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J. Biol. Chem., Vol. 275, Issue 28, 21033-21040, July 14, 2000

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Rapid Ca²⁺ Influx and Diacylglycerol Synthesis in Growth Hormone-mediated Islet -Cell Mitogenesis^*

Åke Sjöholm^§, Qimin Zhang, Nils Welsh^¶, Anders Hansson, Olof Larsson, Michael Tally, and Per-Olof Berggren

From the  Department of Molecular Medicine, Endocrine and Diabetes Unit, Rolf Luft Center for Diabetes Research, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden and the ^¶ Department of Medical Cell Biology, University of Uppsala, S-751 23 Uppsala, Sweden

Received for publication, February 11, 2000, and in revised form, March 21, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth hormone (GH) is an important mitogenic stimulus for the insulin-producing -cell. We investigated the effects of GH on Ca²⁺ handling and diacylglycerol (DAG) and cAMP formation in the -cell. GH elicited a rapid increase in the cytoplasmic free [Ca²⁺], which required extracellular Ca²⁺ and was also blocked by pertussis toxin or protein kinase C (PKC) inhibition. GH also elevated islet DAG content, which should lead to PKC activation. Pertussis toxin and PKC inhibitors obliterated the mitogenicity of GH, suggesting involvement of GTP-binding proteins. PKC activation stimulated -cell proliferation, and it also activated phospholipase D. Islet cAMP content was not elevated by GH. Addition of a specific protein kinase A antagonist failed to influence the mitogenicity of GH, whereas a stimulatory cAMP agonist stimulated -cell replication. We conclude that GH rapidly increases the -cell cytoplasmic free [Ca²⁺] and also evokes a similar increase in DAG content via a phosphatidylcholine-specific phospholipase C, but does not affect mitogen-activated protein kinases, phospholipase D, or the cAMP signaling pathway. This rise in DAG may be of importance in translation of the stimulatory signal of GH into a proliferative response by the -cell, which seems to occur through GTP-binding proteins and PKC-dependent mechanisms.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Long-term alterations in pancreatic islet -cell mass constitute an important means to accommodate an increased demand for insulin. Since previous studies have established a defective insulin secretory response to glucose as well as a decreased -cell volume in diabetic patients (1), further elucidation of factors governing insulin production and -cell proliferation is clearly warranted. In contrast to other tissues (e.g. the liver) that readily regenerate, the adult insulin-producing pancreatic -cell is characterized by a limited proliferative potential (1). Additionally, its capacity to divide diminishes by increasing age, when glucose intolerance becomes more prevalent (1). Conversely, expansion of the pancreatic -cell mass by recruitment of -cells to proliferate may constitute a means by which the organism can compensate for the loss or dysfunction of -cells occurring in diabetes. Thus, if -cells could be induced to replicate at a higher rate, this may prove beneficial in maintaining normoglycemia. Importantly, when the adult -cell population is expanded in vivo by "cellophane wrapping" of the pancreas, this not only induces islet hyperplasia resembling nesidioblastosis, but also ameliorates experimental diabetes in hamsters (2).

Despite the potential importance of an insufficient extent of -cell replication in diabetes, not much is known about the intracellular mechanisms that normally govern this event, although some extracellular factors stimulating -cell DNA synthesis in vitro have been identified (3, 4). One of the most potent mitogenic factors is growth hormone (GH),¹ but not much is known about the molecular events that convey the stimulatory signal of GH into a mitogenic response by the -cell (5). In other cell systems, increases in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i), polyphosphoinositide and phosphatidylcholine hydrolysis, and protein kinase activation are among the earliest changes taking place after mitogenic stimulation (reviewed in Refs. 6-9), events occurring also in the -cell subsequent to stimulation (4). Hence, in this study, we have utilized an in vitro system of fetal rat islets enriched in rapidly proliferating -cells to study the putative role of [Ca²⁺]_i, GTP-binding proteins, polyphosphoinositide and phosphatidylcholine hydrolysis, and protein kinase activation in the proliferative signal induced by GH. In addition, we have employed various pharmacological probes to pin down the locus in the signal transduction pathway where GH acts and have furthermore attempted to elucidate the functional significance of this event.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Recombinant human GH was kindly provided by Dr. Anna Skottner (Pharmacia & Upjohn Corp., Stockholm, Sweden). The S_p- and R_p-diastereomers of cAMP-S ((S_p)-cAMP-S and (R_p)-cAMP-S, respectively) were provided by Biolog Life Science Institute (Bremen, Germany). They were dissolved in RPMI 1640 medium and stored at 4 °C as 1000-fold concentrated stock solutions. [methyl-³H]Thymidine (5 Ci/mmol), [-³²P]ATP (100-500 cpm/pmol), [9,10-³H]myristate, and the inositol 1,4,5-trisphosphate and cAMP assay kits were purchased from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom). Unisolve was from NEN Life Science Products, and Soluene was provided by Packard Instrument Co. Collagenase type CLS (EC 3.4.24.3) was obtained from Roche Molecular Biochemicals (Mannheim, Germany). RPMI 1640 medium, fetal calf serum, L-glutamine, benzylpenicillin, and streptomycin were from Flow Laboratories (Irvine, United Kingdom). Fura-2/AM, ATP (disodium salt), TPA, 4-phorbol dibutyrate, D-1,2-dipalmitin, and PTX were from Sigma. Forskolin, DAG kinase, calmidazolium, protein kinase A inhibitor protein , calphostin C, and H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) were obtained from Calbiochem. Rabbit anti-phosphotyrosine IgG was from Zymed Laboratories Inc. D-600 was a generous gift from Knoll AG (Ludwigshafen-am-Rhein, Germany), and Kieselgel 60 plates and all other chemicals of analytical grade were obtained from E. Merck AG (Darmstadt, Germany).

