Inositol hexakisphosphate and sulfonylureas regulate ...

BBRC Biochemical and Biophysical Research Communications 316 (2004) 893–897 www.elsevier.com/locate/ybbrc

Inositol hexakisphosphate and sulfonylureas regulate b-cell protein phosphatasesq
Sj€ Mikael Lehtihet,a Richard E. Honkanen,b and Ake oholma,* b

a Karolinska Institutet, Department of Internal Medicine, Stockholm South Hospital, SE-118 83 Stockholm, Sweden Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA

Received 20 February 2004

Abstract In human type 2 diabetes, loss of glucose-stimulated insulin exocytosis from the pancreatic b-cell is an early pathogenetic event. Mechanisms controlling insulin exocytosis are, however, not fully understood. We show here that inositol hexakisphosphate (InsP6 ), whose concentration transiently increases upon glucose stimulation, dose-dependently and differentially inhibits enzyme activities of ser/thr protein phosphatases in physiologically relevant concentrations. None of the hypoglycemic sulfonylureas tested affected protein phosphatase-1 or -2A activity at clinically relevant concentrations in these cells. Thus, an increase in cellular phosphorylation state, through inhibition of protein dephosphorylation by InsP6 , may be a novel regulatory mechanism linking glucosestimulated polyphosphoinositide formation to insulin exocytosis in insulin-secreting cells. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Polyphosphoinositides; Islet; Insulin secretion; Diabetes; Sulfonylurea; Protein phosphatase; Okadaic acid; Signal transduction; Exocytosis

Reversible phosphorylation of specific intracellular proteins is believed to be an important and versatile mechanism for regulating their biological activity, which, in turn, controls a variety of cellular functions [1]. For instance, significant changes in protein kinase activities and in protein phosphorylation patterns occur subsequent to stimulation of insulin release by glucose and other important physiological b-cell stimuli [2–4]. Therefore, the molecular mechanisms regulating phosphorylation of proteins involved in the insulin secretory process by the pancreatic b-cell have been extensively investigated. Hypoglycemic sulfonylureas have been widely used clinically in the treatment of patients with type 2 diabetes mellitus for the past 50 years, but there is still q Abbreviations: cAMP, cyclic adenosine-30 ,50 -monophosphate; DTT, dithiothreitol; GTP, guanosine trisphosphate; InsP6, D -myoinositol-1,2,3,4,5,6-hexakisphosphate; PKA, protein kinase A; PKC, protein kinase C; PPase, serine/threonine protein phosphatase; RINm5F, clonal rat insulinoma cells; TPA, 12-O-tetradecanoylphorbol 13-acetate. * Corresponding author. Fax: +46-8-616-31-46. E-mail address: [email protected] (. Sj€ oholm).

0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.144

much controversy about their mode of action [5]. It is currently believed that sulfonylureas, or ATP generated by glucose metabolism, may close Kþ channels in the b-cell resulting in influx of Ca2þ through voltage-activated Ca2þ channels, an event that sets in motion secretory granule translocation and the rapid exocytotic discharge of insulin [6]. Experiments have unveiled that glucose retains the ability to release insulin even in the presence of maximally depolarizing concentrations of Kþ and diazoxide, an opener of ATP-regulated Kþ channels (KATP ) [7,8]. Thus, signaling molecules other than ATP and Ca2þ may be involved in glucose sensing in the b-cell although the precise nature of such signals has remained elusive. Additionally, sulfonylureas promote insulin exocytosis from permeabilized cells [9], suggesting KATP -independent actions also of these drugs. Previous studies have established that (i) the b-cell contains divalent-cation independent serine/threonine protein phosphatase (PPase) activity, most of which is PPase-1 and PPase-2A [10,11]; (ii) stimulation of protein phosphorylation by direct activation of PKA and PKC with forskolin or phorbol ester results in a stimulated

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M. Lehtihet et al. / Biochemical and Biophysical Research Communications 316 (2004) 893–897

insulin secretion [3,12]; (iii) physiological stimuli of insulin secretion increase b-cell phosphorylation state [3]; (iv) insulin secretagogues, glucose metabolites, polyamines, GTP and ATP transiently and dose-dependently suppress b-cell PPase activities [13,14]; and (v) shortterm treatment of b-cells or permeabilized pancreatic islets with the specific PPase inhibitor okadaic acid promotes Ca2þ entry and insulin exocytosis [11,13,15,16]. These combined findings suggest an important functional role for rapidly reversible protein phosphorylation/dephosphorylation cycles in regulation of the stimulus-secretion coupling in the b-cell. Subsequent to stimulation of b-cell insulin exocytosis with glucose or agonists of muscarinic receptors (e.g., neurotransmitters), there is a rapid increase in polyphosphoinositide synthesis [2,3,12]. In this study, we have therefore investigated the effect of specific polyphosphoinositides and hypoglycemic sulfonylureas on divalent-cation independent serine/threonine PPase activities in insulinsecreting cells. Part of this study has been published in abstract form [17].

