JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 JPET FastThis Forward. article has not Published been copyedited on andSeptember formatted. The 10, final 2009 version as may DOI:10.1124/jpet.109.152751 differ from this version.

JPET #152751 1

Kathrin Hatlapatka, Antje Wienbergen, Claudia Kühne, Anne Jörns, Michael Willenborg and

Ingo Rustenbeck

Selective enhancement of nutrient-induced insulin secretion by KATP channel-blocking imidazolines.

Institute of Pharmacology and Toxicology, Technical University of Braunschweig, D-38106

Braunschweig, Germany (K.H., A.W., C.K., M.W., I.R.) and Institute of Anatomy, Hannover

Medical School, D-30623 Hannover, Germany (A.J.) Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021

Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 2

Running title : Insulinotropic effect of imidazolines

Correspondence: Dr. Ingo Rustenbeck, Institute of Pharmacology and Toxicology,

University of Braunschweig, Mendelssohnstr. 1, D-38106 Braunschweig, Germany. Tel: x49/531/391-6570; Fax: x49/531/391-8287; E-mail: [email protected]

Text pages : 20

Tables : 0 Downloaded from

Figures : 8

References : 40 jpet.aspetjournals.org

Abstract : 249 words

Introduction : 622 words at ASPET Journals on September 25, 2021

Discussion : 1672 words

2+ Abbreviations: [Ca ]c: cytosolic free calcium concentration; KATP channel, ATP-sensitive

K+ channel; KIC: -ketoisocaproic acid; SUR: sulfonylurea receptor

Section : Endocrine and Diabetes JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 3

Abstract

The contribution of KATP channel-dependent and -independent signalling to the insulinotropic characteristics of imidazolines was explored using perifused mouse islets and cells. Up to a concentration of 100 µM efaroxan had no insulinotropic effect in the presence of a basal glucose concentration, but enhanced the effect of a stimulatory concentration of glucose or non-glucidic nutrients (ketoisocaproate plus glutamine). The secretion by a non-nutrient (40 mM KCl) was not enhanced. At 500 µM efaroxan stimulated insulin secretion when glucose was basal. Similarly, at 0.1 to 10 µM RX871024 showed a purely enhancing effect, but at 100 Downloaded from

µM it elicited a strong KCl-like secretory response in the presence of basal glucose. At 0.1 and 1 µM RX871024 did not significantly depolarize the cell membrane. However, at a jpet.aspetjournals.org purely enhancing drug concentration (10 µM RX871024 or 100 µM efaroxan) KATP channel activity was strongly reduced, the membrane was depolarized, and the cytosolic Ca2+

concentration was elevated in the presence of basal glucose. The insulin secretion by SUR1 at ASPET Journals on September 25, 2021

KO islets, which have no functional KATP channels, was neither increased by efaroxan (100 or

500 µM) nor by 10 µM RX871024, but by 100 µM RX871024. The imidazolines phentolamine and alinidine (100 µM) were also ineffective on SUR1 KO islets. It is concluded that a significant KATP channel block is compatible with a purely enhancing effect of the imidazolines on nutrient-induced insulin secretion. Only RX871024 has an additional, non-depolarizing effect, which at a high drug concentration is able to elicit a KATP channel- independent secretion. JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 4

Introduction

The insulinotropic effect of the prototypical imidazoline, phentolamine was originally believed to be due to an antagonism at -adrenoceptors of the cell (Robertson and Porte,

1973; Efendic et al, 1975). The observation that the insulinotropic effect of this compound was not shared by other antagonists at -adrenoceptors but was shared by other compounds with imidazoline moieties (Östenson et al., 1988, Schulz and Hasselblatt, 1989), led to the hypothesis that the insulinotropic effect was mediated by a -cell-specific subtype of the imidazoline receptor (Chan et al., 1994). In other tissues at least two non-adrenergic Downloaded from imidazoline binding sites have been identified and consequently the hypothetical -cell

subtype was named I3-receptor (Eglen et al., 1998; Morgan, 1999). In contrast to the I1 and I2 jpet.aspetjournals.org sites where binding occurs with nanomolar affinity, binding to I3 sites requires micromolar concentrations of the imidazoline (Rustenbeck et al., 1997).

The demonstration that phentolamine and other imidazolines block KATP channels in at ASPET Journals on September 25, 2021 pancreatic -cells offered an explanation for their insulinotropic property (Plant and Henquin,

1990; Chan and Morgan, 1991). However, it remained unclear how the effect on the KATP channel was related to the non-adrenergic imidazoline binding sites on -cells (Rustenbeck et al., 1997). The concept of a -cell imidazoline receptor mediating multiple effects by signal transduction cascades was rendered less likely by the observation that the KATP channel block by imidazolines was exerted directly at Kir6.2, the pore-forming subunit of the channel (Proks and Ashcroft, 1997; Grosse-Lackmann et al, 2003). Kir6.2 was then suggested to be the correlate of the I3 receptor (Morgan, 1999, Monks et al., 1999).

The demonstration that the imidazoline RX 871024 increased insulin secretion not

2+ only by blocking KATP channels, but also by acting at a site distal to Ca influx led to the alternative hypothesis that imidazolines exert effects at multiple independent sites (Zaitsev et al., 1996). The experimental model to demonstrate the KATP channel-independent effect was JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 5

2+ the permeabilized cell where the cytosolic calcium concentration ([Ca ]c) was clamped by use of Ca2+-buffered incubation media. RX871024 (Zaitsev et al., 1996) and later on efaroxan

(Chan et al., 2001) were shown to increase the insulin release from such permeabilized cell preparations. It was hypothesized that these imidazolines sensitize the exocytotic machinery

2+ to [Ca ]c, which in turn would be increased by the KATP channel-blocking effect of these compounds. A similar combination of effects, comprizing a PKC-mediated sensitization of the exocytotic machinery in addition to a KATP channel block, has been suggested to explain Downloaded from the insulinotropic characteristics of sulfonylureas (Eliasson et al., 1996). The hypothesis that the dependency on stimulatory glucose concentrations is due to an effect at a site distal to

2+

Ca influx appeared to be confirmed by the development of imidazolines (e.g. BL 11282 or jpet.aspetjournals.org

NNC77-0074) which do not block KATP channels, but stimulate insulin secretion nevertheless

(Efanov et al., 2001; Hoy et al., 2003). These imidazolines were then termed “second at ASPET Journals on September 25, 2021 generation imidazolines“ to distinguish them from the KATP channel-blocking imidazolines which were termed “first generation imidazolines“ (Efendic et al., 2002).

