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Metallothionein induction in islets of Langerhans and insulinoma cells
Molecular and Cellular Endocrinology 165 (2000) 179 – 187 www.elsevier.com/locate/mce
Metallothionein induction in islets of Langerhans and insulinoma cells S.G. Laychock a,*, J. Duzen b, C.O. Simpkins b a
Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, State Uni6ersity of New York at Buffalo, 102 Fanber Hall, Buffalo, NY 14214, USA b Department of Surgery, School of Medicine and Biomedical Sciences, the State Uni6ersity of New York at Buffalo, Buffalo, NY 14214, USA Received 1 February 2000; accepted 21 March 2000
Abstract Isolated pancreatic islets from rat and mouse and the insulinoma cell lines, bHC9 and RINm5F, were investigated to determine the regulation of metallothionein (MT). Dexamethasone (DEX) increased rat and mouse islet and insulinoma cell MT levels in a time- and concentration-dependent manner. Rat islet MT expression was increased with interleukin-1b (IL-1b), but not tumor necrosis factor-a (TNF). However, MT induction by IL-1b and TNF was synergistic with DEX in rat islets and insulinoma cells. Mouse islet MT failed to respond to IL-1b alone, although IL-1b and TNF were synergistic. IL-1b and TNF did not synergize with DEX for mouse islet MT induction. Zinc sulfate induced MT in rat islets but not mouse islets. MT messenger RNA levels were significantly increased in rat islets in response to DEX and IL-1b plus DEX. The inducible nitric oxide synthase inhibitors N G-monomethyl-L-arginine and aminoguanidine failed to inhibit IL-1b induced MT levels in insulinoma cells, and the nitric oxide generating agent sodium nitroprusside failed to significantly affect MT levels. Phorbol dibutyrate increased MT levels in rat islets and bHC9 cells, but phorbol dibutyrate and IL-1b effects were not additive. Transgenic MT-null and wild-type mouse islets had similar insulin contents, but basal and glucose-stimulated insulin release from MT-null islets were significantly lower than in wild-type islets. Blood glucose levels in MT-null mice were, however, slightly lower than those in wild-type mice. Thus, MT induction in pancreatic islets and b-cells is regulated by cytokines and DEX, and protein kinase C activation may play a role. However, regulation of MT induction in mouse and rat islets differs. MT also appears to modulate insulin release from pancreatic islets. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Metallothionein; Cytokine; Rat pancreatic islet; Insulinoma; Protein kinase C
1. Introduction Metallothioneins (MT) are highly conserved lowmolecular weight and cysteine-rich proteins possessing a very high metal binding capacity (Moffatt and Denizeau 1997; Fischer and Davie, 1998). They are found in vertebrate and invertebrate life forms. MT usually binds seven zinc atoms, but copper, cadmium and traces of other metals can also bind. MT-1 and MT-2 are prevalent isoforms coded for by distinct genes (Uchida et al., 1991; Moffatt and Seguin 1998) inducible by metals, members of the nuclear hormone * Corresponding author. Tel.: +1-716-8292808; fax: + 1-7168292801. E-mail address: [email protected] (S.G. Laychock).
receptor family, cytokines, oxidative and physical stresses, and certain hormones, among others (Nath et al., 1988; Vallee, 1995). Hormonal induction of MT synthesis is especially marked with glucocorticoids, although growth factors and glucagon have also been identified as inducers (Nath et al., 1988; Moffatt and Denizeau, 1997). Regulation of MT expression is both tissue and isoform specific. The function of MT has been long considered to be detoxification, posttranscriptional activation of enzymes and metal metabolism, transport and storage (Nath et al., 1988). While the function of MT 3 has been linked to inhibition of neuronal growth, the physiological significance of types 1 and 2 is less readily defined. While it has been reported that MT-1 and -2 double knock-out mice become obese and hyperleptine-
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mic (Beattie et al., 1998), the mechanism responsible for changes in energy metabolism is not known. Certain properties of MT implicate the proteins in several cellular reactions which potentially impact metabolism and cell function. The high thiol content of MT may allow for scavenging of reactive oxygen species and protecting cell membranes, protein sulfhydryls, or nucleic acids from the effects of nitric oxide, superoxide, peroxynitrite, and hydroxyl radicals, among others (Moffatt and Denizeau, 1997; Kumari et al., 1998). MT is reportedly superior even to reduced glutathione in scavenging superoxide radicals (Hussain et al., 1996), as well as hydroxyl and peroxyl radicals (Miura et al., 1997) and protecting DNA (Chubatsu and Meneghini, 1993; Cai et al., 1995). The pancreas has been reported to contain relatively high levels of MT compared to liver, heart or muscle tissues, but similar levels to kidney, brain and small intestine (Nath et al., 1988). MT has been reported to be induced in mouse pancreatic islets by zinc sulfate (Zn) or streptozotocin (STZ) treatment in vitro (Ohly and Gleichmann, 1995) and in vivo (Onosaka et al., 1988; Zimny et al., 1993). However, in another report, Zn failed to increase mouse islet endocrine cell MT levels (Minami et al., 1999). Other investigators have also failed to identify MT in mouse islets (De Lisle et al., 1996), whereas reports from another laboratory show the immunohistochemical localization of MT in mouse islets (Zimny et al., 1993; Ohly et al., 1999). In addition, there is one report of MT-1 and MT-2 immunohistochemical staining in rat islets (Andrews et al., 1990). Controversy also surrounds the possible role of MT in protecting pancreatic islets from the effects of diabetogenic agents. Although zinc sulfate (Zn) increased the recovery of superoxide dismutase activity in wildtype STZ-treated mice, Zn also suppressed the development of hyperglycemia in wild-type and MT-null transgenic STZ-treated mice, suggesting that MT was not the causative factor in the protective response to Zn (Apostolova et al., 1997). The effect of the superoxide radical generating agent alloxan to induce hyperglycemia was also not antagonized by MT induction in mice (Onosaka et al., 1988; Minami et al., 1999). However, in a low-dose STZ in vivo model of diabetes mellitus in the mouse, Zn administration antagonized the development of hyperglycemia and increased MT levels in isolated pancreatic islets (Ohly et al., 1999). The present study was undertaken to fully characterize MT levels in rat and mouse islets in vitro in response to classical MT inducing agents, diabetogenic agents, and signal transduction modulators. The hypothesis tested was that MT, if it is a b-cell cytoprotective agent, would be responsive to cytokines and modulated by signal transduction pathway activation. In addition, this study is the first to report the reduced insulin secretory response of MT-null mouse islets.
2. Methods and materials
2.1. Materials 109
Cadmium was from Amersham Life Science (Arlington Heights, IL). CMRL-1066 media, RPMI-1640, and Dulbecco’s modified Eagle’s medium (DMEM) (pyruvate free) and other culture media reagents were from GIBCO/Life Technologies (Grand Island, NY). Fetal bovine serum (FBS) was from Atlanta Biologicals (Norcross, GA). Aminoguanidine, phorbol dibutyrate (PDBu), sodium nitroprusside and bovine serum albumin (Fraction V) (BSA) were from Sigma Chemical (St Louis, MO). N G-monomethyl-L-arginine (NMMA) was from Calbiochem (San Diego, CA). Recombinant human interleukin (IL)-1b was obtained from R&D Systems (Minneapolis, MN). Insulin for radioimmunoassay standard was a gift from Eli Lilly (Indianapolis, IN). Collagenase (type P) was from Boehringer Mannheim/Roche (Indianapolis, IN). All other chemicals were reagent grade from commercially available sources. bHC9 cells were a gift from Dr D. Hanahan, the University of California at San Francisco; RINm5F cells were a gift from Dr C. Wollheim, Geneva, Switzerland.