Fetal Islet Preparation and Culture-- Pregnant Harlan Sprague-Dawley rats belonging to a local stock or Wistar rats purchased from B&K Universal (Sollentuna, Sweden) were killed by cervical dislocation on day 21 of gestation, and the fetuses were rapidly removed. Fetal rat islets were prepared from pancreatic glands as described previously (11, 12). Briefly, the pancreata were finely chopped and digested for a short time with collagenase. The carefully washed digest was plated in culture dishes allowing cell attachment (Nunc, Roskilde, Denmark) and cultured for 5 days at 37 °C in a humidified atmosphere of 5% CO₂ in ambient air in RPMI 1640 medium containing 11.1 mM glucose, 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml benzylpenicillin, and 0.1 mg/ml streptomycin. At the end of the culture period, groups of islets were transferred to fresh medium containing 1% fetal calf serum and cultured free-floating overnight, a procedure that minimizes fibroblast proliferation. Spherical islets, free of connective tissue, were then selected under a stereomicroscope and used for the different analyses described below. In each experiment, all test groups received the same amount of solvent.

Islet DNA Synthesis and DNA Content-- Islets in groups of 50 were cultured as described above. During the last 5 h, 1 µCi/ml [methyl-³H]thymidine was present in culture medium. At the end of the labeling period, the islets were rinsed once in PBS, sonicated in 200 µl of redistilled water, and precipitated in 1 ml of ice-cold 10% trichloroacetic acid. The precipitate was washed twice in trichloroacetic acid and dissolved in 50 µl of Soluene. The radioactivity incorporated was determined by scintillation counting after addition of 1 ml of Unisolve. It has been shown previously that fetal rat islets, serum-starved overnight, are able to respond fully mitogenically to GH (12). Importantly, islets prepared by this method appear to be essentially devoid of fibroblast contamination, as visualized by electron microscopy (11); contain >90% -cells; and do not respond to classical fibroblast mitogens such as epidermal growth factor, transforming growth factor , or platelet-derived growth factor with increased DNA synthesis (12). Duplicate samples of the homogenate were analyzed fluorometrically for DNA (13, 14).

[Ca²⁺]_i Measurements-- Islets were cultured as described above. Monolayers of cells were prepared as described (15) by shaking in a Ca²⁺-free medium containing EGTA and cultured overnight on plastic coverslips. They were then loaded for 30 min with 1 µM fura-2/AM in culture medium at 37 °C. After rinsing, the coverslip was placed at the bottom of an open perifusion chamber (150-µl volume) connected to a two-channel peristaltic pump, allowing constant superfusion of cells. The chamber was mounted on a thermostatically controlled stage of an inverted microscope (Zeiss Axiovert 35M) connected to a Spex Fluorolog-2 CM1T11Y system. Fluorescence measurements were performed at 37 °C using excitation and emission wavelengths of 340/380 and 510 nm, respectively. Cells were superfused at a flow rate of 500 µl/min with buffer (pH 7.4) containing 125 mM NaCl, 5.9 mM KCl, 1.3 mM CaCl₂, 1.2 mM MgCl₂, 3 mM D-glucose, 25 mM HEPES, and 1 mg/ml bovine serum albumin. Additions of GH and other substances were made in the same buffer. Clusters of two to four -cells or a single islet was localized with the microscope and selected for [Ca²⁺]_i measurements. Occasional fibroblast-like elements were identified, but were avoided. All traces shown are typical for experiments repeated with at least three different cell preparations, and [Ca²⁺]_i is expressed as the ratio of the fluorescence measured at 340 and 380 nm.