Materials and methods Materials. Okadaic acid was the kind gift of Dr. R. Dickey (US Food and Drug Administration). Glibenclamide and tolbutamide were kindly given by Aventis A.G. (Frankfurt, Germany), whereas Pfizer (New York, NY) graciously donated glipizide. Calyculin-A was obtained from LC Services (Woburn, MA). ATP, cAMP, PKA, D-myoinositol-1,2,3,4,5,6-hexakisphosphate (InsP6 ), and crude histone (type 2-AS) were from Sigma (St. Louis, MO). [c-32 P]ATP (350 Ci/mol) was purchased from Du Pont-New England Nuclear (Boston, MA).

Preparation of phosphohistone. Histone type 2-AS was phosphorylated with rabbit muscle type I PKA as described in [13,14]. Determination of PPase activity. Clonal rat insulinoma RINm5F cells [18] were cultured to subconfluence. Phosphatase activity against phosphohistone was determined by measuring the liberation of [32 P] in cell homogenates [13,14]. Okadaic acid (final concentration 1 nM) or its solvent dimethylformamide was also included. At one nanomolar, okadaic acid will inhibit essentially all PPase-2A activity without appreciably affecting PPase-1 [10]. Statistical analysis. Data are given as means
SEM. The statistical probability for differences between groups was analyzed by Student’s t test or by one-way ANOVA, with post hoc analysis using the Student– Newman–Keuls multiple comparison test. For differences within groups, one-way ANOVA was used. A value of P < 0:05 was considered significant.

Results Okadaic acid and calyculin-A as inhibitors of RINm5F cell ser/thr PPases We first characterized the dose-dependency of okadaic acid, a complex polyether fatty acid produced by certain strains of marine dinoflagellates [1,10], and calyculin-A, a spiroketal isolated from a marine sponge, as inhibitors of RINm5F cell PPases [1,10]. As shown in Fig. 1, the divalent cation-independent PPase activity in dilute RINm5F cell homogenates is completely inhibited by okadaic acid at a concentration of 106 M, with partial inhibition occurring at a concentration of less than 1010 M. This indicates that both PPase-1 and PPase-2A are active in RINm5F cells (i.e., if only PPase-1 was active, then inhibition should not occur at concentrations below 109 M [10]). Similarly,

Fig. 1. Different inhibitory profiles of okadaic acid and calyculin-A on RINm5F PPase activities. Dilute cell homogenates were incubated with the indicated agent using [32 P]histone as a substrate. PPase activity was assayed as detailed in Materials and methods. Values are mean percent of controls
SEM for 4–6 separate experiments.

M. Lehtihet et al. / Biochemical and Biophysical Research Communications 316 (2004) 893–897

if only PPase-2A was active, then complete inhibition would be observed at a concentration of 109 M [10]. Okadaic acid was less potent an inhibitor than was calyculin-A, the IC50 for both okadaic acid and calyculin-A being 109 M (Fig. 1), whereas IC100 was 106 M for okadaic acid and 108 M for calyculin-A (Fig. 1), values that are in good agreement with previously published results [10]. The inflection point noted at 109 M of okadaic acid (Fig. 1) likely reflects the concentration where all PPase-2A activity is inhibited and type 1 activity remains unaffected [10]. Again, this is in agreement with studies with purified enzymes [10] and suggests that PPase-1 and PPase-2A represent the quantitatively most important cation-independent ser/ thr-PPases in RINm5F cells. Inositol hexakisphosphate differentially inhibits ser/thr PPase activities In the second set of experiments, the actions of various polyphosphoinositides that are generated upon glucose stimulation on PPase activities were investi-

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Table 1 PPase activities of RINm5F cells exposed to sulfonylureas Drug added

Glibenclamide (3 lM) Glipizide (3 lM) Tolbutamide (100 lM) ATP (500 lM)

Enzyme activity (% of control) PPase-1

PPase-2A

99
3 (NS) 89
7 (NS) 95
1 (NS) 43
2

111
6 (NS) 98
7 (NS) 107
9 (NS) 1
1

RINm5F cell homogenates were incubated with the above compounds. Values are means
SEM for 3–8 observations. PPase-1 activity was calculated as activity remaining in the presence of 1 nM okadaic acid; PPase-2A activity was calculated as the difference between total activity and PPase-1 activity. NS, not significant. *** denotes P < 0:001 for chance difference vs. controls using Student’s t test.