2.2. Isolation of islets and culture of islets and insulinoma cells Pancreatic islets from adult male Sprague–Dawley rats or mice were isolated using collagenase, essentially as described previously (Xia and Laychock, 1993). Transgenic MT-1 and MT-2 null ( − /−) mice (originally C57/BL6xOLA129, backcrossed to C57/BL6) or wild-type ( + /+) mice were produced by A. Michalska and K.H.A. Choo of the Murdoch Institute, Royal Childrens Hospital, Parkville, Australia (Michalska and Choo, 1993) and were maintained at our institution. All animal procedures were approved by the Institutional Animal Care and Use Committee. Isolated islets were either used immediately as freshly isolated islets, or they were cultured for 24 h in 35 mm culture dishes (:60 islets/dish) with 2 ml CMRL-1066 medium containing 5.5 mM glucose, 9% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) at 5% CO2 –95% air, and 35°C, as described previously (Xia and Laychock, 1993). Culture media was changed every second day. The clonal insulinoma cell line bHC9 was cultured in DMEM, and RINm5F cells were cultured in RPMI1640, as described previously (Lee et al., 1998). Culture media was supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml) at 5% CO2 –95% air, at 35°C. Other additions to the culture media were as described in the text, and the cells were cultured for 24 h except where indicated.
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2.3. Insulin content and release Total islet insulin content was determined by extracting islets with ethanol (70%) in HCl (1 N) overnight at − 20°C, and determining insulin in a small aliquot by insulin radioimmunoassay. Insulin release was determined by incubating islets (10 islets/sample) in Krebs Ringer bicarbonate buffer (pH 7.4) containing 16 mM Hepes (KRBH buffer) and 0.01% BSA at 5.5 mM glucose for 60 min, followed by a 60 min incubation in fresh KRBH buffer with various glucose concentrations indicated in the text. Aliquots of incubation medium were removed at time 0 and after the 60 min incubation for assay of insulin by radioimmunoassay. Insulin release was expressed as insulin released after 60 min minus 0-time insulin levels.
2.4. Metallothionein assay MT levels were determined following lysis of cells or islets with brief sonication in 10 mM Tris buffer at pH 7.4 containing 0.1 or 1 mg cadmium (220 ml). An aliquot of lysate was used to determine protein content by Bio – Rad protein assay, using BSA as standard. Lysates were microcentrifuged briefly to remove particulate matter, and 200 ml were used for MT determination by cadmium–hemoglobin affinity assay, essentially as described previously (Eaton and Cherian, 1991). Lysate or blank buffer was mixed with 109Cd (0.5 mCi/ml Tris buffer without cadmium) and incubated at RT for 10 min. Then, 100 ml 2% bovine hemoglobin solution in Tris buffer without cadmium was added to each sample and vortexed. The samples were subsequently boiled for 4 min, vortexed, and then centrifuged to pellet the denatured hemoglobin. Supernatant (350 ml) was transferred to another tube containing 100 ml fresh hemoglobin solution, votexed and boiled for 3 min, vortexed again, and centrifuged. The supernatant (350 ml) was transferred to tubes for gamma counting. The specific activity of the 109Cd was determined, and the number of nmol cadmium bound in MT was calculated and corrected for to the total starting volume of lysate. Assuming 1 nmol cadmium bound is equivalent to 1 mg MT, data were expressed as number of nanograms MT per microgram protein.
2.5. Semiquantitati6e re6erse-transcriptase polymerase chain reaction (RT-PCR) Relative levels of metallothionein messenger (m)RNA were determined in isolated rat islets (80–120 islets per reaction). Total RNA was extracted and reverse transcribed essentially as reported previously (Lee et al., 1998). The resulting complimentary (c)DNA was then amplified, as indicated with the following (+) and (−) strand oligonucleotide primers, respectively:
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metallothionein 1 and 2 (400 bp) 5%-ctcaagaagaccatctgcctc-3% and 5%-cagcttgacaccaatgttcc-3%; cyclophilin (490 bp) 5%-gtgttcttcgacatcacgg-3% and 5%-gaacttcagtgagagcagag-3%. Polymerization reactions were carried out in a Hybaid Sprint thermalcycler using a 1:5 dilution of cDNAs as templates in a 25 ml reaction volume, as described previously (Lee et al., 1998). The amplification conditions were 26 cycles with denaturation for 4 min at 94°C, annealing for 1 min at 60°C, extension for 2 min at 72°C with the final extension for 7 min. The amounts of cDNA chosen were within a linear range for amplification. The reaction products were separated by agarose gel electrophoresis, and the gel was stained with CYBER Green and viewed by densitometry. The image density of metallothionein mRNA was compared with the density of co-amplified cyclophilin mRNA to determine the relative level of metallothionein expression in each experimental sample. DNA sequencing confirmed the RT-PCR reaction product to be 99% identical to the metallothionein 1 and 2 reported sequence in GenBank.
2.6. Statistical analysis The data are presented as the mean 9 SE and were analyzed by Student’s t test, or one-way analysis of variance (ANOVA) with Student’s/Newman –Keuls multiple comparison test; PB0.05 was accepted as significant.
3. Results
3.1. Isolated islet MT le6els in response to dexamethasone Isolated rat pancreatic islets were cultured in the absence and presence of dexamethasone in order to determine glucocorticoid effects on MT levels. Dexamethasone increased islet MT at all concentrations tested (0.01–1 mM) (Fig. 1A). The effect of dexamethasone was also time-dependent and a significant increase in MT occurred within 8 h (Fig. 1B). It was also determined by RT-PCR that dexamethasone (0.1 mM) increased islet MT mRNA levels by 1289 6% of control (PB 0.05; n= 3) after 6 h. Dexamethasone also induced MT in wild-type MT + /+ mouse islets (Table 1). In contrast, MT levels in islets from MT-null mice were at the limit of detectability for the assay in control islets (0.0059 0.001 ng MT/mg protein) and were only 3% of the levels observed in wild-type islets (PB0.001) (Table 1). Dexamethasone failed to elicit any significant changes in MT levels compared to control in MT − /− islets (data not shown). These data indicate that 97% of the MT measured by the cadmium–hemoglobin affinity assay was
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specific for MT-1 and/or 2. The very low levels of cadmium binding detected in MT-null mouse islets may be due to other isoforms of MT, low levels of other metal binding proteins, or low-molecular weight thiols such as cyteine and glutathione which can affect the cadmium– hemoglobin affinity assay (Eaton and Cherian, 1991).
3.2. Islet MT le6els in response to cytokines
Fig. 1. Rat islet MT response to dexamethasone. (A) Isolated islets were cultured for 24 h in the absence (control) and presence of dexamethasone (DEX) at the concentrations (mM) indicated. (B) Islets were cultured for up to 24 h in the absence (control) and presence of dexamethasone (1 mM). Control islet values were 0.00599 0.0008 ng MT/mg protein. Values are the mean 9 SE for the number of independent determinations shown at the base of each bar. *, PB 0.02 versus control as determined by one-way ANOVA and multiple comparison test.
Table 1 Metallothionein in isolated mouse isletsa Treatment
+/+ mouse islet MT levels
Control DEX IL-1b TNF+IL-1b TNF+IL-1b+DEX Zn
0.1559 0.025 0.4339 0.112** 0.1719 0.040 0.2549 0.065* 0.5129 0.126** 0.1159 0.031
a
(7) (4) (3) (4) (4) (3)
Islet of Langerhans were isolated from pancreata of wild-type (MT +/+) mice. Islets were cultured for 24 h in the absence (control) or presence of dexamethasone (DEX; 1 mM), TNF (10 ng/ml), IL-1b (1 ng/ml), or Zn (0.1–0.5 mM). Values are the mean 9 SE for ng MT/mg protein. Number of independent experiments are shown in parentheses. * PB0.05. ** PB0.01 versus control, as determined by one-way ANOVA and post-hoc analysis.
Rat islets were cultured for 24 h in the absence and presence of the cytokines interleukin-1b (IL-1b) and tumor necrosis factor a (TNF) in order to determine effects on MT expression. IL-1b, but not TNF, elicited an increase in MT levels similar to the increase observed in response to dexamethasone (Fig. 2). However, when IL-1b and TNF were added to islets together with dexamethasone there was a synergistic effect on MT induction and the response was especially evident with the combination of IL-1b and dexamethasone (Fig. 2). When rat islet MT mRNA expression was determined, dexamethasone (1 mM) and IL-1b (1 ng/ml) increased MT mRNA levels to 133911% of control (0.2309 0.047 MT mRNA/cyclophilin mRNA) (PB 0.05; n=5) within 4 h after beginning treatment. Rat islet MT levels also increased in response to the MT inducing agent, Zn (Fig. 2). Mouse islets were also found to contain MT at levels comparable to rat islets (Table 1). Wild-type mouse islets cultured with IL-1b did not show an increase in MT levels compared to control islets (Table 1). The combination of IL-1b and TNF elicited a small increase in MT levels (Table 1). However, the combination of IL-1b, TNF, and dexamethasone failed to significantly increase MT levels above the level elicited by dexamethasone alone (Table 1). Zn failed to elicit a significant change in mouse islet MT (Table 1). In islets from MT-null mice, dexamethasone (1 mM) in the presence of the cytokines IL-1b (1 ng/ml) and TNF (10 ng/ml), failed to elicit any significant changes in MT synthesis after 24 h culture (data not shown). Zn was also not an effective stimulus in MT-null islets (0.003 90.002 ng MT/mg protein).