Lipid Extraction and Quantification of DAG-- Groups of 250-300 islets were cultured free-floating overnight in RPMI 1640 medium supplemented with 1% fetal calf serum. Islets were then swiftly transferred to Eppendorf tubes containing 1 ml of conditioned medium (prewarmed to 37 °C) supplemented with 1 µg/ml GH or 200 µM ATP. In experiments designed to elucidate the dependence of extracellular Ca²⁺ for the ability of GH to raise DAG, islets were washed three times and then incubated for 5 min at 37 °C in buffer (pH 7.4) containing 125 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 3 mM D-glucose, 25 mM HEPES, and 1 mg/ml bovine serum albumin with or without 1.3 mM CaCl₂. Islets were incubated for the indicated time periods, rapidly pelleted, and quickly rinsed once in ice-cold PBS. Tubes were then immediately plunged into liquid nitrogen (196 °C) and kept frozen at 80 °C, pending further analysis of their DAG content. The islets were sonicated in a 500-µl chloroform solution consisting of chloroform/methanol/HCl (100:100:1, v/v/v) and 100 µl of PBS containing 10 mM EDTA. After centrifugation (5 min, 12,000 × g), the aqueous phase was removed and re-extracted with 100 µl of chloroform, which was added to the chloroform phase. The combined chloroform phases were evaporated under a stream of liquid nitrogen and resolubilized in 50 µl of the chloroform solution. This solution was re-extracted with 10 µl of PBS containing 10 mM EDTA and then re-evaporated. Samples were then stored at 70 °C under nitrogen until analyzed for DAG. 1,2-Diacylglycerols were quantified essentially according to Preiss et al. (16). Briefly, dried lipids were solubilized in 20 µl of an octyl -D-glucoside/cardiolipin solution (7.5% octyl -D-glucoside and 5 mM cardiolipin in 1 mM diethylenetriaminepentaacetic acid) by sonication in a bath sonicator. The reaction was then carried out in 100 µl containing 20 µl of sample solution, 50 mM imidazole HCl (pH 6.6), 50 mM NaCl, 12.5 mM MgCl₂, 1 mM EGTA, 2 mM dithiothreitol, 6.6 µg of DAG kinase, and 1 mM [-³²P]ATP for 30 min at room temperature. Lipids were extracted and evaporated as described above. Samples were then run on Kieselgel 60 plates activated by preheating at 120 °C. Plates were developed with chloroform/methanol/acetic acid (65:15:5, v/v/v) and subjected to autoradiography. Standard samples of D-1,2-dipalmitin were run in parallel. The intensities of the spots corresponding to phosphatidic acid were quantified by densitometry and are expressed as arbitrary units (optical density).

Phospholipase D Assay-- Fifty islets were metabolically labeled with [³H]myristate (0.8 µCi/ml) in 4 ml of serum-free RPMI 1640 medium for 24 h. Islets were pretreated with 0.5% ethanol for 5 min and next stimulated with 1 µg/ml GH or 300 nM 4-phorbol dibutyrate for 5 min. The medium was removed by centrifugation, and the islets were suspended in cold MeOH and 0.9% NaCl. Total lipids were extracted after addition of 1 ml of CHCl₃, followed by vigorous vortexing, and the organic phase was dried under a stream of N₂. The residue was applied to a silica gel TLC plate that was developed in the upper phase of H₂O/HAc/isooctane/ethyl acetate (100:20:50:110, v/v/v/v) (17). The starting zone (containing polar lipids) and the zones co-chromatographing with authentic standards of phosphatidic acid and phosphatidylethanol (R_F = 0.1 and 0.2, respectively) were scraped into scintillation vials containing 0.5 ml of MeOH. Radioactivity was determined by liquid scintillation counting. The activities in phosphatidic acid and phosphatidylethanol were normalized to the activity in the starting zone (typically ~30,000 cpm) to account for slight variations in labeling between different experiments. Duplicate sets of islets were analyzed in each experiment.

MAPK Assay-- Batches of 50-200 islets were cultured free-floating overnight in serum-free RPMI 1640 medium and exposed to GH for the indicated time periods. The islets were then rinsed in 5 ml of ice-cold PBS and 0.5 ml of ice-cold extraction buffer (18) containing 50 mM -glycerophosphate (pH 7.3), 1.5 mM EGTA, 0.1 mM Na₃VO₄, 1 mM dithiothreitol, 1 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 2 µg/ml pepstatin A. Islets were homogenized by ultrasonic disruption for 10 s, and a cytosolic fraction was prepared by centrifugation at 10,000 × g for 20 min at 4 °C. The kinase incubation medium contained 0.5 mg/ml myelin basic protein (a substrate for MAPKs (19)), 50 mM -glycerophosphate (pH 7.3), 1.5 mM EGTA, 0.1 mM Na₃VO₄, 1 mM dithiothreitol, 10 mM MgCl₂, 0.1 mM [-³²P]ATP (0.1 µCi/nmol), 2 µM recombinant rabbit PKA inhibitor protein , and 10 µM calmidazolium. Aliquots (37.5 µl) of cytosolic extracts or the chromatographic eluate were used in a final volume of 75 µl. The incubation was allowed to proceed for 20 min at 30 °C, after which the reaction mixture was spotted onto 2-cm² phosphocellulose paper, and the reaction was terminated by immersing the paper in 1% H₃PO₄. After removing unreacted [³²P]ATP by five changes of H₃PO₄, the radioactivity associated with the paper was measured in a scintillation counter as Cerenkov radiation. The radioactivity in extraction buffer in control experiments was subtracted, and the cytosolic extract from each dish was used for a separate point. Kinase assays were performed in triplicate. Protein in cytosolic extracts was measured by the Coomassie dye method according to the manufacturer's instructions (Bio-Rad).