2+ However, a sensitization of the exocytotic machinery to [Ca ]c should also amplify subthreshold signals of non-nutrient secretagogues and thus seems an unlikely explanation for the selective enhancement by efaroxan and RX871024 of the effect of suprathreshold glucose concentrations (Zaitsev et al., 1996; Efendic et al., 2002; Bleck et al., 2004). Here we present evidence that the selective enhancement does not consist in a sensitization of exocytosis to

2+ [Ca ]c increases, but in an enhanced efficacy of amplifying signals derived from the metabolism of suprathreshold concentrations of nutrient secretagogues (Henquin, 2000). In spite of this common property, only RX871024 but not efaroxan, phentolamine or alinidine had a KATP channel independent insulinotropic effect, arguing against an indispensable role of the distal site for the characteristics of imidazoline-induced insulin secretion. JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 6

Methods

Chemicals. Efaroxan was purchased from Tocris-Cookson (Bristol, UK), quinine from Sigma (Taufkirchen, Germany) and tolbutamide from Serva (Heidelberg, Germany).

Phentolamine was kindly donated by Novartis/Ciba-Geigy (Lörrach, Germany), alinidine by

Boehringer Ingelheim (Ingelheim, Germany), and RX871024 by Reckitt and Colman

(Kingston, UK). Fura-PE3/AM was purchased from TEF Labs (Austin, TX, USA).

Collagenase P from Boehringer Mannheim/Roche Diagnostics (Mannheim, Germany), cell Downloaded from culture medium RPMI 1640 from Gibco/Invitrogen (Karlsruhe, Germany) and fetal calf serum from PAA (Cölbe, Germany). All other reagents of analytical grade were from E. jpet.aspetjournals.org Merck (Darmstadt, Germany).

Tissue culture. Islets were isolated from the pancreas of NMRI or SUR1 KO mice (10

to 14 weeks old) by a collagenase digestion technique and hand-picked under a at ASPET Journals on September 25, 2021 stereomicroscope. Single cells were obtained by incubation of the islets for 10 min in a Ca2+- free medium and subsequent vortex-mixing for 1 min. Islets and single islet cells were cultured in cell culture medium RPMI-1640 with 10 % fetal calf serum (5 mM glucose) in a humidified atmosphere of 95% air and 5% CO2 at 37°C.

Electrophysiological recordings. KATP channel activity was measured by a standard patch-clamp technique using the cell-attached configuration (Hamill et al., 1981). Pipettes were pulled from borosilicate glass (2 mm o.d., 1.4 mm i.d., Hilgenberg, Malsfeld, Germany) by a two-stage vertical puller (List Electronic, Darmstadt, Germany ) and had resistances between 3 and 6 MOhm when filled with solution. Currents were recorded by an EPC 7 patch-clamp amplifier (List Electronic), and low pass-filtered by a 4-pole Bessel filter at 2 kHz and stored on a video tape. The pipette holding potential was 0 mV in cell-attached recordings. The membrane potential of -cells was determined using the whole-cell and JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 7 perforated patch modes under current clamp condition (Smith et al., 1990). Exposure to the test compounds and wash-out was done by changing the bath medium with a slow bath perfusion system. The compositions of the bath and pipette media were as given by Zünkler et al. (Zünkler et al., 1988). All experiments were performed at room temperature (20 - 23 °C).

Data were analysed off-line using pClamp 6.03 software (Axon Instruments, Foster City, CA,

USA).

2+ Microfluorimetric measurements of the cytosolic free Ca concentration Downloaded from

2+ ([Ca ]c). Islets were cultured on collagen-coated glass cover slips in Petri dishes and were used from day 2 to 4 after isolation. Fura-2/AM was loaded at a concentration of 2 µM jpet.aspetjournals.org (dissolved in Krebs-Ringer medium with 5 mM glucose) for 45 min at 37 °C. The cover slip with the attached cells or islets was inserted in a purpose-made perifusion chamber on the stage of an epifluorescence microscope fitted with a Zeiss Fluar (40 x) objective. The at ASPET Journals on September 25, 2021 fluorescence (excitation at 340 or 380 nm, emission > 470 nm) was recorded by a slow-scan

CCD camera (TILL Photonics). All perifusions were performed at 35 °C using a HEPES- buffered Krebs-Ringer bicarbonate medium. Image pairs were taken at intervals as indicated in the figures, illumination time for each image was 800 ms. RX871024, but not efaroxan proved to be fluorescent with an excitation maximum at 331 nm and a broad emission peak with a maximum at 460 nm. This necessitated to perform the experiments in duplicate using sham-loaded islets to subtract the RX871024 fluorescence from the Fura fluorescence emission.

Electron microscopy. Collagenase-isolated islets from SUR1 KO mice were pooled and cultured in batches of 50 islets. After culturing for 20 h in RPMI 1640 with 100 µM efaroxan or RPMI 1640 alone (glucose concentration 5 mM), the islets were fixed for electron microscopy by immersing in a solution of para-formaldehyde (2 %) and di-glutaraldehyde (2

%) in cacodylate buffer (0.1 M, pH 7.3), post-fixing in osmium tetroxide (1 %) for 1 h and JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 8 embedding in epoxy resin. Fifty-nm sections of the islets were cut by an ultramicrotome

(Ultracut, Leica-Reichert-Jung, Germany), placed on nickel grids and contrast-stained with uranyl acetate and lead citrate. Transmission electron microscopy was performed using a

Zeiss EM 9 electron microscope. Medium-sized islets (diameters about 150 µm) were chosen for ultrastructural examination of the granulation state. The analyzed islets contained 50 to 90

-cells per section. Two independent incubations were performed for each condition.

Measurement of insulin secretion. Batches of 50 freshly isolated NMRI or SUR1 Downloaded from

KO mouse islets were perifused (1 ml per min) at 37 °C with a HEPES-buffered Krebs-

Ringer medium containing the respective secretagogue. The insulin content in the fractionated jpet.aspetjournals.org effluate was determined by ELISA (Mercodia, Uppsala, Sweden).