3.3. Insulinoma cell MT responses The insulinoma cell lines for rat (RINm5F) and mouse (bHC9) were investigated for MT responsiveness since they represent a homogeneous population of b-cells, unlike islets which are a heterogeneous collection of three types of endocrine cells, other peptide secreting cells, endothelial cells and neuronal tissue. Similar to isolated islets, dexamethasone elicited a concentration-dependent increase in MT levels in bHC9 cells (Fig. 3A). The response to dexamethasone was also maximal within 24 h (Fig. 3B). RINm5F cells also responded to dexamethasone with increased MT levels (Fig. 4).
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Fig. 2. Rat islet MT induction by cytokines and dexamethasone (DEX). Islets were cultured for 24 h in the absence (basal) or presence of IL-1b (IL) (1 ng/ml), TNF (200 ng/ml), dexamthasone (DEX) (1 mM), or Zn (100 mM). Values are the mean 9SE for the number of independent determinations shown at the base of each bar. Significant differences were determined by one-way ANOVA and multiple comparison test. *, P 50.05 versus basal.
IL-1b also increased MT levels in RINm5F and bHC9 cells by about 50% (Figs. 4 and 5), and maximal effects were observed after 24 h (data not shown). RINm5F cells responded to a combination of dexamethasone and IL-1b with a synergistic increase in MT similar to that noted for rat islets (Fig. 4); bHC9 cells showed a similar response (data not shown). Inhibitors of inducible nitric oxide synthase (iNOS), NMMA and aminoguanidine, failed to inhibit the MT response to IL-1b in bHC9 cells (Fig. 5). Neither NMMA (0.5 mM) nor aminoguanidine (0.5 mM) had any significant effect on MT levels in control cells (data not shown). In addition, the nitric oxide generating agent, sodium nitroprusside, failed to significantly affect MT levels in bHC9 cells (Fig. 5).
Fig. 3. Dexamethasone effects on MT in bHC9 cells. (A) Cells were cultured 24 h in the absence (control) or presence of different concentrations (mM) of dexamethasone (DEX) as indicated. (B) Cells were cultured for up to 48 h in the absence (control) or presence of dexamethasone (1 mM). Control values were 0.054 90.027 ng MT/mg protein.Values are the mean 9SE for the number of independent determinations shown at the base of each bar. *, P B0.05 versus control; , PB0.01 versus control, as determined by one-way ANOVA and multiple comparison test.
3.4. Metallothionein expression in response to phorbol ester Since IL-1b effects in islets are accompanied by increased protein kinase C (PKC) activity (Eizirik et al., 1995), islet MT responses to a PKC activating phorbol ester, PDBu, were investigated to determine if PKC mediates changes in MT expression. Rat islets incubated with PDBu for 24 h showed a doubling of MT content compared to control islets (Fig. 6). bHC9 cells also responded to PDBu with increased MT content (Fig. 7). However, the combination of IL-1b and PDBu did not elicit an increase in MT greater than that elicited by either agent alone (Fig. 7).
Fig. 4. MT responses to IL-1b (IL) and dexamethasone (DEX) in RINm5F cells. Cells were cultured for 24 h in the absence (basal) or presence of IL (1 ng/ml) and/or DEX (1 mM). Values are the mean9 SE for three to seven independent determinations. Significant differences were determined by one-way ANOVA and multiple comparison test; *, PB0.05 versus basal.
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Fig. 5. Effects of iNOS inhibitors and sodium nitroprusside (SNP) on bHC9 cell MT levels. Cells were cultured for 24 h in the absence (basal) or presence of IL-1b (IL) (1 ng/ml), NMMA (500 mM), aminoguanidine (AG) (500 mM), and SNP (100 mM) as indicated. Control values were 0.12 9 0.06 ng MT/mg protein. Values are the mean9 SE for the number of independent determinations shown at the base of each bar. *, P B0.05 versus control as determined by paired t-test for each treatment group versus basal.
Fig. 6. Rat islet MT response to PDBu. Islets were cultured for 24 h in the absence (control) or presence of PDBu (1 mM) as indicated. Values are the mean9 SE for the number of independent determinations shown at the base of each bar. *, PB 0.002 versus control as determined by paired t-test.
glucose) and maximally secretagogic glucose (17 mM) concentrations in order to determine if the insulin secretory responses of wild-type and MT-null mouse islets differed. A markedly reduced release of basal insulin was observed for the MT-null mouse islets (Fig. 8). Stimulation with 17 mM glucose increased wild-type mouse islet insulin release by almost 3-fold, whereas stimulation of MT-null islets increased insulin release almost 9-fold (Fig. 8). Even though 17 mM glucose increased MT-null islet insulin release above control, the level remained significantly lower than that released by glucose-stimulated wild-type islets (Fig. 8). 4. Discussion MT is found in high levels in the pancreas (Nath et al., 1988), however, controversy exists regarding the presence and inducibility of MT in endocrine pancreatic islets of Langerhans from the rat and mouse (Onosaka et al., 1988; Andrews et al., 1990; Zimny et al., 1993; Ohly and Gleichmann, 1995; De Lisle et al., 1996; Minami et al., 1999; Ohly et al., 1999). The present study shows not only that rat and mouse isolated pancreatic islets contain comparable amounts of MT, but also that b-cells which synthesize and package insulin for secretion contain MT as well as the mRNA for MT. These findings are in agreement with the immunohistochemical localization of MT in rat and mouse islets (Zimny et al., 1993; Ohly et al., 1999). And, for the first time we show that MT is induced in both rat and mouse islets and rat and mouse b-cell lines in response to dexamethasone, a primary inducer for MT gene transcription (Moffatt and Denizeau, 1997). In addition, MT mRNA expression was rapidly increased in response to dexamethasone. The MT response to dexamethasone was sensitive to low concentrations of the hormone, and showed a time-dependency consistent with the rapid increase in MT gene transcription.
Fig. 7. bHC9 cell MT response to IL-1b (IL) and PDBu. Cells were cultured for 24 h in the absence (basal) or presence of IL (1 ng/ml) and PDBu (1 mM) as indicated. Basal values were 0.138 9 0.074 ng MT/mg protein. Values are the mean 9 SE for the number of independent determinations shown at the base of each bar. *, PB 0.05 versus basal as determined by Student’s paired t-test.
3.5. Insulin release in islets of wild-type mice and MT-null mice The insulin content of mouse islets was quantitated to determine if MT-null mouse islets differed from wild-type islets. The insulin contents of islets from MT-null (1709 16 ng insulin/five islets) and wild-type (168925 ng insulin/5 islets) mouse islets were similar (P \ 0.05). Islets were also incubated at basal (5.5 mM
Fig. 8. Insulin release from MT-null and wild-type mouse islets. Isolated islets were incubated for 60 min in the presence of 5.5 mM glucose (basal) or 17 mM glucose (G17). Insulin release was determined by radioimmunoassay and normalized per five islets. Values are the mean 9 SE for seven determinations. Significant differences were determined by one-way ANOVA and multiple comparison test.