cAMP Measurements-- For cAMP measurements, islets in groups of 50 that had been cultured for 3 days in the presence or absence of GH or the desired test substances were quickly rinsed once in ice-cold PBS and then swiftly transferred to Eppendorf tubes containing 150 µl of ice-cold 6% trichloroacetic acid. These tubes were immediately sealed, plunged into liquid nitrogen, and stored at 80 °C, pending analysis. The samples were thawed by sonication on ice and centrifuged at 2000 × g for 15 min at 4 °C. The pellet was re-sonicated in 200 µl of redistilled water and used for measurements of DNA and [³H]thymidine incorporation as detailed above. The supernatant was washed four times in 5 volumes of water-saturated diethyl ether. The aqueous extract was freeze-dried, and the content of cAMP was measured by radioimmunoassay (using ¹²⁵I-cAMP) exactly as described by the manufacturer of the assay kit. To increase the sensitivity of the method, samples were acetylated.

Statistical Analysis-- Results presented were derived from at least three independent experiments performed on different days. Means ± S.E. were calculated, and groups of data were compared using Student's paired or unpaired t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As shown in Fig. 1, stimulation of fetal rat pancreatic islets with 1 µg/ml GH for 3 days evoked a brisk stimulation of -cell mitogenesis as monitored by [³H]thymidine incorporation into DNA. Given the long cell cycle of the -cell (20) and the fact that unsynchronized cells were studied, it was considered necessary to expose the islets for 1-3 days to the different test substances. This procedure allows DNA synthesis initiated prior to addition of GH or other test substances to be terminated before [³H]thymidine addition.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   GH, PKA, and PKC promote pancreatic -cell mitogenesis. Groups of 50 fetal rat pancreatic islets were cultured free-floating for 3 days in RPMI 1640 medium with 1% fetal calf serum in the presence or absence of the indicated compounds. Concentrations used were as follows: GH, 1 µg/ml; (Sp)-cAMP-S, 50 µM; (Rp)-cAMP-S, 50 µM; H-7, 10 µM; PTX, 50 ng/ml; forskolin, 10 µM; and TPA, 10 nM. Following a 5-h [3H]thymidine labeling period, DNA synthesis rates were measured by quantitation of the radioactivity in the trichloroacetic acid-precipitable fraction of islet homogenates. Values are expressed as mean percent of controls ± S.E. for five to six observations. Single and double asterisks denote p < 0.05 and p < 0.01, respectively, for chance differences versus control islets using Student's paired t test.

Given the mitogenicity of GH, we then set out to clarify by which intracellular signaling systems the hormone acts. Since changes in Ca²⁺ fluxes occur early in growth promotion (6), we first looked into whether GH affected this pathway. As is evident from Fig. 2, stimulation of -cells with 1 µg/ml GH elicited an increase in [Ca²⁺]_i. The [Ca²⁺]_i increase was rapid and occurred at both a substimulatory (3.3 mM; Fig. 2A) and a maximally stimulatory (16.7 mM; Fig. 2B) glucose concentration. The rise in [Ca²⁺]_i evoked by GH was found to be due to influx of Ca²⁺ across the plasma membrane since it did not occur in the absence of extracellular Ca²⁺ (Fig. 2C). The mitogenic response to GH does not seem to require the Ca²⁺ response, however, because it remained intact in the presence of D-600 (50 µM), a blocker of voltage-dependent Ca²⁺ channels (data not shown). Both PKC inhibitors H-7 and calphostin C (1 µM; data not shown) blocked the GH-induced Ca²⁺ elevation by some 50-60% (Fig. 2, D and E).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   A-F, GH stimulates Ca2+ influx in pancreatic -cells. Islet cell monolayers on glass coverslips were loaded for 30 min with 1 µM fura-2/AM in culture medium at 37 °C. Clusters of two to four -cells were identified in the microscope and selected for Ca2+ measurements. Fluorescence measurements were performed at 37 °C in an inverted fluorescence microscope using excitation and emission wavelengths of 340/380 and 510 nm, respectively. Cells were superfused at a flow rate of 0.5 ml/min. All traces shown are typical for experiments repeated with at least three different cell preparations, and [Ca2+]i is expressed as the ratio of the fluorescence measured at 340 and 380 nm. Unless otherwise indicated, the ambient glucose concentration was 3.3 mM. GH (1 µg/ml), glucose (Glu; 16.7 mM), ATP (200 µM), calphostin C (Calph; 1 µM), H-7 (1 µM), and KCl (25 mM) were present as indicated by the horizontal bars. F, cells pretreated with PTX.