Data handling and statistics. Statistical calculations were performed by Prism and

Instat software (GraphPad, San Diego CA, USA). If not specified otherwise, differences were at ASPET Journals on September 25, 2021 considered significant if p  0.05. JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 9

Results

Concentration-dependent changes in the insulintropic characteristics of imidazolines. The glucose dependency of the insulinotropic effect was characterized in dependence of the drug concentration. The experimental protocol was designed to assess the effect of the secretagogue in the presence of a substimulatory glucose concentration and then to raise the glucose concentration to a moderately stimulatory value to assess the enhancing effect of the secretagogue. Efaroxan at 10, 30 or 100 µM did not stimulate insulin secretion Downloaded from when the glucose concentration was 5 mM (Fig. 1A). When glucose was raised to 10 mM, the secretion was only slightly affected by 10 µM efaroxan, whereas 30 µM had a clear jpet.aspetjournals.org enhancing effect (Fig. 1A). After a first phase-like increase a plateau was established with a secretory rate of about 350 % of the prestimulatory level. In the presence of 100 µM efaroxan the stimulatory effect of 10 mM glucose was strongly enhanced, resulting in a first phase-like at ASPET Journals on September 25, 2021 increase of secretion which peaked after 6 min at 710 % of the prestimulatory rate. After 10 min a nadir was reached, thereafter the secretion rate increased again until to the end of the efaroxan perifusion (Fig. 1A). 500 µM efaroxan, in contrast, was markedly effective in the presence of 5 mM glucose, within 8 min a plateau of 520 % of the prestimulatory level was established. Raising the glucose concentration to 10 mM led to a further increase to 930 % which remained stable until to the end of the efaroxan exposure. Very low concentrations of efaroxan (0.1 and 1.0 µM) were entirely ineffective (Fig. 1A).

The insulinotropic effect of 10 µM RX871024 resembled that of 30 or 100 µM efaroxan in that no stimulation of secretion was seen in the presence of 5 mM glucose, but a marked enhancement of secretion with a prominent first phase occurred when the glucose concentration was raised to 10 mM (Fig. 1B). At 100 µM RX871024 however, a completely different pattern emerged. After 4 min of exposure in the presence of 5 mM glucose a massive increase of secretion started, which peaked after 10 min at 1080 %. This was followed by a JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 10 steady decline of secretion which was only transiently reversed when glucose was raised to 10 mM (Fig. 1B). This response pattern resembled that of a strongly depolarising concentration of KCl (40 mM). Again, a very high peak value was quickly reached in the presence of 5 mM glucose and thereafter the secretion rate declined steadily and was only transiently increased when glucose concentration was raised to 10 mM (Fig. 1B). In contrast to efaroxan very low concentrations of RX871024 (0.1 and 1.0 µM) clearly enhanced the stimulatory effect of 10 mM glucose (Fig. 1B). Downloaded from KATP channel block and plasma membrane depolarization by efaroxan and

RX871024. The concentration-dependent change in the insulinotropic characteristics was compared with the concentration-dependent block of KATP channel activity. Efaroxan reduced jpet.aspetjournals.org the KATP channel activity in intact -cells to 45.4  4.9 % at 10 µM, 34.0  5.8 % at 30 µM,

19.1  5.3 % at 100 µM and 8.2  2.4 % at 300 µM. Under the same condition, RX 871024 reduced channel activity to 17.5  6.7 % at 10 µM and 4.3.  1.9 % at 100 µM. Thus, the at ASPET Journals on September 25, 2021

KATP channel-blocking effect of 10 µM RX 871024 was equivalent with that of 100 µM efaroxan and the effect of 100 µM RX was not significantly different ( p = 0.23, t-test) from that of 300 µM efaroxan.

Consistent with these data it was found that 10 µM RX871024 was significantly more effective than 10 µM efaroxan to depolarize the -cell membrane. RX871024 depolarized from a resting potential of -69.3  2.3 mV to -40.3  2.8 mV (n = 7), whereas efaroxan depolarized from -72.4  2.6 mV to -54.6  5.1 mV (n = 5). At 100 µM however, the membrane depolarization by RX 871024 (-26.3  2.5 mV, n = 5) was not significantly different from that by efaroxan (-28.6  3.4 mV, n = 7). At a very low concentration (0.1 µM)

RX was unable to significantly depolarize the -cell membrane. In conclusion, RX871024 is clearly more potent as a KATP channel blocker than efaroxan, but at concentrations above 100

µM, both compounds were practically equi-effective . JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 11

2+ The role of [Ca ]c signalling in the imidazoline enhancement of glucose-induced

2+ insulin secretion. The relevance of [Ca ]c signalling for the glucose-dependency of the insulinotropic effect was measured using the the same experimental protocol as for the secretion measurements. A preceding perifusion with a high K+ concentration (40 mM) for 10 min served as internal standard. In the presence of 5 mM glucose 10 µM efaroxan had a

2+ negligible effect on [Ca ]c (Fig. 2A). As under control condition, raising the glucose

2+ concentration to 10 mM evoked an immediate increase of [Ca ]c which developed into an Downloaded from

2+ oscillatory pattern. Similar slow oscillations of [Ca ]c were induced by 100 µM efaroxan in the presence of 5 mM glucose and continued with a higher frequency (0.31 min-1 vs 0.16 min- jpet.aspetjournals.org 1, n = 6 each) when the glucose concentration was raised to 10 mM (Fig. 2B). A transition

2+ 2+ from an oscillatory [Ca ]c increase to a sustained [Ca ]c increase occurred reproducibly by increasing the efaroxan concentration from 100 to 500 µM in the presence of 5 mM glucose at ASPET Journals on September 25, 2021 (Fig. 2C).

100 µM RX led to massive sustained increase of the fura fluorescence ratio (Fig. 3A).

However, the extraordinary magnitude of this increase was largely due to the fluorescence of

RX871024 itself, as could be shown by performing the same experiment with islets which had not been loaded with Fura (Fig. 3B). Calculating a net Fura fluorescence ratio revealed that

2+ + RX871024 increased the [Ca ]c to about the same values as the preceding K depolarization

(Fig. 3C). Because of the autofluorescence of RX871024 and the necessary correction

2+ procedure, the occurrence of [Ca ]c oscillations could not be observed.

Efaroxan enhancement of insulin secretion induced by non-glucidic nutrients and non-nutrient stimuli. To test whether the enhancement of secretion by efaroxan was specifically dependent upon a stimulatory glucose concentration or more generally dependent upon a stimulatory concentration of any fuel secretagogue, the glucose content in the perifusion medium was replaced by the combination of ketoisocaproic acid (KIC) and JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 12 glutamine. At a concentration of 1 mM each, insulin secretion was basal, but raising the KIC concentration to 3 mM produced a moderate monophasic increase (Fig. 4A). 100 µM efaroxan had no effect in the presence of 1 mM KIC plus 1 mM glutamine, but the increase of secretion elicited by 3 mM KIC was markedly enhanced in the presence of 100 µM efaroxan

(Fig. 4A).

