Báo cáo khoa học: Pituitary adenylate cyclase-activating polypeptide attenuates streptozotocin-induced apoptotic death of RIN-m5F cells through regulation of Bcl-2 family protein mRNA expression pdf

Pituitary adenylate cyclase-activating polypeptide attenuates streptozotocin-induced apoptotic death of RIN-m5F cells through regulation of Bcl-2 family protein mRNA expression Satomi Onoue, Junko Hanato and Shizuo Yamada Department of Pharmacokinetics and Pharmacodynamics and Global Center of Excellence Program, School of Pharmaceutical Sciences, University of Shizuoka, Japan Type 2 diabetes has been identified as a constellation of metabolic disorders, including insulin resistance, impaired control of hepatic glucose production, and b cell dysfunction [1]. In particular, b cell apoptosis is core to the pathophysiology of diabetes mellitus [2] because a loss of b cell mass resulting from an increase in b cell death was reported to be an important contributor to the evolution of the diabetic state [3]. Islets are more susceptible to damage from oxidative stress than other tissues due to their extremely low expression of oxygen radical metabolizing enzymes, such as manganese superoxide dismutase, catalase and glutathione peroxidase [4]. In this context, the protection of pancreatic b cells from glucotoxicity, Keywords apoptosis; PACAP; RIN-m5F cells; streptozotocin Correspondence S. Onoue, Department of Pharmacokinetics and Pharmacodynamics and Global Center of Excellence Program, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan Fax: +81 569 74 4748 Tel: +81 569 74 4855 E-mail: onoue@u-shizuoka-ken.ac.jp (Received 21 July 2008, revised 2 September 2008, accepted 8 September 2008) doi:10.1111/j.1742-4658.2008.06672.x Oxidative stress, followed by the apoptotic death of pancreatic b cells, is considered to be one of causative agents in the evolution of the type 2 dia- betic state; therefore, the protection of b cells can comprise an efficacious strategy for preventing type 2 diabetes. In the present study, RIN-m5F cells (i.e. the rat insulinoma b cell line) were stimulated with streptozotocin, resulting in a time- and concentration-dependent release of lactate dehydrogenase. There appeared to be significant apoptotic cell death after 2 h of treatment with streptozotocin at 10 mm, as demonstrated by termi- nal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining and 2.6-fold activation of cellular caspase-3, an apoptotic enzyme. By con- trast, some neuropeptides of the glucagon-secretin family and coenzyme Q 10 , an endogenous mitochondrial antioxidant, could attenuate streptozo- tocin cytotoxicity, and especially pituitary adenylate cyclase-activating polypeptide (PACAP), at a concentration of 10 )7 m, exhibited 34% attenu- ation of lactate dehydrogenase release from streptozotocin-treated RIN- m5F cells. Quantitative RT-PCR experiments indicated the inhibitory effect of PACAP on streptozotocin-evoked up-regulation of pro-apoptotic factor (Noxa and Bax) and a 2.3-fold enhancement of Bcl-2 mRNA expression, a pro-survival protein, was also observed after addition of PACAP. The data obtained suggest the anti-apoptotic role of PACAP in streptozotocin- treated RIN-m5F cells through the regulation of pro-apoptotic and pro-survival factors. Abbreviations CoQ 10 , coenzyme Q 10 ; GLP, glucagon-like peptide; LDH, lactate dehydrogenase; PACAP, pituitary adenylate cyclase-activating polypeptide; STZ, streptozotocin; TdT, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end- labeling; VIP, vasoactive intestinal peptide. 5542 FEBS Journal 275 (2008) 5542–5551 ª 2008 The Authors Journal compilation ª 2008 FEBS lipotoxicity, inflammation and oxidant stress is thought to be effective in preventing or delaying the onset of the disease [5]. Oxidative stress followed by apoptotic cell death is a dynamic process that reflects an imbalance between pro-oxidant and antioxidant factors [6] and is partly involved in the pathogenesis of cancer, cardiovascular diseases, and even the amyloidoses, which include b-amyloid (Ab) in Alzheimer’s disease [7], amylin in type 2 (non-insulin-dependent) diabetes mellitus [8], and prion protein in Creutzfeldt–Jakob disease