gated. InsP6 was the only polyphosphoinositide exerting significant and consistent effects, the results being shown in Fig. 2. As shown in Fig. 2A, InsP6 dose-dependently inhibited PPases in RINm5F cell homogenates, showing IC50 values of 6 lM and 1 lM for PPase-1 and PPase2A, respectively. Assaying enzymes purified to apparent homogeneity revealed that PPase-2A was most susceptible to InsP6 inhibition, whereas PPase-1 was least sensitive (Fig. 2B). Hypoglycemic sulfonylureas do not affect PPase activity In an attempt to elucidate the possible molecular mechanism behind the stimulatory effect of sulfonylureas upon exocytosis, the enzymatic activities of intracellular PPase-1 and -2A were measured in the presence and absence of 3 lM glibenclamide, 3 lM glipzide or 100 lM tolbutamide. The results presented in Table 1 show no significant effect of any of the three sulfonylureas on the activity of any of these PPases. ATP (500 lM), which was included as a positive control, caused an approximate 50% inhibition of PPase-1 and abolished PPase-2A activity completely (Table 1) which is in agreement with previous studies [14].

Discussion

Fig. 2. (A,B) InsP6 dose-dependently and differentially inhibits divalentcation independent PPase activities. Dilute RINm5F cell homogenates (A) or purified PPases (B) were incubated with desired concentrations of InsP6 . In (A), dose–response curves are plotted. In (B), purified PPases 1 and 2A were used and InsP6 was tested at a fixed concentration of 2 lM. PPase activity was assayed as detailed in Materials and methods. Values are mean percent of controls
SEM for 4–6 separate experiments. *, **, and *** denote P < 0:05, P < 0:01, and P < 0:001, respectively, for chance differences vs. controls using Student’s t test. Abbreviation: InsP6 , D -myo-inositol-1,2,3,4,5,6-hexakisphosphate.

In this paper, a novel mechanism of linking glucosestimulated polyphosphoinositide formation to the promotion of insulin exocytosis is described. In human type 2 diabetes mellitus, loss of glucose-sensitive insulin secretion is an early pathogenetic event resulting in hyperglycemia [5]. Initially, this may be compensated for by stimulating insulin secretion by sulfonylurea drugs as is frequently done in diabetic patients [5,6]. The mechanisms involved in the regulation of insulin secretion, either by natural or pharmacological stimuli, are, however, not fully understood. Experiments indicate that glucose retains an excellent ability to secrete insulin even in the presence of maximally effective concentrations of

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Kþ and diazoxide, an opener of Kþ channels [7,8]. Thus, signaling molecules other than ATP and Ca2þ must be involved in glucose sensing in the b-cell, but the precise nature by which these complementary signals promote secretion and the KATP -independent signaling pathways activated by glucose has remained elusive. Additionally, sulfonylureas promote insulin exocytosis from permeabilized cells [19], suggesting KATP -independent actions also of these drugs. Recently, we reported that one such mechanism may involve inhibition of carnitine palmitoyltransferase 1 activity by glibenclamide, thereby switching b-cell fatty acid metabolism to the synthesis of diacylglycerol and subsequent PKC-dependent, KATP independent insulin exocytosis [9]. Furthermore, reports show that >90% of glibenclamide binding sites are localized intracellularly in the b-cell [9]. Others have suggested that tolbutamide, glibenclamide, and gliclazide act as inhibitors of islet ser/thr-PPase activities [20], findings that were not confirmed in our present studies. The reasons for this obvious discrepancy remain unclear, but may possibly be attributable to substratedirected effects. Insulin-secreting cells also have a number of receptors whose activation regulates the intracellular concentration of polyphosphoinositides [2,3,12,18,21]. Although a large number of polyphosphoinositides have been identified in eukaryotic cells, except for the inositol 1,4,5-trisphosphate-induced mobilization of Ca2þ from intracellular stores, little is known about their roles in cell regulation [3]. In insulin-secreting b-cells, rapidly reversible protein phosphorylation/dephosphorylation cycles are believed to play an essential role in integrating and coordinating incoming stimuli into an appropriate rate of insulin exocytosis [3,4,11,13–16]. A great deal of interest has been focused on how reversible phosphorylation of proteins is involved in regulation of cellular functions [1]. The phosphorylation/dephosphorylation cycle is now known to be a dynamic process, with cellular levels of protein phosphorylation being determined by the combined actions of numerous protein kinases and PPases. Compared with protein kinases, relatively little attention has been paid to the role of PPases in the b-cell [3,4]. However, divalent-cation independent serine/threonine PPases have been identified in b-cells by Western blotting and by enzymatic assay [10,11, 13,14,16,17]. Additionally, the specific PPase inhibitor, okadaic acid [1], was shown to promote Ca2þ entry and insulin exocytosis, possibly through hyperphosphorylation (and thereby activation) of voltage-activated L-type Ca2þ channels [10,11,13,15,16]. Conversely, suppression of insulin secretion by inhibitory neurotransmitters was reported to occur through activation of PPase-2B [22]. In intact b-cells several insulin secretagogues evoked a rapid and transient inhibition of PPase activity, thereby presumably contributing to a