At a non-stimulatory concentration of glutamine plus KIC (1 mM each) repeated

2+ [Ca ]c waves were elicited by 100 µM efaroxan, this pattern continued when KIC was raised

2+ Downloaded from to 3 mM (Fig. 4B). When mean values were calculated, the transient increase of [Ca ]c elicited by 3 mM KIC was clearly discernible above the plateau resulting from the phase-

2+ jpet.aspetjournals.org shifted [Ca ]c waves elicited by efaroxan (Fig. 4C). Wash-out of efaroxan led to a return to basal levels (Fig. 4C).

To measure the effect of efaroxan on the insulin secretion elicited by a non-nutrient at ASPET Journals on September 25, 2021 stimulus the glucose concentration was kept at 5 mM throughout. Exposure to 40 mM KCl for

10 min gave a marked secretory response which returned to baseline values within 15 min after the K+ concentration was reduced to the physiological level. Repeating the 10 min exposure to high K+ after an interval of 40 min gave a secretory response (area under the curve) which amounted to 64 % of the initial response (Fig 5A). When the second exposure to high K+ was performed in the presence of 100 µM efaroxan, the percentage was not significantly increased (85 % of the initial secretory response to KCl). In these experiments the exposure to efaroxan preceded the second K+ depolarization for 20 min, during this time no increase in secretion was observed (Fig. 5A). The 10 min K+ depolarization led to a square

2+ + wave-like [Ca ]c increase which was essentially unchanged when the K depolarization was repeated after a 40 min interval (data not shown). When 100 µM efaroxan was added to the

+ 2+ perifusion medium prior to the second K depolarisation, an oscillatory increase of [Ca ]c

2+ resulted. The amplitude of the mean value corresponded to the plateau value of the [Ca ]c JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 13 increase by K+ depolarization (Fig. 5B). The second K+ depolarization which was performed

2+ in the presence of efaroxan resulted in transient [Ca ]c peak discernible above the mean

2+ [Ca ]c value established by efaroxan (Fig. 5B).

The contribution of KATP channel-independent signalling of the imidazolines to the glucose-dependency. To assess the role of KATP channel-independent effects in the secretory characteristics of efaroxan and RX 871024, isolated islets from SUR1 KO mice were perifused using the same protocol as for the initial secretion measurements (see Fig. 1). Downloaded from Neither at 5 mM glucose nor at 10 mM glucose did 100 µM efaroxan increase secretion by

SUR1 KO islets. Even at 500 µM efaroxan was completely ineffective (Fig. 6A). Similarly,

RX871024 at 0.1 and 10 µM was unable to significantly increase insulin secretion when the jpet.aspetjournals.org glucose concentration was raised to 10 mM. In contrast, a clear increase of secretion (2.5 fold) was produced by 100 µM RX871024. However, this increase occurred already when the at ASPET Journals on September 25, 2021 islets were perifused with 5 mM glucose. When the glucose concentration was raised to 10 mM, a marked transient decrease down to the prestimulatory level occurred (Fig. 6B). To decide which of the response patterns can be regarded as typical for the class of KATP channel- blocking imidazolines the effect of phentolamine and alinidine (100 µM each) on insulin secretion by SUR1 KO islets was characterized (Fig. 7) The secretory response of SUR1 KO islets to both compounds was negligible as compared to that of NMRI islets (Fig. 7A and B).

Quinine, which like imidazolines blocks KATP channels at the pore-forming subunit had a moderate insulinotropic effect on SUR1 KO islets (Fig. 7C), but similar to RX871024 this effect was already visible in the presence of 5 mM glucose. The inefficiency of efaroxan to stimulate insulin secretion by SUR1 KO islets was confirmed by ultrastructural examination of the granulation state. A 20 h exposure to 100 µM efaroxan, which strongly degranulated - cells in NMRI mouse islets, did not affect the content of insulin granules as compared with control-cultured SUR1 KO islets (Fig. 8). JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 14

Discussion

In the present investigation we confirm the earlier observation, that the imidazoline RX

871024 enhances insulin secretion at stimulatory, but not at subthreshold glucose concentrations and that this selectivity is lost at a higher drug concentration (Zaitsev et al.,

1996; Efendic et al., 2002). Our other observations, however, lead us to a reinterpretation as to the underlying mechanism of action. It has been proposed that the loss of selectivity at high concentrations of RX871024 ( 50 µM) was due to the KATP channel-blocking effect of this Downloaded from imidazoline (Efendic et al., 2002), since it is widely accepted that a block of KATP channels is sufficient to stimulate insulin secretion. Our recent observation that the imidazoline efaroxan jpet.aspetjournals.org did not stimulate insulin secretion of normal mouse islets at a basal glucose concentration, even though it reduced KATP channel activity, depolarized the plasma membrane and induced

2+ oscillatory increases in [Ca ]c under the same condition (Bleck et al., 2004), was not at ASPET Journals on September 25, 2021 compatible with this explanation and prompted us to reassess the mechanism of action of

KATP channel-blocking imidazolines.

The secretion measurements in this study showed that similar to the insulinotropic effect of RX871024, that of efaroxan lost its selectivity for stimulatory glucose concentrations at a high drug concentration. The potency of the insulinotropic effect was paralleled by the potency of the KATP channel block, in both respects RX871024 being about 5 to 10fold more potent than efaroxan. 10 µM RX871024 and 100 µM efaroxan were similarly effective to inhibit KATP channel activity (by about 80 % in intact cells) and produced an insulinotropic effect of similar strength, which was selective for stimulatory glucose. With both compounds a reduction of KATP channel activity by about 95 % was paralled by a stimulatory effect at a basal glucose concentration.

However, there was a remarkable qualitative difference between the stimulatory effects of high concentrations of efaroxan and of RX871024. While the secretion plateau JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 15 induced by 500 µM efaroxan in the presence of 5 mM glucose could be further increased by raising glucose to 10 mM, 100 µM RX871024 produced a very high secretion peak in the presence of 5 mM glucose, which spontaneously turned into a steady decline, which could only transiently reverted by raising glucose to 10 mM. This pattern resembled that produced by a strongly depolarizing KCl concentration and is reminiscent of the earlier observations that the insulinotropic action of non-nutrient secretagogues leads to a decreased responsiveness upon renewed stimulation, whereas the opposite, a potentiation, results from nutrient stimulation (Nesher and Cerasi, 1987; Nesher and Cerasi, 2002). Downloaded from

As a non-glucidic nutrient we used KIC in combination with glutamine. KIC requires a transamination partner to continuously produce both triggering and amplifying signals in the jpet.aspetjournals.org

Krebs cycle (Gao et al., 2003; Panten and Rustenbeck, 2008). This way, high concentrations of KICcould be avoided, which have a direct blocking effect on KATP channels of the -cells

2+ at ASPET Journals on September 25, 2021 (Heissig et al., 2005). As with glucose, efaroxan raised [Ca ]c, but not insulin secretion when the KIC concentration was non-stimulatory. Raising the nutrient concentration to a stimulatory level revealed the enhancing effect of efaroxan on secretion. Apparently, efaroxan enhances not only glucose-induced secretion, but more generally, nutrient-induced insulin secretion, whereas it did not enhance the secretion elicited by a non-nutrient (40 mM KCl).