and spongi- form encephalopathy [9]. We have previously demon- strated that NMDA-type glutamate-receptor agonists [10] and misfolded b-amyloid and prion protein frag- ments [11,12] are potent neurotoxins in neuronal cells, with the mechanism of their effects possibly being related to oxidative stress and apoptosis. Interestingly, the neurotoxicity of these toxic agents in neuronal cells was attenuated by neuropeptides, including vasoactive intestinal peptide (VIP) [13] and pituitary adenylate cyclase-activating polypeptide (PACAP) [14–16], and their neuroprotective effects were associated with the deactivation of caspase-3, an apoptotic enzyme. In humans and rats, VIP and related neuropeptide- immunoreactive nerves have been identified in intra- pancreatic ganglia as well as in close association with the islets of Langerhans [17]. PACAP-like immuno- reactivity was also observed in rat pancreatic tissues, including nerve fibers, blood capillaries and islets [18]. These previous observations provide motivation to conduct studies into the protective effects of these neuropeptides, as well as the family peptides, on the apoptotic death of b cells. We previously demonstrated that exposure to streptozotocin (STZ) induced signifi- cant cytotoxicity and apoptotic cell death in RIN-m5F cells (i.e. the rat insulinoma cells) in which some recep- tors for glucagon-secretin family peptides were being expressed (Fig. 1). In addition, the protective effects of neuropeptides on STZ-induced cell death have been further characterized in relation to the participation of anti-apoptotic signaling pathways in these cells. The anti-apoptotic effects of coenzyme Q 10 (CoQ 10 ), as a positive control, were also evaluated and compared with those of neuropeptides because CoQ 10 could pre- vent high glucose- or STZ-induced oxidative stress in several tissues and cells [19,20]. Results and Discussion Cytotoxicity of streptozotocin in RIN-m5F cells In the present study, we initially investigated the early mechanisms implied in b cell death with use of the RIN-m5F cell line, a model currently used for the study of pancreatic cell death [21]. RIN-m5F cells have been identified as continuous cell lines that retain many features indicative of these pancreatic islet b cell lineage, including toxic responses [21,22]. STZ has been identified as a potent DNA methylating agent and acts as a free radical donor in the pancreas in which b cells are sensitive to oxidative damage because of low expression of free radical scavenging enzymes [4]. To induce oxidative stress and subsequent apopto- tic cell death, RIN-m5F cells were exposed to STZ for various periods, followed by the measurement of cyto- toxicity as determined by released lactate dehydro- genase (LDH) as a simple and reproducible screening method. The extent of cell death could be assessed by the measurement of LDH released from dead cells, due to the loss of cell membrane integrity observed in both necrotic and apoptotic cells. The long-term exposure of RIN-m5F cells to STZ at concentration of > 10 mm induced marked cell death by as early as 12 h, and there was no significant increase of LDH release from RIN-m5F cell thereafter (data not shown). Shorter incubation times were then under- taken to examine whether STZ might be deleterious for RIN-m5F cells following a shorter period of incu- bation. Preincubation of RIN-m5F cells with STZ at various concentrations for 2 h, followed by replace- Fig. 1. Amino acid sequences of glucagon- secretin family peptides. All peptides were chemically synthesized and amidated at the C-terminus, except for glucagon. S. Onoue et al. PACAP attenuates streptozotocin-induced apoptotic death FEBS Journal 275 (2008) 5542–5551 ª 2008 The Authors Journal compilation ª 2008 FEBS 5543 ment of medium by freshly prepared STZ-free med- ium, resulted in the concentration (1.25–20 mm)- and time (12–48 h)-dependent release of cellular LDH activity into the culture medium (Fig. 2A). However, no significant cytotoxicity was observed in RIN-m5F cells treated with STZ (1.25–20 mm) for 1 h (data not shown). The extracellular LDH activity released by STZ at a concentration of 10 mm for 48 h was equi- valent