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Rat islet MT levels were also shown to be modestly responsive to the cytokine IL-1b, but not to TNF. This is consistent with the marked effects of IL-1b on increasing nitric oxide (NO) synthesis and inhibiting rat islet insulin secretion responses, but also of the relative lack of effect of TNF alone in isolated islets (Corbett et al., 1993a). RINm5F cells also showed a small but significant increase in MT in response to IL-1b. However, both the rat islet and RINm5F cells showed evidence that IL-1b and TNF act synergistically with dexamethasone in eliciting a large increase in MT levels. An early increase in metallothionein mRNA confirmed the effect of these agents on metallothionein expression. Cytokines have been implicated previously in inflammation-associated increases in MT (Schroeder and Cousins, 1990; Moffatt and Denizeau, 1997). It has also been reported that dexamethasone caused a significant protection against IL-1b in terms of maintaining insulin secretion in rats treated with the cytokine (Shimizu et al., 1992). Whether this protective effect could be due to enhanced MT induction is not known. However, copper, which induces MT synthesis (Moffatt and Denizeau, 1997), can prevent the inhibitory effects of IL-1b on islet insulin secretion in vitro (Vinci et al., 1995). Dexamethasone also antagonizes the inhibitory effects of IL-1b on insulin secretion from islets in vitro (Corbett et al., 1993b), and whether this is due to a direct effect on inducible nitric oxide synthase (iNOS) or to a combined effect on other protective proteins such as MT can only be speculated upon at this time. In contrast to rat islets, mouse islets did not respond to individual cytokine exposure with changes in MT and only the combination of IL-1b and TNF increased MT. TNF has previously been reported to potentiate IL-1b effects in islets (Corbett et al., 1993a). It has been reported that islets contain low levels of antioxidant enzymes compared to other tissues (Lenzen et al., 1996; Tiedge et al., 1997) and that mouse islets are lower in catalase and superoxide dismutase activities than rat islets (Welsh et al., 1995). It is perhaps differences between the species ability to generate or dispose of certain free radicals which accounts for differences in MT induction in response to cytokines in islets. One of the most prominent differences between mouse and rat islets was the failure of mouse islets to display any synergy between dexamethasone and IL-1b and TNF on MT levels. Another prominent feature of mouse islets was their failure to respond to high concentrations of Zn with changes in MT levels. Other investigators have observed a similar lack of response by mouse islets (Minami et al., 1999). Clearly, the cytokines induce changes in MT levels by a different mechanism than glucocorticoids in rat islets, and there are major differences in MT regulation in rat and mouse islets related to cytokines and metals. The murine bHC9 cell line appeared more responsive to cytokine stimulation
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than did the mouse islets, and IL-1b not only increased MT levels to a small extent but also potentiated the dexamethasone response in both rat and mouse cell lines. Differences between mouse primary islet cells and tumor cell line responses may be due to transcriptional factor differences or differences in antioxidant enzymes which might impact on MT induction (Tiedge et al., 1997). Since lysosomes degrade MT (Moffatt and Denizeau, 1997) it is possible that MT levels are also regulated in this manner, although there is no reported evidence of this being a regulated phenomenon. The transcriptional regulation of MT is not well known. Metals such as Zn stimulate the binding of protein transcription factors to a metal regulatory element (MRE) to enhance MT synthesis (Moffatt and Denizeau, 1997). Based upon results in the present study, the mouse MT promoter MRE appears to be less responsive to Zn stimulation than that of the rat islet. Glucocorticoid receptor activation of MT synthesis relies on binding to a glucocorticoid responsive element (GRE) sequence of the MT promoter. Another sequence, 12-o-tetradecanoylphorbol-13-acetate responsive element (TRE), mediates induction of MT by phorbol esters which activate PKC. In addition, TRE binds AP-1 which probably mediates phorbol-induced MT transcription. Another recognition site sequence is for activator protein AP-2, whose transcriptional activity is stimulated by phorbol ester and cyclic AMP through stimulation of protein kinase A. The antioxidant response element (ARE) mediates induction of MT by hydrogen peroxide. However, the TRE sequence is an integral part of the ARE site and confers crossover among the stimulatory ligands. In an effort to determine the mechanism behind the synergy between cytokines and dexamethasone in rat islets, the mechanism accounting for cytokine stimulation of MT synthesis was sought. It is well known that a major response to IL-1b stimulation of b-cells is an increase in iNOS which increases the free radical NO production in these cells (Corbett et al., 1993a). However, in the present study two inhibitors of iNOS, the L-arginine analog NMMA and an enzyme inhibitor aminoguanidine (Misko et al., 1993; Corbett et al., 1993b), failed to significantly alter the MT response to IL-1b even though they have been reported to inhibit iNOS activity in islets (Corbett et al., 1991, 1992). Moreover, the generation of NO with sodium nitroprusside did not significantly alter insulinoma cell MT levels, suggesting that NO is not an important mediator of the MT response. Sodium nitroprusside at the concentration used in this study has previously been shown to induce another stress protein, heme oxygenase-1, within 20 h in islets (Ye and Laychock, 1998). Thus, a mechanism other than NO generation appears to mediate the MT response in b-cells.
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A proposed mechanism of action of IL-1b and TNF is the activation of PKC and nuclear factor (NF)-kB (Barnes and Karin 1997). IL-1b increases NF-kB activation in b-cells (Kwon et al., 1995). Pathways mediating IL-1b and TNF action include the activation of PKC (Schu¨tze et al., 1990; Bergmann et al., 1998; Boussouar et al., 1999), perhaps including atypical forms of PKC (Bonizzi et al., 1999) and the phosphorylation of the inhibitory IkB (Scherer et al., 1995) with subsequent NF-kB activation. In islets, IL-1b stimulates PKC activation (Eizirik et al., 1995). Phorbol ester stimulation of PKC activation in this study was undertaken to determine if this enzyme mediates changes in MT synthesis observed in islets. As the results showed, PDBu increased MT synthesis in rat islets and bHC9 cells to about the level observed with IL-1b. However, the combination of IL-1b and PDBu were not additive, suggesting that they were affecting MT synthesis by a similar mechanism. Presumably, both agents elicit a response through the TRE element mediating PKC responses. MT-null mice were investigated for their insulin secretory responses in order to determine if MT might play a physiological role in this process. Although isolated islets from wild-type and MT-null mice had similar insulin content, the islets from the MT-null animals showed much lower levels of basal insulin release. In addition, although glucose stimulated insulin release from islets of MT-null mice, the maximal insulin secreted was significantly lower than that released by wild-type islets. MT as a target for reactive oxygen species might scavenge superoxide and hydroxyl radicals and preserve membrane integrity, protein sulfhydryls or nucleic acids, and so also secretory responsiveness. In summary, MT has been identified in rat and mouse islets to be regulated by dexamethasone. MT induction in rat islets also appeared to be responsive to cytokine and Zn stimulation, whereas mouse islets lacked sensitivity to these agents in this study. There also appears to be a unique mechanism in rat islets which allows for synergy between dexamethasone and cytokines for MT induction which is lacking in mouse islets. In addition, MT appears to be required for normal b-cell insulin secretory responsiveness. It remains to be determined how MT impacts on insulin secretory mechanisms, however, the antioxidant properties of MT (Ebadi et al., 1996) and its proposed target in mitochondial function (Simpkins et al., 1996, 1998) may play a role in insulin secretion and glucose responsiveness.
Acknowledgements This work was supported by NIH grant DK-25705 (awarded to S.G.L.). The technical assistance of Maureen Adolf and Shawn Sessanna is appreciated.