Thus the earlier hypothesis that the characteristics of the imidazoline effect are due to a

2+ sensitization of the exocytotic machinery to [Ca ]c (Zaitsev et al., 1996) may need revision.

Concurrent with their KATP channel-blocking activity, both efaroxan and RX871024

2+ produced substantial increases of [Ca ]c in the presence of 5 mM glucose, which raises the

2+ question as to why this [Ca ]c increase was not transduced into an increased secretion. The

2+ characteristic feature of the [Ca ]c increase by efaroxan is its oscillatory nature. It has a

-1 2+ frequency (0.3 min ) like the slow [Ca ]c oscillation typically induced by moderate

2+ stimulatory glucose concentrations (Liu et al., 1998). Most likely, the [Ca ]c oscillation is the JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 16 direct consequence of the oscillatory cell membrane depolarisation elicited by efaroxan in the presence of 5 mM glucose (Bleck et al., 2005). The repolarization in spite of the continuous presence of efaroxan may result from to the marked susceptibility of the KATP channel block by efaroxan to the opening effect of nucleoside diphosphates (Wienbergen et

2+ al., 2007). The [Ca ]c oscillation caused by efaroxan may thus reflect an underlying oscillation of the ATP/ADP ratio at basal glucose levels (Dryselius et al., 1994).

The observation that raising the efaroxan concentration from a nutrient-selective (100 Downloaded from 2+ µM) to a nonselective level (500 µM) transformed the [Ca ]c oscillation into a sustained

2+ increase suggested a critical role of the oscillatory [Ca ]c pattern. On the other hand, a short

2+ jpet.aspetjournals.org term [Ca ]c increase by 40 mM KCl elicited a strong secretory response. Our earlier assumption that imidazolines may exert an inhibitory action distal to Ca2+ influx which is overcome by increased energy metabolism (Bleck et al., 2004, Bleck et al., 2005) cannot at ASPET Journals on September 25, 2021 explain this discrepancy, since the insulinotropic effect of the short KCl pulse persisted practically unchanged in the presence of efaroxan. The difference may rather be due to the fact that 40 mM KCl has about the double depolarizing strength than a KATP channel closure

(about 40 mV vs. 20 mV). Recently, it was described that a prolonged 20 mV depolarization by 15 mM KCl had only a modest transient effect on secretion (Hatlapatka et al., 2009). So,

2+ the oscillatory pattern may keep the [Ca ]c signal at a subthreshold level for secretion when the nutrient concentration is insufficient to activate metabolic amplifying pathways.

2+ RX871024-induced [Ca ]c oscillations could not be observed because of the fluorescence of aqueous solutions of this compound, which was not cancelled out by the ratiometric Fura measurements. This could be overcome by calculating a net Fura ratio from

2+ the mean values. Some earlier conclusions concerning the effects of RX871024 on [Ca ]c may need reevaluation in light of this observation. Nevertheless, effects on intracellular Ca2+ handling by RX871024 could contribute to the different insulinotropic characteristics as JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 17

compared to the other KATP channel-blocking imidazolines (Efanov et al., 1998; Efanova et al., 1998).

The role of KATP channel-independent signalling was tested by using islets of SUR1

KO mice, which have no functional KATP channels in the endocrine pancreas (Seghers et al.,

2000). In an earlier investigation, 50 µM RX871024 increased insulin secretion from such islets three- to fourfold in the presence of both 3 or 17 mM glucose (Efanov et al., 2001). The increase of secretion by SUR1 KO mice in the present investigation was of a comparable magnitude, but it occurred only at the high (100 µM), but not at the low (10 µM) or very low Downloaded from

(0.1 µM) concentration, which selectively enhance glucose-stimulated insulin secretion. In marked contrast to RX871024, efaroxan had practically no insulinotropic effect on SUR1 KO jpet.aspetjournals.org islets. This applied to both the nutrient-selective concentration (100 µM) and the non- selective concentration (500 µM). The lack of effect was confirmed by the inability of 100

µM efaroxan to affect the -cell granule content of SUR1 KO islets during a 20 h tissue at ASPET Journals on September 25, 2021 culture, whereas cells in NMRI islets were markedly degranulated by this procedure

(Rustenbeck et al., 2004).

Phentolamine and alinidine, two other KATP channel-blocking imidazolines which were used in early studies on this group of compounds (Schulz and Hasselblatt, 1989;

Rustenbeck et al., 1997; Proks and Ashcroft, 1997), were also unable to stimulate insulin secretion from SUR1 KO mice. On the other hand, quinine which similar to imidazolines blocks KATP channels at the pore-forming subunit and stimulates insulin secretion (Grosse-

Lackmann et al., 2003), but does not have an imidazoline moiety, had a moderate insulinotropic effect on SUR1 KO islets both in the presence of 5 and 10 mM glucose, similar to RX871024. Taken together, the present results suggest that the ability to elicit a KATP channel-independent insulin secretion is neither a general property of imidazoline JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 18 compounds, nor is it essential for the marked glucose dependency of their insulinotropic effect on normal mouse islets.

In view of this heterogeneity, is the imidazoline moiety a pharmacophore with regard to the insulinotropic effect? The existence of the second generation imidazolines (Efanov et al., 2001; Hoy et al., 2003) and the inability of the imidazole analogue of efaroxan, KU14R, to enhance insulin secretion in spite of a KATP channel-blocking and depolarizing effect

(Bleck et al., 2005) suggested that this is the case. In the latter report it was hypothesized that Downloaded from in addition to the KATP channel two imidazoline-specific sites of action would exist: one site

2+ which mediates the larger [Ca ]c increase of efaroxan as compared to KU14R and another

site which mediates the enhanced response to nutrients. The present observations are best jpet.aspetjournals.org explained by assuming that this may be one and the same effect: the imidazoline induced

2+ increase in [Ca ]c is practically insufficient to stimulate secretion on its own, but enhances the effect of nutrient stimuli, which activate the metabolic amplifying pathway(s) (Henquin, at ASPET Journals on September 25, 2021

2000; Panten and Rustenbeck, 2008). Such a model would also explain why the insulin secretion by 40 mM KCl, which has no metabolic amplifying effect, was not enhanced.