to 62.3 ± 6.3% of the total LDH activity in RIN-m5F cells. Type 1 and 2 diabetes are characterized by progres- sive b cell failure, and b cell necrosis and apoptosis are thought to be key regulatory elements in the patho- physiology of diabetes. Type 1 diabetes mellitus is associated with b cell necrosis, whereas type 2 diabetes is associated more with accelerated apoptosis [2]. The biochemical features of apoptosis include the activa- tion of one or more cysteine proteases of the caspase family [23]. To examine the possible involvement of caspase-3 in the STZ-induced cell death in RIN-m5F cells, we measured caspase-3-like activity in cell lysates via cleavage of the fluorometric caspase-3 substrate Z-DEVD-rhodamine 110 [24]. Following the pre-treat- ment of RIN-m5F cells with STZ (10 mm) for 2 h, caspase-3 activity increased significantly prior to the loss of membrane integrity, and maximal enhancement (260% of control) was observed after 9 h of incubation (Fig. 2B). Caspase-3 activity returned to the basal level after 48 h of incubation with STZ. These results indi- cated that 2 h of exposure of RIN-m5F cells to STZ induced a rapid and significant elevation in the cas- pase-3 activity within 9 h, which preceded the loss of cell viability. The activation of caspase-3 is required for the early stage of apoptosis, and this could be one reason for the time lag between activation of caspase-3 and loss of cell viability. In addition, RIN-m5F cells exposed to 10 mm STZ for 2 h clearly showed the morphological hallmarks of apoptosis, such as cellular shrinkage, cell surface smoothing, nuclear compaction, chromatin condensation at the periphery of the nuclear envelope, and fragmentation of nuclei as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining (Fig. 3). In the control condition, these events were rare or absent. Fig. 2. STZ-cytotoxicity in RIN-m5F cells. (A) LDH release from RIN-m5F cells pretreated with STZ. RIN-m5F cells were preincubat- ed with STZ at several concentrations for 2 h, then changed by freshly prepared culture medium. Extracellular LDH was assessed at 12, 24 and 48 h. d, Control (vehicle alone); 4, STZ at 1.25 m M; ,, 2.5 m M; ), 5.0 mM; s,10mM; and h,20mM. The results are the mean ± SE of four experiments. **P < 0.01 and *P < 0.05 with respect to the control group. (B) Time course of caspase-3 activity in cytosolic protein extracts from RIN-m5F cells pretreated with STZ. RIN-m5F cells were exposed to STZ (10 m M) and lysed for the indicated period. Caspase-3-like protease activity was deter- mined by the cleavage of the fluorometrical caspase-3 substrate, Z-DEVD-rhodamine 110. The data are expressed as a percentage of the control value (mean ± SE of four experiments). *P < 0.05 and **P < 0.01 with respect to the control group. AB Fig. 3. Induction of apoptosis in RIN-m5F cells by STZ. After pre- treatment with STZ at 10 m M, RIN-m5F cells were incubated for 48 h. Apoptosis was evaluated with the TUNEL method using the 3,3¢-diaminobenzidine reaction. (A) Control and (B) pretreated with STZ. Scale bar = 50 lm. PACAP attenuates streptozotocin-induced apoptotic death S. Onoue et al. 5544 FEBS Journal 275 (2008) 5542–5551 ª 2008 The Authors Journal compilation ª 2008 FEBS On the basis of the data presented, STZ has an ability to induce both necrotic and apoptotic cell death in RIN-m5F cells; however, apoptotic cell death might occur in RIN-m5F cells that are exposed to STZ for short period. Protective effects of neuropeptides on STZ-induced cell death VIP and related-peptides have been shown to potentiate glucose- and arginine-induced insulin release in anaesthetized rats, as well as insulin release from perfused newborn rat pancreas [25,26]. In particular, PACAP in the subpicomolar range activates islet b cells and stimulates insulin release in a glucose-dependent manner, suggesting the role of PACAP as neuronal and ⁄ or hormonal regulator of the glucose-induced insu- lin secretion [18]. These observations were indicative of expression of some specific receptors for glucagon- secretin family peptides in RIN-m5F cells. An RT-PCR experiment was performed to demonstrate the expres- sion of the neuropeptide receptors in RIN-m5F