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S.G. Laychock et al. / Molecular and Cellular Endocrinology 165 (2000) 179–187 Kumari, M.V.R., Hiramatsu, M., Ebadi, M., 1998. Free radical scavenging actions of metallothionein isoforms I and II. Free Rad. Res. 29, 93 – 101. Kwon, G., Corbett, J.A., Rodi, C.P., Sullivan, R., McDaniel, M.L., 1995. Interleukin-1b-induced nitric oxide synthase expression by rat pancreatic b-cells: evidence for the involvement of nuclear factor kB in the signaling mechanism. Endocrinology 136, 4790 – 4795. Lee, B., Bradford, P.G., Laychock, S.G., 1998. Characterization of inositol 1,4,5-trisphosphate receptor mRNA expression and regulation in rat pancreatic islets, RINm5Fcells and bHC9 cells. J. Mol. Endocrinol. 21, 31–39. Lenzen, S., Drinkgern, J., Tiedge, M., 1996. Low antioxidant enzme gene expression in pancreatic islets compared with various other mouse tissues. Free Rad. Biol. Med. 20, 463–466. Michalska, A.E., Choo, K.H.A., 1993. Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc. Natl. Acad. Sci. USA 90, 8088–8092. Minami, T., Tanaka, H., Okazaki, Y., Tohno, S., Tohno, Y., 1999. MT does not protect endocrine cell damage induced by alloxan in pancreas of mice. In: Klassen, C. (Ed.), Metallothionein IV. Birkhauser Verlag, Basel, Switzerland, pp. 429–431. Misko, T.P., Moore, W.M., Kasten, T.P., et al., 1993. Selective inhibition of the inducible nitric oxide synthase by aminoguanidine. Eur. J. Physiol. 233, 119–125. Miura, T., Muraoka, S., Ogiso, T., 1997. Antioxidant activity of metallothionein compared with reduced glutathione. Life Sci. 60, 301 – 309. Moffatt, P., Denizeau, F., 1997. Metallothionein in physiological and physiopathological processes. Drug Metab. Rev. 29, 261– 307. Moffatt, P., Seguin, C., 1998. Expression of the gene encoding metallothionein-3 in organs of the reproductive system. DNA Cell Biol. 17, 501 – 510. Nath, R., Kambadur, R., Gulati, S., Paliwal, V.K., Sharma, M., 1988. Molecular aspects, physiological function, and clinical significance of metallothioneins. Crit. Rev. Food Sci. Nutr. 27, 41– 85. Ohly, P., Gleichmann, H., 1995. Metallothionein: in vitro induction with zinc and streptozotocin in pancreatic islets of mice. Exp. Clin. Endocrinol. 103, 79–82. Ohly, P., Wang, Z., Dohle, C., Abel, J., Gleichmann, H., 1999. Zinc sulphate protects from streptozotocin-induced diabetes mellitus in mice: a function of metallothionein? In: Klassen, C. (Ed.), Metallothionein IV. Birkhauser Verlag, Basel, Switzerland, pp. 421 – 428. Onosaka, S., Min, K.S., Fukita, Y., et al., 1988. High concentration of pancreatic metallothionein in normal mice. Toxicology 50, 27– 35.
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Metallothionein induction in islets of Langerhans and insulinoma cells S.G. Laychock a,*, J. Duzen b, C.O. Simpkins b a
Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, State Uni6ersity of New York at Buffalo, 102 Fanber Hall, Buffalo, NY 14214, USA b Department of Surgery, School of Medicine and Biomedical Sciences, the State Uni6ersity of New York at Buffalo, Buffalo, NY 14214, USA Received 1 February 2000; accepted 21 March 2000
Abstract Isolated pancreatic islets from rat and mouse and the insulinoma cell lines, bHC9 and RINm5F, were investigated to determine the regulation of metallothionein (MT). Dexamethasone (DEX) increased rat and mouse islet and insulinoma cell MT levels in a time- and concentration-dependent manner. Rat islet MT expression was increased with interleukin-1b (IL-1b), but not tumor necrosis factor-a (TNF). However, MT induction by IL-1b and TNF was synergistic with DEX in rat islets and insulinoma cells. Mouse islet MT failed to respond to IL-1b alone, although IL-1b and TNF were synergistic. IL-1b and TNF did not synergize with DEX for mouse islet MT induction. Zinc sulfate induced MT in rat islets but not mouse islets. MT messenger RNA levels were significantly increased in rat islets in response to DEX and IL-1b plus DEX. The inducible nitric oxide synthase inhibitors N G-monomethyl-L-arginine and aminoguanidine failed to inhibit IL-1b induced MT levels in insulinoma cells, and the nitric oxide generating agent sodium nitroprusside failed to significantly affect MT levels. Phorbol dibutyrate increased MT levels in rat islets and bHC9 cells, but phorbol dibutyrate and IL-1b effects were not additive. Transgenic MT-null and wild-type mouse islets had similar insulin contents, but basal and glucose-stimulated insulin release from MT-null islets were significantly lower than in wild-type islets. Blood glucose levels in MT-null mice were, however, slightly lower than those in wild-type mice. Thus, MT induction in pancreatic islets and b-cells is regulated by cytokines and DEX, and protein kinase C activation may play a role. However, regulation of MT induction in mouse and rat islets differs. MT also appears to modulate insulin release from pancreatic islets. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Metallothionein; Cytokine; Rat pancreatic islet; Insulinoma; Protein kinase C
1. Introduction Metallothioneins (MT) are highly conserved lowmolecular weight and cysteine-rich proteins possessing a very high metal binding capacity (Moffatt and Denizeau 1997; Fischer and Davie, 1998). They are found in vertebrate and invertebrate life forms. MT usually binds seven zinc atoms, but copper, cadmium and traces of other metals can also bind. MT-1 and MT-2 are prevalent isoforms coded for by distinct genes (Uchida et al., 1991; Moffatt and Seguin 1998) inducible by metals, members of the nuclear hormone * Corresponding author. Tel.: +1-716-8292808; fax: + 1-7168292801. E-mail address: [email protected] (S.G. Laychock).
receptor family, cytokines, oxidative and physical stresses, and certain hormones, among others (Nath et al., 1988; Vallee, 1995). Hormonal induction of MT synthesis is especially marked with glucocorticoids, although growth factors and glucagon have also been identified as inducers (Nath et al., 1988; Moffatt and Denizeau, 1997). Regulation of MT expression is both tissue and isoform specific. The function of MT has been long considered to be detoxification, posttranscriptional activation of enzymes and metal metabolism, transport and storage (Nath et al., 1988). While the function of MT 3 has been linked to inhibition of neuronal growth, the physiological significance of types 1 and 2 is less readily defined. While it has been reported that MT-1 and -2 double knock-out mice become obese and hyperleptine-
0303-7207/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 0 ) 0 0 2 4 7 - 1
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mic (Beattie et al., 1998), the mechanism responsible for changes in energy metabolism is not known. Certain properties of MT implicate the proteins in several cellular reactions which potentially impact metabolism and cell function. The high thiol content of MT may allow for scavenging of reactive oxygen species and protecting cell membranes, protein sulfhydryls, or nucleic acids from the effects of nitric oxide, superoxide, peroxynitrite, and hydroxyl radicals, among others (Moffatt and Denizeau, 1997; Kumari et al., 1998). MT is reportedly superior even to reduced glutathione in scavenging superoxide radicals (Hussain et al., 1996), as well as hydroxyl and peroxyl radicals (Miura et al., 1997) and protecting DNA (Chubatsu and Meneghini, 1993; Cai et al., 1995). The pancreas has been reported to contain relatively high levels of MT compared to liver, heart or muscle tissues, but similar levels to kidney, brain and small intestine (Nath et al., 1988). MT has been reported to be induced in mouse pancreatic islets by zinc sulfate (Zn) or streptozotocin (STZ) treatment in vitro (Ohly and Gleichmann, 1995) and in vivo (Onosaka et al., 1988; Zimny et al., 1993). However, in another report, Zn failed to increase mouse islet endocrine cell MT levels (Minami et al., 1999). Other investigators have also failed to identify MT in mouse islets (De Lisle et al., 1996), whereas reports from another laboratory show the immunohistochemical localization of MT in mouse islets (Zimny et al., 1993; Ohly et al., 1999). In addition, there is one report of MT-1 and MT-2 immunohistochemical staining in rat islets (Andrews et al., 1990). Controversy also surrounds the possible role of MT in protecting pancreatic islets from the effects of diabetogenic agents. Although zinc sulfate (Zn) increased the recovery of superoxide dismutase activity in wildtype STZ-treated mice, Zn also suppressed the development of hyperglycemia in wild-type and MT-null transgenic STZ-treated mice, suggesting that MT was not the causative factor in the protective response to Zn (Apostolova et al., 1997). The effect of the superoxide radical generating agent alloxan to induce hyperglycemia was also not antagonized by MT induction in mice (Onosaka et al., 1988; Minami et al., 1999). However, in a low-dose STZ in vivo model of diabetes mellitus in the mouse, Zn administration antagonized the development of hyperglycemia and increased MT levels in isolated pancreatic islets (Ohly et al., 1999). The present study was undertaken to fully characterize MT levels in rat and mouse islets in vitro in response to classical MT inducing agents, diabetogenic agents, and signal transduction modulators. The hypothesis tested was that MT, if it is a b-cell cytoprotective agent, would be responsive to cytokines and modulated by signal transduction pathway activation. In addition, this study is the first to report the reduced insulin secretory response of MT-null mouse islets.
