocytosis (16). Considering that the PI3K inhibitors wortmannin and LY-294002 have no selectivity for individual PI3K isoforms (28) and that different isoforms of PI3K may play different roles in insulin secretion (16, 37), the PI3K isoform involved in BLX-1002-induced insulin secretion requires further investigation.

Activation of the β-cell by BLX-1002 also induced a sustained activation of AMPK, a cellular energy sensor that plays an important role in the regulation of glucose and lipid homeostasis (38, 47). AMPK is also considered a potential pharmacological target for the treatment of metabolic disorders, including obesity and type 2 diabetes (22). In β-cells, AMPK was shown to be involved in both preproinsulin gene expression (8) and insulin secretion (8, 34). The role of AMPK in insulin secretion remains controversial. Activation of AMPK in the β-cell was shown to suppress glucose-stimulated insulin secretion (8), supported by the fact that glucose-stimulated insulin secretion was accompanied by an inhibition of AMPK activity (8, 34, 48). In agreement with this notion, glucose-stimulated insulin secretion was impaired in islets overexpressing AMPK (46) or in islet cells subjected to a chronic activation of the enzyme (50). However, inhibition of AMPK activity by amino acids did not correlate with insulin secretion (17). Activation of the kinase by 5-amino-imidazole carboxamide riboside (AICAR) was associated with an increased insulin secretion (1). In addition, glucose-stimulated insulin secretion was not affected in islets from transgenic mice lacking AMPK (51). A recent study suggested that AMPK may not be part of the signaling pathways regulating glucose-stimulated insulin secretion, but rather a critical factor in nutritional regulation of the mammalian target of rapamycin (mTOR) signaling (17). It was proposed that changes in β-cell AMPK activity evoked by nutrients may facilitate regulation of energy availability, protein synthesis (29), cell growth (33), or apoptosis (8, 17, 33). The present study shows that metformin, which may affect insulin secretion in the long term (50), failed to acutely influence glucose-induced insulin secretion (45) or insulin secretion stimulated by BLX-1002 despite an activation of AMPK by both effectors. Similarly, the BLX-1002 metabolite, BLX-1015, activated AMPK in the cells without influencing insulin secretion. The suppression BLX-1002-induced [Ca2+]i by the AMPK inhibitor compound C may reflect an unspecific effect of the inhibitor, as glucose-stimulated rise in [Ca2+]i was also suppressed. We interpret this to mean that activation of AMPK does not seem to play a major role in the BLX-1002-induced insulin secretion over the short term. Although the role of activation of AMPK in the present study remains unknown, the sustained AMPK activation elicited by BLX-1002 may suggest an involvement in long-term effects of the drug, an issue that should be pursued.

In conclusion, BLX-1002 selectively potentiates insulin secretion induced by high glucose in normal and diabetic murine islets, an effect that is dependent on PI3K activity and is associated with an increased [Ca2+]i and sustained AMPK activation in the β-cell. The glucose-sensitive stimulatory impact of BLX-1002 on β-cell function may translate into substantial clinical benefits of the drug in the management of type 2 diabetes, by avoidance of hypoglycemia.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Financial support was provided by Karolinska Institutet, Stiftelsen Olle Engkvist Byggmästare, the Janne Elgqvist Family Foundation, the Swedish Society of Medicine, the Sigurd and Elsa Golje Memorial Foundation, Svenska Försäkringsföreningen, Svenska Diabetesstiftelsen, Magn. Bergvall Foundation, Stiftelsen Samariten, Barndiabetesfonden, Åke Wiberg's Foundation, Torsten and Ragnar Söderberg's Foundations, Berth von Kantzow's Foundation, Trygg-Hansa's Research Foundation, Tore Nilson's Foundation for Medical Research, Fredrik and Inger Thuring's Foundation, Syskonen Svensson's Fund, and the Research Center at Södersjukhuset, Stockholm.


   ACKNOWLEDGMENTS
 
We thank Torun Bergström for assisting in AMPK measurements.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Q. Zhang and Å. Sjöholm, Diabetes Research Center, Dept. of Internal Medicine, Karolinska Institutet, South Hospital, SE-11883 Stockholm, Sweden (e-mail: qimin.zhang{at}ki.se; ake.sjoholm{at}ki.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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