Pretreatment of islets for 24 h with PTX (50 ng/ml), known to influence signal transduction through heterotrimeric GTP-binding proteins connected to PLC and adenylyl cyclase, curtailed the mitogenicity of GH (Fig. 1), indicating that the mitogenic action of GH involves its interaction with such GTP-binding proteins. Additionally, PTX blocked the GH-induced Ca²⁺ elevation by ~40% (Fig. 2F).

GH also significantly elevated islet DAG content ~2.3-fold from 5 min of exposure, an increase that remained for at least up to 30 min (Fig. 3) and that should activate PKC. After 72 h of exposure to GH, the elevated DAG content could no longer be detected (data not shown). For comparison, after 5 min, islet DAG content was elevated ~3.7-fold by 200 µM ATP, which also stimulated -cell mitogenesis by 212 ± 18%. It should be noted that the observed DAG levels (~300 µM) are well in agreement with those reported previously, assuming an islet volume of 2 nl (21). One possible source of DAG, besides breakdown of phosphatidylinositol 4,5-bisphosphate or phosphatidylcholine via specific PLC enzymes, is through the action of PLD and phosphatidate phosphatase. However, when the activity of this latter pathway was measured using the transphosphatidylation technique (17), no increase in the formation of phosphatidylethanol was observed in islets treated with GH (1 µg/ml) for 5 min (Fig. 4). By contrast, addition of phorbol ester (300 nM 4-phorbol dibutyrate) elicited a 2-fold stimulatory effect of PLD under these conditions (Fig. 4), thus confirming previous results (22) and indicating a functional PLD system in the -cell. Because GH was unable to raise [Ca²⁺]_i in the absence of extracellular Ca²⁺ (Fig. 2C), it is unlikely that GH would signal through a phosphatidylinositol 4,5-bisphosphate-specific PLC, which catalyzes formation of inositol 1,4,5-trisphosphate, releasing Ca²⁺ from intracellular stores (8). We also measured the generation of inositol 1,4,5-trisphosphate and found no effect of GH at 20 min (data not shown), when GH-induced DAG accumulation was maximally enhanced. This indicates that DAG was generated through a phosphatidylcholine-specific PLC being activated by GH.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   GH rapidly increases islet DAG content. Groups of 250-300 islets were cultured free-floating overnight in RPMI 1640 medium supplemented with 1% fetal calf serum. Islets were then swiftly transferred to Eppendorf tubes containing 1 ml of medium (with or without Ca2+) containing 1 µg/ml GH or 200 µM ATP. Islets were incubated for the indicated time periods, rapidly pelleted, and quickly washed once in ice-cold PBS. Tubes were then immediately plunged into liquid nitrogen (196 °C) and kept frozen at 80 °C, pending further analysis of their DAG content as under "Experimental Procedures." Bars indicate means ± S.E. for four to six experiments. Single and double asterisks denote p < 0.05 and p < 0.01, respectively, for chance differences versus controls using Student's unpaired t test.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Phorbol ester, but not GH, activates -cell phospholipase D. Groups of 50 fetal rat pancreatic islets were prelabeled with [3H]myristate and pretreated with 0.5% ethanol prior to stimulation with GH (1 µg/ml) or 4-phorbol dibutyrate (PDBu; 300 nM) for 5 min. Phosphatidylethanol and phosphatidic acid were isolated, and their associated radioactivity was measured as described under "Experimental Procedures." Bars represent means ± S.E. for four separate experiments. The double asterisks denote p < 0.01 for a chance difference versus control islets using Student's paired t test.

Specific activation of PKC with the phorbol ester TPA (10 nM) for 3 days stimulated -cell proliferation to the same extent as GH (Fig. 1). Conversely, the PKC inhibitor H-7 (10 µM) blocked the mitogenicity of TPA and GH (Fig. 1), suggesting that GH promotes -cell mitogenesis through PKC activation. Both PKC inhibitors H-7 (Fig. 1) and calphostin C (data not shown) blocked the mitogenicity of GH, glucose, and TPA, but not that of cAMP. It should be noted that not only GH- and TPA-stimulated DNA syntheses were markedly decreased by H-7, but also base-line DNA synthesis to ~14% of control. Nonetheless, the finding that forskolin could still stimulate DNA synthesis by the same magnitude in H-7-treated islets as in controls (data not shown) argues against a nonspecific general cytotoxicity of H-7, but does not address the question of H-7 specificity.