cells. Using specific primers for the receptors of glucagon- secretin family peptides, including glucagon-like peptide (GLP)-1, glucagon, secretin and PACAP ⁄ VIP receptors (PAC1, VPAC1, VPAC2), distinct RT-PCR products of predicted size for the PAC1 (290 and 371 ⁄ 374 bp), VPAC1 (299 bp), VPAC2 (326 bp), GLP-1 (190 bp), and glucagon (407 bp) receptors were obtained from RIN-m5F cells (Fig. 4). PCR products were barely detectable with primers for the secretin receptor, whereas the primers were effective in generating prod- ucts for the secretin receptors in the rat brain [27] and lung (data not shown). In parallel control experiments without reverse transcription, PCR products for the b actin and neuropeptide receptors were barely detect- able, indicating that the amplified receptor product was not derived from contaminating genomic or mitochon- drial DNA. This result is consistent with previous reports on numerous functions of glucagon-secretin family peptides in b cells [25,26]. It is well established that PACAP and some family peptides protected neuronal cells against apoptosis induced by different neurotoxins, such as free radicals [28] and glutamate receptor agonists [29]. With respect to the free radicals, it was shown that neuropeptides can serve as an effective scavenger ⁄ quencher of some radicals, including singlet oxygen and peroxyl radicals. Herein, the modulation of radical-induced oxidative tissue injury was assumed to be involved in the protec- tive effect of neuropeptides against the oxidative stress. In the present study, the effects of glucagon-secretin family peptides on the STZ-induced cytotoxicity were examined with the LDH assay. Although preincuba- tion with STZ (10 mm) alone for 2 h resulted in a marked decrease in cell viability, the co-exposure of RIN-m5F cells with some neuropeptides at concentra- tions of 10 )7 m attenuated the cytotoxicity of STZ (Table 1). Of all glucagon-secretin-family peptides tested, PACAP38 at 10 )7 m exhibited the highest activ- ity, with approximately 34% protection against the STZ-induced LDH release (Fig. 5). PACAP27 at 10 )7 m also showed 22% attenuation of STZ-induced cytotoxicity. Although PACAP27 and PACAP38 exhibited similar affinity with PAC1 receptor, PACAP38 sometimes had a longer-lasting biological activities [30,31]. Several factors might be associated with the pharmacological differences between Fig. 4. RT-PCR analysis of neuropeptide receptor mRNAs in RIN- m5F cells. Total RNA was reverse transcribed in the absence (RT)) and presence (RT+) of reverse transcriptase and PCR amplified with primer pairs specific for the PAC1, VPAC1, VPAC2, GLP-1, glu- cagon and secretin receptors, and for b-actin (control). Ethidium bromide-stained 2% agarose gels are shown. The data shown are representative of three experiments. Table 1. Protective effects of glucagon-secretin family peptides in STZ-treated RIN-m5F cells. Each data represents the mean ± SE of four experiments. *, < 0.05, **, < 0.01 with respect to control (vehicle alone). Inhibition of LDH release (%) Neuropeptides (10 )7 M) PACAP38 33.7 ± 2.4** PACAP27 22.1 ± 5.7** VIP 7.3 ± 4.0* Secretin 12.9 ± 3.9** Glucagon 14.2 ± 3.0** Helodermin 14.9 ± 4.3** GLP-1 19.2 ± 5.3** GLP-1(7–37) 14.2 ± 7.1* Control (5 · 10 )6 M) Coenzyme Q 10 25.1 ± 5.0** S. Onoue et al. PACAP attenuates streptozotocin-induced apoptotic death FEBS Journal 275 (2008) 5542–5551 ª 2008 The Authors Journal compilation ª 2008 FEBS 5545 PACAPs, and Nokihara et al. [32] demonstrated that differences in the duration of action can be attributed to the structural differences, particularly the C-termi- nal extended helical structure. In a previous study, PACAP27 demonstrated a bell-shaped concentration– response curve for the neuroprotective effect on the b-amyloid- and prion protein fragment-induced apop- tosis of PC12 cells, whereas VIP displayed far weaker neuroprotection [11,12]. In addition to the neuro- peptides, the protective effect of CoQ 10 against STZ-cytotoxicity was also assessed as positive control because of its antioxidant properties with significant attenuation of STZ-evoked oxidative stress in some tissues and cultured cells [19]. Incubation of RIN-m5F