2. Methods and materials
2.1. Materials 109
Cadmium was from Amersham Life Science (Arlington Heights, IL). CMRL-1066 media, RPMI-1640, and Dulbecco’s modified Eagle’s medium (DMEM) (pyruvate free) and other culture media reagents were from GIBCO/Life Technologies (Grand Island, NY). Fetal bovine serum (FBS) was from Atlanta Biologicals (Norcross, GA). Aminoguanidine, phorbol dibutyrate (PDBu), sodium nitroprusside and bovine serum albumin (Fraction V) (BSA) were from Sigma Chemical (St Louis, MO). N G-monomethyl-L-arginine (NMMA) was from Calbiochem (San Diego, CA). Recombinant human interleukin (IL)-1b was obtained from R&D Systems (Minneapolis, MN). Insulin for radioimmunoassay standard was a gift from Eli Lilly (Indianapolis, IN). Collagenase (type P) was from Boehringer Mannheim/Roche (Indianapolis, IN). All other chemicals were reagent grade from commercially available sources. bHC9 cells were a gift from Dr D. Hanahan, the University of California at San Francisco; RINm5F cells were a gift from Dr C. Wollheim, Geneva, Switzerland.
2.2. Isolation of islets and culture of islets and insulinoma cells Pancreatic islets from adult male Sprague–Dawley rats or mice were isolated using collagenase, essentially as described previously (Xia and Laychock, 1993). Transgenic MT-1 and MT-2 null ( − /−) mice (originally C57/BL6xOLA129, backcrossed to C57/BL6) or wild-type ( + /+) mice were produced by A. Michalska and K.H.A. Choo of the Murdoch Institute, Royal Childrens Hospital, Parkville, Australia (Michalska and Choo, 1993) and were maintained at our institution. All animal procedures were approved by the Institutional Animal Care and Use Committee. Isolated islets were either used immediately as freshly isolated islets, or they were cultured for 24 h in 35 mm culture dishes (:60 islets/dish) with 2 ml CMRL-1066 medium containing 5.5 mM glucose, 9% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) at 5% CO2 –95% air, and 35°C, as described previously (Xia and Laychock, 1993). Culture media was changed every second day. The clonal insulinoma cell line bHC9 was cultured in DMEM, and RINm5F cells were cultured in RPMI1640, as described previously (Lee et al., 1998). Culture media was supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml) at 5% CO2 –95% air, at 35°C. Other additions to the culture media were as described in the text, and the cells were cultured for 24 h except where indicated.
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2.3. Insulin content and release Total islet insulin content was determined by extracting islets with ethanol (70%) in HCl (1 N) overnight at − 20°C, and determining insulin in a small aliquot by insulin radioimmunoassay. Insulin release was determined by incubating islets (10 islets/sample) in Krebs Ringer bicarbonate buffer (pH 7.4) containing 16 mM Hepes (KRBH buffer) and 0.01% BSA at 5.5 mM glucose for 60 min, followed by a 60 min incubation in fresh KRBH buffer with various glucose concentrations indicated in the text. Aliquots of incubation medium were removed at time 0 and after the 60 min incubation for assay of insulin by radioimmunoassay. Insulin release was expressed as insulin released after 60 min minus 0-time insulin levels.
2.4. Metallothionein assay MT levels were determined following lysis of cells or islets with brief sonication in 10 mM Tris buffer at pH 7.4 containing 0.1 or 1 mg cadmium (220 ml). An aliquot of lysate was used to determine protein content by Bio – Rad protein assay, using BSA as standard. Lysates were microcentrifuged briefly to remove particulate matter, and 200 ml were used for MT determination by cadmium–hemoglobin affinity assay, essentially as described previously (Eaton and Cherian, 1991). Lysate or blank buffer was mixed with 109Cd (0.5 mCi/ml Tris buffer without cadmium) and incubated at RT for 10 min. Then, 100 ml 2% bovine hemoglobin solution in Tris buffer without cadmium was added to each sample and vortexed. The samples were subsequently boiled for 4 min, vortexed, and then centrifuged to pellet the denatured hemoglobin. Supernatant (350 ml) was transferred to another tube containing 100 ml fresh hemoglobin solution, votexed and boiled for 3 min, vortexed again, and centrifuged. The supernatant (350 ml) was transferred to tubes for gamma counting. The specific activity of the 109Cd was determined, and the number of nmol cadmium bound in MT was calculated and corrected for to the total starting volume of lysate. Assuming 1 nmol cadmium bound is equivalent to 1 mg MT, data were expressed as number of nanograms MT per microgram protein.
2.5. Semiquantitati6e re6erse-transcriptase polymerase chain reaction (RT-PCR) Relative levels of metallothionein messenger (m)RNA were determined in isolated rat islets (80–120 islets per reaction). Total RNA was extracted and reverse transcribed essentially as reported previously (Lee et al., 1998). The resulting complimentary (c)DNA was then amplified, as indicated with the following (+) and (−) strand oligonucleotide primers, respectively:
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metallothionein 1 and 2 (400 bp) 5%-ctcaagaagaccatctgcctc-3% and 5%-cagcttgacaccaatgttcc-3%; cyclophilin (490 bp) 5%-gtgttcttcgacatcacgg-3% and 5%-gaacttcagtgagagcagag-3%. Polymerization reactions were carried out in a Hybaid Sprint thermalcycler using a 1:5 dilution of cDNAs as templates in a 25 ml reaction volume, as described previously (Lee et al., 1998). The amplification conditions were 26 cycles with denaturation for 4 min at 94°C, annealing for 1 min at 60°C, extension for 2 min at 72°C with the final extension for 7 min. The amounts of cDNA chosen were within a linear range for amplification. The reaction products were separated by agarose gel electrophoresis, and the gel was stained with CYBER Green and viewed by densitometry. The image density of metallothionein mRNA was compared with the density of co-amplified cyclophilin mRNA to determine the relative level of metallothionein expression in each experimental sample. DNA sequencing confirmed the RT-PCR reaction product to be 99% identical to the metallothionein 1 and 2 reported sequence in GenBank.
2.6. Statistical analysis The data are presented as the mean 9 SE and were analyzed by Student’s t test, or one-way analysis of variance (ANOVA) with Student’s/Newman –Keuls multiple comparison test; PB0.05 was accepted as significant.
3. Results
3.1. Isolated islet MT le6els in response to dexamethasone Isolated rat pancreatic islets were cultured in the absence and presence of dexamethasone in order to determine glucocorticoid effects on MT levels. Dexamethasone increased islet MT at all concentrations tested (0.01–1 mM) (Fig. 1A). The effect of dexamethasone was also time-dependent and a significant increase in MT occurred within 8 h (Fig. 1B). It was also determined by RT-PCR that dexamethasone (0.1 mM) increased islet MT mRNA levels by 1289 6% of control (PB 0.05; n= 3) after 6 h. Dexamethasone also induced MT in wild-type MT + /+ mouse islets (Table 1). In contrast, MT levels in islets from MT-null mice were at the limit of detectability for the assay in control islets (0.0059 0.001 ng MT/mg protein) and were only 3% of the levels observed in wild-type islets (PB0.001) (Table 1). Dexamethasone failed to elicit any significant changes in MT levels compared to control in MT − /− islets (data not shown). These data indicate that 97% of the MT measured by the cadmium–hemoglobin affinity assay was
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specific for MT-1 and/or 2. The very low levels of cadmium binding detected in MT-null mouse islets may be due to other isoforms of MT, low levels of other metal binding proteins, or low-molecular weight thiols such as cyteine and glutathione which can affect the cadmium– hemoglobin affinity assay (Eaton and Cherian, 1991).
3.2. Islet MT le6els in response to cytokines
Fig. 1. Rat islet MT response to dexamethasone. (A) Isolated islets were cultured for 24 h in the absence (control) and presence of dexamethasone (DEX) at the concentrations (mM) indicated. (B) Islets were cultured for up to 24 h in the absence (control) and presence of dexamethasone (1 mM). Control islet values were 0.00599 0.0008 ng MT/mg protein. Values are the mean 9 SE for the number of independent determinations shown at the base of each bar. *, PB 0.02 versus control as determined by one-way ANOVA and multiple comparison test.