hyperphosphorylated state of b-cell regulatory proteins [14]. Furthermore, glucose metabolites, GTP and ATP, dose-dependently suppress b-cell PPase activities [13,14]. Entirely consistent with this model is the finding that PPase-2A can be activated in the b-cell by ceramide [23], a second messenger molecule that inhibits b-cell function and insulin secretion [24]. Moreover, in b-cells the 36 kDa catalytic subunit of PPase-2Ac undergoes carboxyl methylation, an effect accompanied by increased PPase-2A activity and suppressed insulin secretion [25]. Inositol hexakisphosphate (InsP6 ), the dominant polyphosphoinositide in insulin-secreting pancreatic bcells [16], inhibited ser/thr PPases type 1 and type 2A activities in a concentration-dependent manner. The activity of voltage-gated L-type Ca2þ channel is increased in hamster HIT-T15 cells treated with inhibitors of ser/thr PPases [11,16]. Thus, the increased Ca2þ channel activity obtained in the presence of InsP6 might result from the inhibition of PPase activity. Glucose elicited a transient increase in InsP6 concentration [16], which indicates that this polyphosphoinositide may modulate Ca2þ influx over the plasma membrane and serve as a physiologic signal in the pancreatic b-cell stimulus-secretion coupling. Whereas PPase-2A was the most selective for InsP6 , PPase-1 was the least selective (Fig. 2B). It should be noted that PPases-1 and -2A constitute the bulk of cation-insensitive ser/thr-PPases in RINm5F cells [10,11,13,16], implying that suppression of their activity contributes significantly to an increase in overall cellular phosphorylation. InsP6 is more abundant than any of the InsP5 isomers in b-cells and is thus the only polyphosphoinositide that is likely to affect the activities of the PPases in vivo. Because 2 lM InsP6 had a stimulatory effect on the Ca2þ current [16], only a slight suppression of PPase activity may be sufficient to modulate Ca2þ channel activity. Because InsP6 is localized to membranes [16], it is also topographically disposed to regulate ion channels. Besides inhibiting ser/ thr-PPases and activating Ca2þ currents, InsP6 has been shown to stimulate non-Ca2þ -mediated and prime Ca2þ mediated insulin secretion through activation of PKC-e [26,27]. Thus, by being both under metabolic control and regulated by muscarinic receptor activation of phospholipase C, InsP6 may have a physiologically important role in orchestrating b-cell insulin exocytosis. It is concluded that a specific polyphosphoinositide under metabolic and receptor-operated control, InsP6 , dose-dependently and differentially inhibits b-cell divalent-cation independent serine/threonine PPase activities at physiological concentrations. An increase in b-cell phosphorylation state, through the inhibition of protein dephosphorylation by InsP6 , may thus represent a novel and important regulatory mechanism in linking glucose sensing and phospholipase activation to insulin exocytosis.

M. Lehtihet et al. / Biochemical and Biophysical Research Communications 316 (2004) 893–897

Acknowledgments Financial support was received from the Swedish Medical Research Council (# 72X-12550, 72X-14507, and 72P-14787), SmithKline Beecham Pharma AB, Petrus and Augusta Hedlund’s Foundation, the Nutricia Research Foundation, the European Foundation for the Study of Diabetes, Centrala F€ ors€ oksdjursn€amnden, the Swedish Society of Medicine, the Sigurd and Elsa Golje Memorial Foundation, Svenska F€ ors€ akringsf€ oreningen, Svenska Diabetesstiftelsen, Magn.
Wiberg’s Foundation, Bergvall Foundation, Barndiabetesfonden, Ake Torsten and Ragnar S€ oderberg’s Foundations, Berth von Kantzow’s Foundation, Tore Nilson’s Foundation for Medical Research, Fredrik and Inger Thuring’s Foundation, and Syskonen Svensson’s Fund (all
to A.S.) and from and NIH CA 60750 (to R.E.H.).

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