cells with CoQ 10 for 48 h resulted in a significant con- centration-dependent increase in viability of up to 25% at 5 · 10 )6 m (Fig. 5). Considering these findings together with the effective concentration of CoQ 10 in the current assay, some glucagon-secretin family peptides, especially PACAPs, were found to be effica- cious in preventing STZ-evoked cytotoxicity in RIN-m5F cells, possibly due to their anti-apoptotic potentials at extremely low concentration. PACAP-evoked alteration of apoptotic signaling in STZ-treated RIN-m5F cells With respect to the expression level of PAC1 receptor in STZ-treated RIN-m5F cells, there was no significant change in the expression level of PAC1 receptors with or without STZ-stimulation (data not shown). These observations are consistent with a previous study [33] showing that expression of the PAC1 receptor gene was not significantly changed in STZ-induced diabetic rats. In apoptotic cascades, Bcl-2 family members are major regulators of mitochondrial integrity and mito- chondrion-initiated caspase activation [34]. The Bcl-2 protein family consists of pro-apoptotic and anti-apop- totic members that interact at both the physical and functional level to regulate apoptotic cell death [35]. The pro-survival members (Bcl-2 and Bcl-X L ) oppose two pro-apoptotic groups, such as the Bax group (Bax, Bak and Bok) and the BH3-only proteins (Bim, Bad, Bid, Bik, Bmf, Noxa, Puma and Hrk). To clarify the role of PACAPs in anti-apoptotic signaling pathways, the expression of mRNA for apoptosis-related proteins (Noxa and Bax) and pro-survival proteins (Bcl-X L and Bcl-2) was compared with or without PACAPs by real-time PCR (Fig. 6). Pre-incubation of RIN-m5F cells with STZ (10 mm) resulted in a 3.3-fold increase of Noxa and a slight increase of Bax mRNA (Fig. 6A,D), suggesting the activation of apoptotic sig- naling. However, the addition of PACAP27 led to a 41% and 43% decrease, respectively, in the expression of Noxa and Bax mRNA compared to STZ alone. PACAP38 could also attenuate STZ-evoked expression of Noxa mRNA by 36%, and the expression level of Bax mRNA in the presence of PACAP38 was found to be almost same as the control. In addition, there appeared to be a significant difference between the control and STZ-treated groups with respect to the expression of Bcl-2 and Bcl-X L , showing a 63% and 74% decrease of Bcl-2 and Bcl-X L , respectively (Fig. 6B,C). Although both PACAP27 and PACAP38 were unable to attenuate down-regulation of Bcl-X L mRNA, they promoted expression of Bcl-2 mRNA significantly. According to these novel findings, the regulation of Bcl-2, Noxa and Bax expression would be involved in the possible signaling pathways of PACAP-evoked anti-apoptotic effects on STZ-treated RIN-m5F cells. A number of efforts have been made to clarify the mechanisms of STZ-induced diabetes both in vitro as well as in an in vivo experimental model [36]. As shown in Fig. 7, STZ acts as a strong alkylating agent, caus- ing direct alkylation of DNA by methyl radical via decomposition of STZ. In addition, generation of reactive oxygen species from STZ is responsible for STZ-induced toxicity in diabetogenesis. Upon these cytotoxic mediators, STZ may damage DNA, which leads to stimulation of apoptotic signaling pathways. After activation, pro-apoptotic family members, such as Bax and Bak, target the mitochondria, and cause membrane permeabilization and the release of cyto- chrome c, a pro-apoptotic effecter, eventually leading Fig. 5. Protective effect of PACAP38 on the STZ-induced cytotoxic- ity in RIN-m5F cells. After pretreatment with STZ at 10 m M, RIN- m5F cells were incubated with PACAP38 (s), VIP (h) or coenzyme Q 10 ()) at the indicated concentrations for 48 h, and extracellular LDH was assessed. Each point represents the mean ± SE of three or four experiments. PACAP attenuates streptozotocin-induced apoptotic death S. Onoue et al. 5546 FEBS Journal 275 (2008) 5542–5551 ª 2008 The Authors Journal compilation ª 2008 FEBS to apoptotic cell death by activated caspases. The apoptotic death of b cells was also observed in patients with type 2 diabetes, which was induced by chronic oxidative stress, glucotoxicity and