So is the selective enhancing effect of KATP channel-blocking imidazolines on nutrient-induced insulin secretion only due to their limited depolarizing effect? This appears to be true for efaroxan, phentolamine and alinidine, but not for RX871024. At 0.1 µM,

RX871024 enhanced glucose-induced insulin secretion, but did not yet significantly depolarize -cells. Apparently, RX871024 has two mechanisms of action with different concentration dependencies, one depolarizing and one non-depolarizing, both of which are compatible with a purely enhancing characteristic at the lower concentration range. The accumulation of PLA2 reaction products like arachidonic acid might be compatible with such a non-depolarizing effect (Sharyoko et al., 2007, Jones et al., 2004). At a high concentration of RX871024 the combination of both effects may be responsible for the massive stimulation JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 19 of secretion which exceeded the maximal effect of efaroxan. The nutrient-selective insulinotropic effect of the second generation imidazolines, the molecular mechanism of which has eluded clarification up to now, would then be phenomenologically similar, but mechanistically unrelated to that of the first generation imidazolines. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 20

Acknowledgments

Joseph Bryan and Lydia Aguilar-Bryan kindly provided the SUR1 KO mice. Skilful technical assistance by Petra Weber, Verena Lier-Glaubitz and Sabine Warmbold is gratefully acknowledged. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 21

References Bleck C, Wienbergen A, and Rustenbeck I (2005) Essential role of the imidazoline moiety for

the insulinotropic effect but not the KATP channel-blocking effect of imidazolines.

Diabetologia 48:2567-2575

Bleck C, Wienbergen A, and Rustenbeck I (2004) Glucose-dependence of imidazoline-

induced insulin secretion. Diabetes 53:S135-S139

Chan SLF, Brown CA, Scarpello K, and Morgan NG (1994) The imidazoline site involved in Downloaded from insulin secretion: characteristics that distinguish it from I1 and I2 sites. Br J Pharmacol

112:1065-1070

Chan SLF and Morgan NG (1991) Stimulation of insulin secretion by efaroxan may involve jpet.aspetjournals.org

interaction with potassium channels. Eur J Pharmacol 176:97-101

Chan SLF, Mourtada M, and Morgan NG (2001) Characterization of a KATP channel-

independent pathway involved in potentiation of insulin secretion by efaroxan. Diabetes at ASPET Journals on September 25, 2021

50:340-347

Dryselius S, Lund P, Gylfe E, and Hellman B (1994) Variations in ATP-sensitive K+ channel

activity provide evidence for inherent metabolic oscillations in pancreatic b-cells.

Biochem Biophys Res Commun 205:880-885

Efanov AM, Hoy M, Bränström R, Zaitsev S, Magnuson M, Efendic S, Gromada J, and

Berggren P-O (2001) The imidazoline RX871024 stimulates insulin secretion in

pancreatic -cells from mice deficient in KATP channel function. Biochem Biophys Res

Commun 284:918-922, 2001

Efanov AM, Zaitsev SV, Efanova IB, Zhu S, Östenson CG, Berggren PO, and Efendic S

(1998) Signaling and sites of interaction for RX-871024 and sulfonylurea in the

stimulation of insulin release. Am J Physiol 274:E751-E757 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 22

Efanov AM, Zaitsev SV, Mest HJ, Raap A, Appelskog IB, Larsson O, Berggren PO, and

Efendic S (2001) The novel imidazoline compound BL11282 potentiates glucose-induced

insulin secretion in pancreatic -cells in the absence of modulation of KATP channel

activity. Diabetes 50:797-802

Efanova IB, Zaitsev SV, Brown G, Berggren PO, and Efendic S (1998) RX871024 induces

Ca2+ mobilization from thapsigargin-sensitive stores in mouse pancreatic b-cells. Diabetes

47:211-218 Downloaded from Efendic S, Cerasi E, and Luft R (1975) Effect of phentolamine and preperfusion with glucose

on insulin release from the isolated perfused pancreas from fasted and fed rats.

Diabetologia 11:407-410 jpet.aspetjournals.org

Efendic S, Efanov A, Berggren PO, and Zaitsev A (2002) Two generations of insulinotropic

imidazoline compounds. Diabetes 51:S448-S454

Eglen RM, Hudson AL, Kendall DA, Nutt DJ, Morgan NG, Wilson VG, and Dillon MP at ASPET Journals on September 25, 2021

(1998) Seeing through a glass darkly: casting light on imidazoline “I“ sites. Trends

Pharmacol Sci 19:381-390

Eliasson L, Renström E, Ämmälä C, Berggren PO, Bertorello A, Bokvist K, Chibalin A,

Deeney JT, Flatt P, Gäbel J, Gromada J, Larsson O, Lindström P, Rhodes C, and Rorsman

P (1996) PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic B-cells.

Science 271:813-815

Gao Z, Young RA, Li G, Najafi H, Buettger C, Sukumvanich SS, Wong RK, Wolf BA, and

Matschinsky FM (2003) Distinguishing features of leucine and alpha-ketoisocaproate

sensing in pancreatic beta-cells. Endocrinology 144:1949-1957

Grosse Lackmann T, Zünkler BJ, and Rustenbeck I (2003) Specificity of nonadrenergic

imidazoline binding sites in insulin-secreting cells and relation to the block of ATP-

sensitive K+ channels. Ann NY Acad Sci 1009:371-377 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 23

Hatlapatka K, Willenborg M, and Rustenbeck I (2009) Plasma membrane depolarization as a

determinant of the first phase of insulin secretion. Am J Physiol Endocrinol Metab.

297:E315-22.

Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ (1981) Improved patch-clamp

techniques for high-resolution current recordings from cells and cell-free membrane

patches. Pflügers Arch 391:85-100

Heissig H, Urban KA, Hastedt K, Zünkler BJ, and Panten U (2005) Mechanism of the insulin-

releasing action of alpha-ketoisocaproate and related alpha-keto acid anions. Mol Downloaded from

Pharmacol. 68:1097-1105

Henquin JC (2000) Triggering and amplifying pathways of regulation of insulin secretion by jpet.aspetjournals.org

glucose. Diabetes 49:1751-1760

Hoy M, Olsen HL, Andersen HS, Bokvist K, Buschard K, Hansen J, Jacobsen P, Petersen JS,

Rorsman P, and Gromada J (2003) Imidazoline NNC77-0074 stimulates insulin secretion at ASPET Journals on September 25, 2021

and inhibits glucagon release by control of Ca2+-dependent exocytosis in pancreatic -

and -cells. Eur J Pharmacol 446:213-221

Jones PM, Burns CJ, Belin VD, Roderigo-Milne HM, and Persaud SJ (2004) The role of

cytosolic phospholipase A(2) in insulin secretion. Diabetes. 53:S172-8.