A quantitatively similar mitogenic effect as that of GH and PKC stimulation was obtained with a 10 µM concentration of the adenylyl cyclase activator forskolin or with a 50 µM concentration of the stimulatory cAMP agonist (S_p)-cAMP-S, which directly activates PKA types I and II (Fig. 1), confirming that PKA activation promotes -cell mitogenesis. The islet cAMP content was, however, not changed after 3 days of exposure to 1 µg/ml GH (Fig. 5). In contrast, the adenylyl cyclase activator forskolin (10 µM) evoked a 4-5-fold increase in cAMP content, and raising the glucose concentration from 3.3 to 16.7 mM elicited a 94% increase in cAMP concentration as assessed by radioimmunoassay (Fig. 5). Addition of the specific PKA antagonist (R_p)-cAMP-S (50 µM) prior to stimulating islets with GH failed to influence the mitogenicity of the hormone (Fig. 1), suggesting that GH does not stimulate -cell proliferation through the cAMP pathway. (R_p)-cAMP-S blocked the mitogenic effects of forskolin and (S_p)-cAMP-S (data not shown), but not that of GH (Fig. 1).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   GH does not affect islet cAMP content. Groups of 50 fetal rat pancreatic islets were cultured free-floating for 3 days in RPMI 1640 medium with 1% fetal calf serum in the presence or absence of the indicated compounds. Concentrations used were as follows: glucose, 16.7 mM; forskolin, 10 µM; and GH, 1 µg/ml. The cAMP content of islets cultured in 16.7 mM glucose was compared with that of islets maintained in 3.3 mM glucose, and 11.1 mM glucose was used in experiments with GH and forskolin. Islet cAMP was measured by radioimmunoassay as described under "Experimental Procedures." Values are expressed as mean percent of controls ± S.E. for five to six observations. Single and double asterisks denote p < 0.05 and p < 0.01, respectively, for chance differences versus control islets using Student's paired t test.

When cytosolic MAPK activity was measured in four different preparations of islets, no discernible effects of GH (1 µg/ml) were observed during 1-60 min of incubation, nor was tyrosine phosphorylation affected in electrophoretic bands corresponding to the mobility of p42 and p44 MAPK (Ref. 23 and data not shown). This finding indicates that, in contrast to other tissues, the mitogenicity of GH in the -cell does not involve activation of these MAPKs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The factors that control the process of pancreatic -cell replication remain largely unknown, although it has been shown that glucose, amino acids, GH, and polypeptide growth factors can stimulate -cell replication in vitro (reviewed in Refs. 3-5). One of the most potent mitogenic stimuli for the -cell is GH, a physiologically important hormone whose levels in blood rise during pregnancy and lactation, states that are associated with an expansion of the -cell mass. In addition, in patients with acromegaly and in rats bearing GH-producing tumors, -cell growth is promoted (1). Compared with other growth factors, surprisingly little is known about the intracellular events that convey the mitogenic signal of GH into a proliferative response in the -cell. Nonetheless, it has been shown that insulin-secreting cells possess a 115-kDa GH receptor resembling the long form of the cloned hepatic GH receptor, which consists of a 638-650-amino acid protein with a hydrophobic transmembrane-spanning domain (24). In rodent islets, human GH seemingly works mainly through the prolactin receptor (25). According to the contemporary conceptual framework, binding of GH to its receptor induces receptor dimerization and phosphorylation of tyrosyl residues in the receptor, although the receptor itself does not exhibit any canonical sequence homology to known tyrosine kinases (26-30). This mechanism is also shared by the newly described cytokine receptor superfamily (31-33). One such non-receptor tyrosine kinase is JAK2 that was identified in pancreatic islets along with STAT5, which seems to be translocated into the nucleus upon mitogenic stimulation with prolactin (34). Interestingly, both GH and its receptor were shown to undergo ligand-activated translocation into the nucleus through an endosomal route (35).

One of the earliest events taking place following mitogenic cell activation is an increase in [Ca²⁺]_i (6, 36). This can be achieved by two major routes: either by influx of Ca²⁺ through voltage-dependent Ca²⁺ channels located in the plasma membrane or by mobilization of Ca²⁺ from intracellular stores. In this study and in RINm5F insulinoma cells,² GH evoked a rapid increase in [Ca²⁺]_i, the shape and size of which resembled that obtained in response to the cardinal -cell mitogen glucose. This increase vanished in the absence of extracellular Ca²⁺, implying that GH promotes influx of Ca²⁺. Similar effects of GH were also reported in the clonal insulinoma INS-1 cell line, in which the mitogenicity of GH was not antagonized by Ca²⁺ channel blockers (37). Possibly this occurs through direct interaction between the GH receptor and the plasma membrane Ca²⁺ channel. Such a scenario has been proposed for insulin-like growth factor I and platelet-derived growth factor in other tissues (36, 38-41). Additionally, it was reported that C-terminal domains of the GH receptor are required for GH-induced [Ca²⁺]_i oscillations and gene transcription and that these events are blocked by the Ca²⁺ channel antagonist verapamil (42), suggesting the involvement of L-type voltage-dependent Ca²⁺ channels (43). Furthermore, GH reportedly also increases [Ca²⁺]_i in adipocytes (44), possibly by regulating the number of functional Ca²⁺ channels in the plasma membrane (45).