Table 1 Metallothionein in isolated mouse isletsa Treatment
+/+ mouse islet MT levels
Control DEX IL-1b TNF+IL-1b TNF+IL-1b+DEX Zn
0.1559 0.025 0.4339 0.112** 0.1719 0.040 0.2549 0.065* 0.5129 0.126** 0.1159 0.031
a
(7) (4) (3) (4) (4) (3)
Islet of Langerhans were isolated from pancreata of wild-type (MT +/+) mice. Islets were cultured for 24 h in the absence (control) or presence of dexamethasone (DEX; 1 mM), TNF (10 ng/ml), IL-1b (1 ng/ml), or Zn (0.1–0.5 mM). Values are the mean 9 SE for ng MT/mg protein. Number of independent experiments are shown in parentheses. * PB0.05. ** PB0.01 versus control, as determined by one-way ANOVA and post-hoc analysis.
Rat islets were cultured for 24 h in the absence and presence of the cytokines interleukin-1b (IL-1b) and tumor necrosis factor a (TNF) in order to determine effects on MT expression. IL-1b, but not TNF, elicited an increase in MT levels similar to the increase observed in response to dexamethasone (Fig. 2). However, when IL-1b and TNF were added to islets together with dexamethasone there was a synergistic effect on MT induction and the response was especially evident with the combination of IL-1b and dexamethasone (Fig. 2). When rat islet MT mRNA expression was determined, dexamethasone (1 mM) and IL-1b (1 ng/ml) increased MT mRNA levels to 133911% of control (0.2309 0.047 MT mRNA/cyclophilin mRNA) (PB 0.05; n=5) within 4 h after beginning treatment. Rat islet MT levels also increased in response to the MT inducing agent, Zn (Fig. 2). Mouse islets were also found to contain MT at levels comparable to rat islets (Table 1). Wild-type mouse islets cultured with IL-1b did not show an increase in MT levels compared to control islets (Table 1). The combination of IL-1b and TNF elicited a small increase in MT levels (Table 1). However, the combination of IL-1b, TNF, and dexamethasone failed to significantly increase MT levels above the level elicited by dexamethasone alone (Table 1). Zn failed to elicit a significant change in mouse islet MT (Table 1). In islets from MT-null mice, dexamethasone (1 mM) in the presence of the cytokines IL-1b (1 ng/ml) and TNF (10 ng/ml), failed to elicit any significant changes in MT synthesis after 24 h culture (data not shown). Zn was also not an effective stimulus in MT-null islets (0.003 90.002 ng MT/mg protein).
3.3. Insulinoma cell MT responses The insulinoma cell lines for rat (RINm5F) and mouse (bHC9) were investigated for MT responsiveness since they represent a homogeneous population of b-cells, unlike islets which are a heterogeneous collection of three types of endocrine cells, other peptide secreting cells, endothelial cells and neuronal tissue. Similar to isolated islets, dexamethasone elicited a concentration-dependent increase in MT levels in bHC9 cells (Fig. 3A). The response to dexamethasone was also maximal within 24 h (Fig. 3B). RINm5F cells also responded to dexamethasone with increased MT levels (Fig. 4).
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Fig. 2. Rat islet MT induction by cytokines and dexamethasone (DEX). Islets were cultured for 24 h in the absence (basal) or presence of IL-1b (IL) (1 ng/ml), TNF (200 ng/ml), dexamthasone (DEX) (1 mM), or Zn (100 mM). Values are the mean 9SE for the number of independent determinations shown at the base of each bar. Significant differences were determined by one-way ANOVA and multiple comparison test. *, P 50.05 versus basal.
IL-1b also increased MT levels in RINm5F and bHC9 cells by about 50% (Figs. 4 and 5), and maximal effects were observed after 24 h (data not shown). RINm5F cells responded to a combination of dexamethasone and IL-1b with a synergistic increase in MT similar to that noted for rat islets (Fig. 4); bHC9 cells showed a similar response (data not shown). Inhibitors of inducible nitric oxide synthase (iNOS), NMMA and aminoguanidine, failed to inhibit the MT response to IL-1b in bHC9 cells (Fig. 5). Neither NMMA (0.5 mM) nor aminoguanidine (0.5 mM) had any significant effect on MT levels in control cells (data not shown). In addition, the nitric oxide generating agent, sodium nitroprusside, failed to significantly affect MT levels in bHC9 cells (Fig. 5).
Fig. 3. Dexamethasone effects on MT in bHC9 cells. (A) Cells were cultured 24 h in the absence (control) or presence of different concentrations (mM) of dexamethasone (DEX) as indicated. (B) Cells were cultured for up to 48 h in the absence (control) or presence of dexamethasone (1 mM). Control values were 0.054 90.027 ng MT/mg protein.Values are the mean 9SE for the number of independent determinations shown at the base of each bar. *, P B0.05 versus control; , PB0.01 versus control, as determined by one-way ANOVA and multiple comparison test.
3.4. Metallothionein expression in response to phorbol ester Since IL-1b effects in islets are accompanied by increased protein kinase C (PKC) activity (Eizirik et al., 1995), islet MT responses to a PKC activating phorbol ester, PDBu, were investigated to determine if PKC mediates changes in MT expression. Rat islets incubated with PDBu for 24 h showed a doubling of MT content compared to control islets (Fig. 6). bHC9 cells also responded to PDBu with increased MT content (Fig. 7). However, the combination of IL-1b and PDBu did not elicit an increase in MT greater than that elicited by either agent alone (Fig. 7).
Fig. 4. MT responses to IL-1b (IL) and dexamethasone (DEX) in RINm5F cells. Cells were cultured for 24 h in the absence (basal) or presence of IL (1 ng/ml) and/or DEX (1 mM). Values are the mean9 SE for three to seven independent determinations. Significant differences were determined by one-way ANOVA and multiple comparison test; *, PB0.05 versus basal.
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Fig. 5. Effects of iNOS inhibitors and sodium nitroprusside (SNP) on bHC9 cell MT levels. Cells were cultured for 24 h in the absence (basal) or presence of IL-1b (IL) (1 ng/ml), NMMA (500 mM), aminoguanidine (AG) (500 mM), and SNP (100 mM) as indicated. Control values were 0.12 9 0.06 ng MT/mg protein. Values are the mean9 SE for the number of independent determinations shown at the base of each bar. *, P B0.05 versus control as determined by paired t-test for each treatment group versus basal.
Fig. 6. Rat islet MT response to PDBu. Islets were cultured for 24 h in the absence (control) or presence of PDBu (1 mM) as indicated. Values are the mean9 SE for the number of independent determinations shown at the base of each bar. *, PB 0.002 versus control as determined by paired t-test.
glucose) and maximally secretagogic glucose (17 mM) concentrations in order to determine if the insulin secretory responses of wild-type and MT-null mouse islets differed. A markedly reduced release of basal insulin was observed for the MT-null mouse islets (Fig. 8). Stimulation with 17 mM glucose increased wild-type mouse islet insulin release by almost 3-fold, whereas stimulation of MT-null islets increased insulin release almost 9-fold (Fig. 8). Even though 17 mM glucose increased MT-null islet insulin release above control, the level remained significantly lower than that released by glucose-stimulated wild-type islets (Fig. 8). 4. Discussion MT is found in high levels in the pancreas (Nath et al., 1988), however, controversy exists regarding the presence and inducibility of MT in endocrine pancreatic islets of Langerhans from the rat and mouse (Onosaka et al., 1988; Andrews et al., 1990; Zimny et al., 1993; Ohly and Gleichmann, 1995; De Lisle et al., 1996; Minami et al., 1999; Ohly et al., 1999). The present study shows not only that rat and mouse isolated pancreatic islets contain comparable amounts of MT, but also that b-cells which synthesize and package insulin for secretion contain MT as well as the mRNA for MT. These findings are in agreement with the immunohistochemical localization of MT in rat and mouse islets (Zimny et al., 1993; Ohly et al., 1999). And, for the first time we show that MT is induced in both rat and mouse islets and rat and mouse b-cell lines in response to dexamethasone, a primary inducer for MT gene transcription (Moffatt and Denizeau, 1997). In addition, MT mRNA expression was rapidly increased in response to dexamethasone. The MT response to dexamethasone was sensitive to low concentrations of the hormone, and showed a time-dependency consistent with the rapid increase in MT gene transcription.
Fig. 7. bHC9 cell MT response to IL-1b (IL) and PDBu. Cells were cultured for 24 h in the absence (basal) or presence of IL (1 ng/ml) and PDBu (1 mM) as indicated. Basal values were 0.138 9 0.074 ng MT/mg protein. Values are the mean 9 SE for the number of independent determinations shown at the base of each bar. *, PB 0.05 versus basal as determined by Student’s paired t-test.