Liu Y, Tengholm A, Grapengiesser E, Hellman B, and Gylfe E (1998) Origin of slow and fast

oscillations of Ca2+ in mouse pancreatic islets. J Physiol 508:471-481

Monks LK, Cosgrove KE, Dunne M, Ramsden C, Morgan NG, and Chan SLF (1999) Affinity

isolation of imidazoline binding proteins from rat brain using 5-amino-efaroxan as a

ligand. FEBS Lett 447:61-64

Morgan NG (1999) Imidazoline receptors: targets for novel antihyperglycemic agents. Exp

Opin Invest Drugs 8:575-584 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 24

Nesher R and Cerasi E (1987) Biphasic insulin release as the expression of combined

inhibitory and potentiating effects of glucose. Endocrinology 121:1017-1024

Nesher R and Cerasi E (2002) Modeling phasic insulin secretion. Immediate and time-

dependent effects of glucose. Diabetes 51:S53-S59

Östenson CG, Pigon J, Doxey JC, and Efendic S (1988) 2-Adrenoceptor blockade does not

enhance glucose-induced insulin release in normal subjects or patients with non insulin-

dependent diabetes. J Clin Endocrinol Metab 67:1054-1059 Downloaded from Panten U and Rustenbeck I (2008) Fuel-induced amplification of insulin secretion in mouse

pancreatic islets exposed to a high sulfonylurea concentration: role of the NADPH/NADP+

ratio.2008 Jan; Diabetologia 51:101-109 jpet.aspetjournals.org

Plant TD and Henquin JC (1990): Phentolamine and yohimbine inhibit ATP-sensitive K+-

channels in mouse pancreatic -cells. Br J Pharmacol 101:115-120 at ASPET Journals on September 25, 2021 Proks P and Ashcroft F (1997) Phentolamine block of KATP channels is mediated by Kir6.2.

Proc Natl Acad Sci USA 94:11716-11720

Robertson RP and Porte D (1973) Adrenergic modulation of basal insulin secretion in man.

Diabetes 22:1-8

Rustenbeck I, Herrmann C, Ratzka P and Hasselblatt A (1997) Imidazoline/guanidinium

binding sites and their relation to inhibition of KATP channels in pancreatic B-cells.

Naunyn-Schmiedeberg's Arch Pharmacol 356:410-417

Rustenbeck I, Wienbergen A, Bleck C and Jörns, A (2004) Desensitization of insulin

secretion by depolarizing insulin secretagogues. Diabetes, 53:S140-S150

Schulz A and Hasselblatt A (1989) An insulin-releasing property of imidazoline derivatives is

not limited to compounds that block receptors. Naunyn-Schmiedeberg's Arch

Pharmacol 340:712-714 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 25

Sharyoko VV, Zaitsev I, Leibiger B, Efendic S, Berggren PO, and Zaitsev SV (2007)

Arachidonic acid signalling is involved in the mechanism of imidazoline-induced KATP

channel-independent stimulation of insulin secretion. Cell Mol Life Sci 64:2985-2993

Smith PA, Ashcroft FM, and Rorsman P (1990) Simultaneous recordings of glucose-

dependent electrical activity and ATP-regulated K+-currents in isolated mouse pancreatic

-cells. FEBS Lett 261:187-190

Wienbergen A, Bleck C, Grosse-Lackmann T, and Rustenbeck I (2007) Antagonism of the Downloaded from insulinotropic effect of first-generation imidazolines by openers of KATP channels.

Biochem Pharmacol 73:94-102

Zaitsev SV, Efanov AM, Efanova IB, Larsson O, Östenson C-G, Gold G, Berggren PO, and jpet.aspetjournals.org

Efendic S (1996) Imidazoline compounds stimulate insulin release by inhibition of KATP

channels and interaction with the exocytotic machinery. Diabetes 45:1610-1618

Zünkler BJ, Lins S, Ohno-Shosaku T, and Panten U (1988) Cytosolic ADP enhances the at ASPET Journals on September 25, 2021

sensitivity to tolbutamide of ATP-dependent K+-channels from pancreatic B-cells. FEBS

Lett 239:241-244 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

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Footnotes

This work was supported by grants from the Deutsche Forschungsgemeinschaft [Ru 368/4-1,

Ru 368/5-1] and the German Diabetes Society. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 27

Legends for Figures:

FIG. 1. Concentration-dependent change in the glucose-dependency of the insulinotropic effect of imidazolines (A) Freshly isolated islets were perifused with Krebs-Ringer medium containing 5 mM glucose for 90 min. From 60 min to 120 min the medium contained either 1

µM (open circles), 30 µM (closed circles), 100 µM (open squares) or 500 µM (closed squares) efaroxan. From 90 min to 130 min the glucose concentration was increased to 10 mM. The effect of 1 µM efaroxan was not significantly different from that of glucose alone Downloaded from (control = solid line) as was that of 10 µM (not shown). Only at 500 µM, efaroxan was effective in the presence of 5 mM glucose. Values are means ± SEM of 3 to 5 experiments.

(B) Freshly isolated islets were perifused with Krebs-Ringer medium containing 5 mM jpet.aspetjournals.org glucose for 90 min. From 60 min to 120 min the medium contained 0.1 µM (open circles), 1.0

µM (closed circles), 10 µM (open squares) or 100 µM (closed squares) of RX871024. From

90 min to 130 min the glucose concentration was increased to 10 mM. Note that the strong at ASPET Journals on September 25, 2021 stimulatory effect of 100 µM RX871024 is different from the effect of 500 µM efaroxan in that the massive increase was followed by a decline which could only be transiently overcome by raising the glucose concentration to 10 mM. This characteristic resembles the effect of 40 mM KCl (dotted line) under the same condition. Values are means ± SEM of 4 or 5 experiments.

2+ FIG. 2. Concentration-dependent effects of efaroxan on [Ca ]c of perifused pancreatic islets.

Fura 2-loaded islets were perifused with Krebs-Ringer medium containing 5 mM glucose.