One way by which an extracellular stimulus can be translated into a functional response is through interference with heterotrimeric GTP-binding proteins that connect occupancy of a cell-surface receptor to regulatory effector systems, mechanisms that have been described for both adenylyl cyclase and PLC (46, 47). Certain of these GTP-binding proteins are sensitive to the toxin of Bordetella pertussis, which inactivates them by means of ADP-ribosylation (46-48). Thus, PTX pretreatment is a useful way to study whether a given stimulus conveys its actions through such GTP-binding proteins. The present data indicate that in PTX-exposed islets, the mitogenic action of GH is abrogated, suggesting that GH induces its biological effects at least in part via interaction with GTP-binding protein(s). The identity of these proteins will have to await future characterization. The Ca²⁺ response to GH of IM-9 lymphocytes is insensitive to PTX (49). In contrast, in our -cells, both PTX (Fig. 2F) and PKC inhibitors (Fig. 2, D and E) partially blocked the GH-induced Ca²⁺ elevation.

Upon hydrolysis of plasma membrane phospholipids, there is a rapid synthesis of cellular DAG (9). Because GH was unable to raise [Ca²⁺]_i in the absence of extracellular Ca²⁺, it is unlikely that GH would signal through a phosphatidylinositol 4,5-bisphosphate-specific PLC, in contrast to what has been reported in kidney proximal tubule (50). Since PLD was not activated by GH (but by phorbol ester) and GH failed to promote inositol 1,4,5-trisphosphate accumulation, this indicates that DAG was generated through a phosphatidylcholine-specific PLC being activated by GH in the -cell, as has been reported in other tissues (51). Interestingly, in OB1771 preadipocytes and hepatocytes, GH elicits a rapid accumulation of DAG without a corresponding synthesis of inositol polyphosphates, implying involvement of phospholipid species other than phosphatidylinositol 4,5-bisphosphate (52, 53) and thus giving support to our present results. Hence, this might be one important pathway activated by GH that participates in the mitogenic action of the hormone. The finding that extracellularly applied ATP stimulated not only DAG formation but also -cell mitogenesis adds further credence to this concept, although these two observations may be merely correlative. Likewise, although not activated by GH, PLD activation may be an important event in transducing the mitogenic and secretory signals of phorbol ester recorded in this system.

Another key regulator in signal transduction is the Ca²⁺- and phospholipid-dependent serine/threonine kinase PKC, which has emerged as a pleiotropic regulator of various cellular functions, including cell proliferation and hormone secretion (reviewed in Ref. 54). This enzyme has been identified as the cellular receptor for DAG and is activated by tumor-promoting phorbol esters such as TPA (54). The significance of PKC in regulation of cell proliferation is amply illustrated by the findings of potent mitogenic (and in some cases, tumorigenic) actions of TPA and inhibitors of DAG degradation and the large increases in PKC activity occurring in response to natural mitogens (6, 55). Further evidence for a crucial role of PKC in normal and neoplastic proliferation is derived from findings of elevated DAG levels or PKC activity in c-Ha-ras-transformed cells (56, 57) and that a mutant PKC obtained from fibrosarcoma cells is able to induce neoplastic transformation of normal fibroblasts (58). Although prolonged exposure to high concentrations of TPA eventually will lead to PKC down-regulation, such changes in enzyme activity are difficult to conclusively relate temporally to changes in -cell replication, which occur over 10-24 h. Nonetheless, PKC overexpression confers an enhanced growth rate (and in some cases, neoplastic transformation) in other tissues (55), and known -cell mitogens transiently activate PKC (55). Additionally, pharmacological inhibition of PKC with H-7 or calphostin C results in cessation of -cell proliferation (the study and Refs. 59 and 60). These combined findings make it likely that the presently observed mitogenicity of TPA indeed reflects PKC activation. GH stimulates PKC activity in hepatocytes (61), and the insulin-like effects of GH on adipocytes can be blocked by certain inhibitors of PKC, e.g. sphingosine (62) and acridine orange (63), suggesting the involvement of PKC in the action of GH. Moreover, additional support in favor of a crucial role for PKC in this context is provided by our finding that GH- and TPA-induced proliferation of -cells can be blocked by H-7. Although H-7 will inhibit PKC and PKA with similar potency, the finding that the specific PKA antagonist (R_p)-cAMP-S was inactive makes it likely that the effects evoked by H-7 reflect inhibition of PKC.