3.5. Insulin release in islets of wild-type mice and MT-null mice The insulin content of mouse islets was quantitated to determine if MT-null mouse islets differed from wild-type islets. The insulin contents of islets from MT-null (1709 16 ng insulin/five islets) and wild-type (168925 ng insulin/5 islets) mouse islets were similar (P \ 0.05). Islets were also incubated at basal (5.5 mM
Fig. 8. Insulin release from MT-null and wild-type mouse islets. Isolated islets were incubated for 60 min in the presence of 5.5 mM glucose (basal) or 17 mM glucose (G17). Insulin release was determined by radioimmunoassay and normalized per five islets. Values are the mean 9 SE for seven determinations. Significant differences were determined by one-way ANOVA and multiple comparison test.
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Rat islet MT levels were also shown to be modestly responsive to the cytokine IL-1b, but not to TNF. This is consistent with the marked effects of IL-1b on increasing nitric oxide (NO) synthesis and inhibiting rat islet insulin secretion responses, but also of the relative lack of effect of TNF alone in isolated islets (Corbett et al., 1993a). RINm5F cells also showed a small but significant increase in MT in response to IL-1b. However, both the rat islet and RINm5F cells showed evidence that IL-1b and TNF act synergistically with dexamethasone in eliciting a large increase in MT levels. An early increase in metallothionein mRNA confirmed the effect of these agents on metallothionein expression. Cytokines have been implicated previously in inflammation-associated increases in MT (Schroeder and Cousins, 1990; Moffatt and Denizeau, 1997). It has also been reported that dexamethasone caused a significant protection against IL-1b in terms of maintaining insulin secretion in rats treated with the cytokine (Shimizu et al., 1992). Whether this protective effect could be due to enhanced MT induction is not known. However, copper, which induces MT synthesis (Moffatt and Denizeau, 1997), can prevent the inhibitory effects of IL-1b on islet insulin secretion in vitro (Vinci et al., 1995). Dexamethasone also antagonizes the inhibitory effects of IL-1b on insulin secretion from islets in vitro (Corbett et al., 1993b), and whether this is due to a direct effect on inducible nitric oxide synthase (iNOS) or to a combined effect on other protective proteins such as MT can only be speculated upon at this time. In contrast to rat islets, mouse islets did not respond to individual cytokine exposure with changes in MT and only the combination of IL-1b and TNF increased MT. TNF has previously been reported to potentiate IL-1b effects in islets (Corbett et al., 1993a). It has been reported that islets contain low levels of antioxidant enzymes compared to other tissues (Lenzen et al., 1996; Tiedge et al., 1997) and that mouse islets are lower in catalase and superoxide dismutase activities than rat islets (Welsh et al., 1995). It is perhaps differences between the species ability to generate or dispose of certain free radicals which accounts for differences in MT induction in response to cytokines in islets. One of the most prominent differences between mouse and rat islets was the failure of mouse islets to display any synergy between dexamethasone and IL-1b and TNF on MT levels. Another prominent feature of mouse islets was their failure to respond to high concentrations of Zn with changes in MT levels. Other investigators have observed a similar lack of response by mouse islets (Minami et al., 1999). Clearly, the cytokines induce changes in MT levels by a different mechanism than glucocorticoids in rat islets, and there are major differences in MT regulation in rat and mouse islets related to cytokines and metals. The murine bHC9 cell line appeared more responsive to cytokine stimulation
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than did the mouse islets, and IL-1b not only increased MT levels to a small extent but also potentiated the dexamethasone response in both rat and mouse cell lines. Differences between mouse primary islet cells and tumor cell line responses may be due to transcriptional factor differences or differences in antioxidant enzymes which might impact on MT induction (Tiedge et al., 1997). Since lysosomes degrade MT (Moffatt and Denizeau, 1997) it is possible that MT levels are also regulated in this manner, although there is no reported evidence of this being a regulated phenomenon. The transcriptional regulation of MT is not well known. Metals such as Zn stimulate the binding of protein transcription factors to a metal regulatory element (MRE) to enhance MT synthesis (Moffatt and Denizeau, 1997). Based upon results in the present study, the mouse MT promoter MRE appears to be less responsive to Zn stimulation than that of the rat islet. Glucocorticoid receptor activation of MT synthesis relies on binding to a glucocorticoid responsive element (GRE) sequence of the MT promoter. Another sequence, 12-o-tetradecanoylphorbol-13-acetate responsive element (TRE), mediates induction of MT by phorbol esters which activate PKC. In addition, TRE binds AP-1 which probably mediates phorbol-induced MT transcription. Another recognition site sequence is for activator protein AP-2, whose transcriptional activity is stimulated by phorbol ester and cyclic AMP through stimulation of protein kinase A. The antioxidant response element (ARE) mediates induction of MT by hydrogen peroxide. However, the TRE sequence is an integral part of the ARE site and confers crossover among the stimulatory ligands. In an effort to determine the mechanism behind the synergy between cytokines and dexamethasone in rat islets, the mechanism accounting for cytokine stimulation of MT synthesis was sought. It is well known that a major response to IL-1b stimulation of b-cells is an increase in iNOS which increases the free radical NO production in these cells (Corbett et al., 1993a). However, in the present study two inhibitors of iNOS, the L-arginine analog NMMA and an enzyme inhibitor aminoguanidine (Misko et al., 1993; Corbett et al., 1993b), failed to significantly alter the MT response to IL-1b even though they have been reported to inhibit iNOS activity in islets (Corbett et al., 1991, 1992). Moreover, the generation of NO with sodium nitroprusside did not significantly alter insulinoma cell MT levels, suggesting that NO is not an important mediator of the MT response. Sodium nitroprusside at the concentration used in this study has previously been shown to induce another stress protein, heme oxygenase-1, within 20 h in islets (Ye and Laychock, 1998). Thus, a mechanism other than NO generation appears to mediate the MT response in b-cells.
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A proposed mechanism of action of IL-1b and TNF is the activation of PKC and nuclear factor (NF)-kB (Barnes and Karin 1997). IL-1b increases NF-kB activation in b-cells (Kwon et al., 1995). Pathways mediating IL-1b and TNF action include the activation of PKC (Schu¨tze et al., 1990; Bergmann et al., 1998; Boussouar et al., 1999), perhaps including atypical forms of PKC (Bonizzi et al., 1999) and the phosphorylation of the inhibitory IkB (Scherer et al., 1995) with subsequent NF-kB activation. In islets, IL-1b stimulates PKC activation (Eizirik et al., 1995). Phorbol ester stimulation of PKC activation in this study was undertaken to determine if this enzyme mediates changes in MT synthesis observed in islets. As the results showed, PDBu increased MT synthesis in rat islets and bHC9 cells to about the level observed with IL-1b. However, the combination of IL-1b and PDBu were not additive, suggesting that they were affecting MT synthesis by a similar mechanism. Presumably, both agents elicit a response through the TRE element mediating PKC responses. MT-null mice were investigated for their insulin secretory responses in order to determine if MT might play a physiological role in this process. Although isolated islets from wild-type and MT-null mice had similar insulin content, the islets from the MT-null animals showed much lower levels of basal insulin release. In addition, although glucose stimulated insulin release from islets of MT-null mice, the maximal insulin secreted was significantly lower than that released by wild-type islets. MT as a target for reactive oxygen species might scavenge superoxide and hydroxyl radicals and preserve membrane integrity, protein sulfhydryls or nucleic acids, and so also secretory responsiveness. In summary, MT has been identified in rat and mouse islets to be regulated by dexamethasone. MT induction in rat islets also appeared to be responsive to cytokine and Zn stimulation, whereas mouse islets lacked sensitivity to these agents in this study. There also appears to be a unique mechanism in rat islets which allows for synergy between dexamethasone and cytokines for MT induction which is lacking in mouse islets. In addition, MT appears to be required for normal b-cell insulin secretory responsiveness. It remains to be determined how MT impacts on insulin secretory mechanisms, however, the antioxidant properties of MT (Ebadi et al., 1996) and its proposed target in mitochondial function (Simpkins et al., 1996, 1998) may play a role in insulin secretion and glucose responsiveness.
Acknowledgements This work was supported by NIH grant DK-25705 (awarded to S.G.L.). The technical assistance of Maureen Adolf and Shawn Sessanna is appreciated.
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