After a depolarization with 40 mM K+ for 10 min the islets were perifused with 10 µM (A) or

100 µM (B) of efaroxan. In the continued presence of the drug, the glucose concentration was raised from 5 to 10 mM. This perifusion protocol corresponds to that of the secretion measurements shown in Fig.1. The response pattern obtained with 10 µM efaroxan - no

2+ 2+ [Ca ]c increase in the presence of 5 mM glucose and oscillatory [Ca ]c increase after raising JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 28 glucose to 10 mM - corresponded to that of a control experiment. (C) Raising the efaroxan concentration from 100 to 500 µM in the continuous presence of 5 mM glucose led to a

2+ transition from an oscillatory to a sustained [Ca ]c increase. Shown are traces from 6

2+ subregions of a single islet to demonstrate the synchronous oscillatory character of the [Ca ]c increase. Representative recordings of 4 to 5 experiments each.

2+ FIG. 3. Effect of RX871024 on [Ca ]c of perifused pancreatic islets. Fura 2-loaded islets were perifused with Krebs-Ringer medium containing 5 mM glucose. (A) After a Downloaded from depolarization with 40 mM K+ for 10 min the islets were perifused with 100 µM RX871024.

In the continued presence of the drug, the glucose concentration was raised from 5 to 10 mM.

This perifusion protocol corresponds to that of the secretion measurements shown in Fig.1. jpet.aspetjournals.org

Note the massive increase in the Fura fluorescence ratio by addition of RX871024. (B) The same experiment as in (A) performed without Fura-loading of the islet. Note there is no at ASPET Journals on September 25, 2021 fluorescence ratio increase during K+ depolarization, but after addition of 100 µM RX871024, suggestive of autofluorescence of this compound. (C) The net Fura fluorescence ratio by subtracting the recordings obtained without Fura loading from the recordings of the regular

2+ experiments. Note that the amplitude of the [Ca ]c increase is now in the range of that of the preceding K+ depolarization. Representative traces of 4 to 5 experiments each.

FIG. 4. Nutrient-dependency of the insulinotropic effect of efaroxan. (A) Freshly isolated islets were perifused with Krebs-Ringer medium containing non-glucidic nutrients (1 mM glutamine and 1 mM KIC) for 110 min. From 80 min to 140 min the medium contained 100

µM efaroxan (closed circles). From 110 min to 140 min the KIC concentration was increased to 3 mM. In the control experiment (open circles) efaroxan was absent. Efaroxan had a clear

2+ enhancing effect when the KIC concentration was raised. (B) Effect of efaroxan on [Ca ]c of a Fura 2-loaded islet using the same perifusion protocol as for the secretion measurements shown in (A). Shown are traces from 6 subregions of a single islet to demonstrate the JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 29

2+ synchronous oscillatory character of the [Ca ]c increase. (C) Mean values ± SEM of 5

2+ experiments as shown in (B). Note the increase of [Ca ]c in the presence of basal concentrations of the nutrients and the reversibility after efaroxan washout.

FIG. 5. Inability of efaroxan to enhance non-nutrient induced insulin secretion. (A) Freshly isolated islets were perifused with Krebs-Ringer medium containing 5 mM glucose throughout. From 60 min to 70 min and again from 110 min to 120 min the K+ concentration was raised to 40 mM. 100 µM efaroxan (closed circles) was present from 90 min to 130 min, Downloaded from i.e. the second K+ depolarization took place in the presence of efaroxan. In the control experiment (open circles) efaroxan was absent throughout. Values are means ± SEM of 4

2+ jpet.aspetjournals.org experiments each. (B) Effect of efaroxan and KCl on [Ca ]c of perifused islets. A Fura 2- loaded islet was perifused with Krebs-Ringer medium containing 5 mM glucose throughout.

KCl was raised to 40 mM first in the absence, then in the presence of efaroxan. Note the at ASPET Journals on September 25, 2021 2+ efaroxan-induced increase of [Ca ]c in the presence of 5 mM glucose. Values are means ±

SEM of 5 experiments.

FIG. 6. Comparison of the effect of efaroxan and RX871024 on insulin secretion by SUR1

KO islets. (A) Freshly isolated SUR1 KO islets were perifused with Krebs-Ringer medium containing 5 mM glucose for 90 min. From 60 min to 120 min the medium contained 100 µM

(open circles) or 500 µM (closed circles) of efaroxan. From 90 min to 130 min the glucose concentration was increased to 10 mM.. Values are means ± SEM of 4 experiments. (B)

Freshly isolated SUR1 KO islets were perifused with Krebs-Ringer medium containing 5 mM glucose for 90 min. From 60 min to 120 min the medium contained 100 µM (closed squares),

10 µM (open squares), 0.1 µM (closed circles), or no RX871024 (control = open circles),

From 90 min to 130 min the glucose concentration was increased to 10 mM. The effect size

(pg / min x islet) of glucose stimulation alone can be estimated from Fig. 7C. Values are means ± SEM of 4 experiments. JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #152751 30

FIG. 7. Lack of insulinotropic effect of imidazolines on islets from SUR1 KO mice. (A)

Freshly isolated SUR1 KO islets (closed circles) and NMRI islets (open circles) were perifused with Krebs-Ringer medium containing 5 mM glucose for 90 min. From 60 min to

120 min the medium contained 100 µM phentolamine. From 90 min to 130 min the glucose concentration was increased to 10 mM. Values are means ± SEM of 4 experiments. (B)

Freshly isolated SUR1 KO islets (closed circles) and NMRI islets (open circles) were perifused with Krebs-Ringer medium containing 5 mM glucose for 90 min. From 60 min to

120 min the medium contained 100 µM alinidine. From 90 min to 130 min the glucose Downloaded from concentration was increased to 10 mM.. Values are means ± SEM of 4 experiments. (C)

Freshly isolated SUR1 KO islets were perifused with Krebs-Ringer medium containing 5 mM jpet.aspetjournals.org glucose for 90 min. From 60 min to 120 min the medium contained 100 µM quinine (closed circles) or no further addition (control = open circles, same as in Fig. 6B). From 90 min to

130 min the glucose concentration was increased to 10 mM. In the presence of quinine the at ASPET Journals on September 25, 2021 secretion (area under the curve from 60 to 120 min) was significantly increased. Values are means ± SEM of 4 to 5 experiments.

FIG. 8. Effect of a long-term exposure to efaroxan on the ultrastructure of pancreatic -cells in SUR1 KO islets. Isolated pancreatic islets were cultured for 20 h in RPMI 1640 with 5 mM glucose in the presence (A) or absence (B) of 100 M efaroxan. The cells in control- cultured SUR1 KO islets had a content of insulin granules which was comparable to that of normal NMRI mouse -cells. There was no degranulation by the exposure to efaroxan.

Magnification 4000 x. JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on September 10, 2009 as DOI: 10.1124/jpet.109.152751 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021