The role of cAMP in regulation of -cell proliferation has become a cornucopia of controversial literature, and based on the use of different cAMP analogs, stimulatory (64) as well as inhibitory (65) effects have been reported. However, recent data based on the use of highly selective cAMP analogs favor a stimulatory role of this second messenger in -cell proliferation (3, 4, 66). An elevated cAMP content was presently noted in response to the adenylyl cyclase activator forskolin or a high glucose concentration (both of which also enhance -cell mitogenesis), but not to GH, confirming previous studies in insulinoma cells (37). However, since cAMP was measured only at one time point, we wanted to exclude the remote possibility that an early transient cAMP surge, which went undetected, would be involved in the mitogenicity of GH. To this end, the specific PKA antagonist (R_p)-cAMP-S (67) was added prior to GH. This maneuver failed to influence the rates of basal and glucose- or GH-stimulated DNA syntheses. From these data, we conclude that, although cAMP increases in -cells upon mitogenic stimulation with glucose, this increase does not seem mandatory for the mitogenic response to the sugar and is not involved in conveying the effects of GH in this cell type, in contrast to previously studied insulinoma cells (37). Nonetheless, direct stimulation of PKA by forskolin or (S_p)-cAMP-S appears sufficient to trigger a mitogenic response by the -cell.

In other cell systems (23), stimulation of MAPKs, a family of downstream signaling threonine/tyrosine kinases, occurs early in mitogenic stimulation and is considered to be an important event in translating a mitogenic signal into a proliferative response. A signaling cascade has been described in which MAPKs regulate S6 kinase activity and increase c-myc, c-fos, and c-jun proto-oncogene expression (23). However, this appears not to be the case in the -cell since no effects were observed on MAPK activity over a 60-min incubation period. This finding concurs with a recent report (68) indicating that -cell MAPK activity (the 42-kDa ERK2) is not altered by glucose or phorbol ester, two known -cell mitogens. Additionally, in the insulin-secreting cell line INS-1, GH and prolactin, probably acting through the same lactogenic receptors as human GH, promoted cell proliferation without affecting p44 MAPK activity over a 30-min incubation period (69). Thus, our data rule out MAPK and implicate TPA-sensitive PKC as one GH-activated downstream signaling serine/threonine kinase.

It is concluded that GH causes a rapid increase in -cell [Ca²⁺]_i and also evokes a similar increase in the DAG content via a phosphatidylcholine-specific PLC, but does not affect MAPKs, PLD, or the cAMP signaling pathway. This rise in DAG may be of importance in translation of the stimulatory signal of GH into a proliferative response by the -cell, which seems to occur through GTP-binding proteins and PKC-dependent mechanisms.

	ACKNOWLEDGEMENTS

We thank Pharmacia & Upjohn Corp. for the donation of GH. We thank Dr. Charlotte Öberg-Welsh for kindly providing fetal islets and Professor Kerstin Hall and Drs. Per Arkhammar and Christer Möller for valuable and constructive discussions. We thank Elvi Sandberg for excellent technical assistance.

	FOOTNOTES

^* This work was supported by Karolinska Institutet; Swedish Medical Research Council Grants 03X-12550, 12X-11564, 72P-12995, 19X-00034, 03X-09890, 03XS-12708, 03X-09891, and 12P-10151; the Swedish Diabetes Association; the Swedish Society of Medicine; the Nordic Insulin Foundation Committee; the Barndiabetesfonden; the Magnus Bergvall's Foundation; the Torsten and Ragnar Söderberg's Foundations; Novo-Nordisk Sweden Pharma AB; the Harald Jeansson's and Harald and Greta Jeansson's Foundations; the Tore Nilsson's Foundation for Medical Research; the Åke Wiberg's Foundation; the Syskonen Svensson's Fund; and the Fredrik and Inger Thuring's Foundation. Parts of this work have been published in abstract form (10).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.

^§ Recipient of an Eli Lilly IEASD (European Association for the Study of Diabetes) Research Fellowship Award in Diabetes and Metabolism. To whom correspondence and reprint requests should be addressed: Dept. of Molecular Medicine, Endocrine and Diabetes Unit, Rolf Luft Center for Diabetes Research, Karolinska Inst., Karolinska Hospital (L6:01B), S-171 76 Stockholm, Sweden. Tel.: 46851775782/46705234057; Fax: 46851773658/468303458; E-mail: ake@enk.ks.se.

Published, JBC Papers in Press, March 28, 2000, DOI 10.1074/jbc.M001212200

² Å. Sjöholm, unpublished data.

	ABBREVIATIONS

The abbreviations used are: GH, growth hormone; DAG, sn-1,2-diacylglycerol; [Ca²⁺]_i, cytoplasmic free Ca²⁺ concentration; cAMP-S, adenosine cyclic 3':5'-monophosphorothioate; TPA, 12-O-tetradecanoylphorbol-13-acetate; PTX, pertussis toxin; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; JAK, Janus kinase; STAT, signal transducer and activator of transcription; ERK, extracellular signal-regulated kinase.

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