lipotoxicity. A recent clinical investigation of 124 autopsies suggested that the frequency of b cell apoptosis was increased ten-fold in the lean and three-fold in the obese cases of type 2 diabetes [37]. Thus, it has been suggested that an increased rate of cell death by apoptosis was responsible for the evolution of the diabetic state. In addition to oxidative stress, the impairment of islet b cell responsiveness to glucose, induced by chronic hyperglycemia, is also involved in the evolution of the type 2 diabetic state. Interestingly, PACAP at low con- centration could prevent KCl-induced impairment of the response to glucose in rat islet b cells, where the mechanism is thought to be the attenuation of KCl-evoked Ca 2+ elevation [38]. In previous studies, PACAPs were thought to be effective for the clinical treatment of type 2 diabetes due to stimulation of Fig. 6. Quantitative RT-PCR analysis of pro-apoptotic and anti-apoptotic protein mRNAs. After 2 h of pretreatment with STZ at 10 mM, RIN- m5F cells were incubated with PACAP27 and PACAP38 for 48 h. The relative mRNA expression alterations of (A) Noxa mRNA, (B) Bcl-2 mRNA, (C) Bcl-X L mRNA and (D) Bax mRNA were quantified in RIN-m5F cells using real-time quantitative RT-PCR. The target genes were calculated, normalized with their corresponding b-actin values and expressed as percent changes of control. Data represented the mean ± SE of three or four experiments. #P < 0.05 and ##P < 0.01 with respect to the control group; *P < 0.05 and **P < 0.01 with respect to STZ only. Fig. 7. Schematic summary of STZ-induced apoptotic signaling cas- cade in b cells and its prevention by PACAP. ATM, ataxia telangiec- tasia-mutated; ATR, ATM and Rad3-related. S. Onoue et al. PACAP attenuates streptozotocin-induced apoptotic death FEBS Journal 275 (2008) 5542–5551 ª 2008 The Authors Journal compilation ª 2008 FEBS 5547 insulin secretion [39] and increased proliferation and differentiation of b cells [40]. Furthermore, PACAP was found to serve as an endogenous amplifier of glucose-induced insulin secretion, leading to the reduc- tion of circulating glucose in GK rats and C57B ⁄ 6J mice, which are animal models for type 2 diabetes and glucose intolerance, respectively [41]. In the present study, PACAPs could attenuate the STZ-induced apoptotic death of RIN-m5F cells through the regula- tion of pro-apoptotic proteins. These novel findings, taken together with previous observations, suggest that the anti-apoptotic effects of PACAPs or other PACAP receptor agonists might be of therapeutic value for the treatment of diabetes. Conclusions In the present study, short-term exposure of RIN-m5F cells to STZ induced the delayed cell death mediated by activation of the apoptotic enzyme caspase-3 in vitro. We also demonstrated the protective effects of gluca- gon-secretin family peptides on STZ-induced cell death, and the protection of PACAP was significantly effective at low concentrations (10 )11 m). CoQ 10 also showed a similar protective effect but only at higher concentra- tions (> 2.5 · 10 )6 m). Treatment of RIN-m5F with STZ resulted in the increased expression of pro-apop- totic Noxa and Bax mRNA and the suppressed expres- sion of anti-apoptotic Bcl-2 and Bcl-X L mRNA. However, PACAPs could suppress the expression of Noxa mRNA and enhance Bcl-2 mRNA expression, leading to down-regulation of Bax mRNA. Thus, PACAPs appear to be a potent regulatory element in the STZ-evoked apoptotic signaling pathways, and PAC1 receptor may, at least in part, mediate the protec- tive effect of PACAPs in RIN-m5F cells. Further inves- tigations on PACAP derivatives and ⁄ or nonpeptidic ligands for PAC1 receptor might provide efficacious drug discovery strategies to identify novel medication for the clinical treatment of diabetes. Experimental procedures Chemicals Glucagon-secretin family peptides (Fig. 1) were chemically synthesized by the solid-phase strategy employing optimal side-chain protection, as described previously [42]. The purity (> 98%) of each tested peptide was checked by the Waters Acquity UPLC ⁄ MS system (Waters, Milford, MA, USA), which includes the binary solvent manager, sampler man- ager, column compartment, Tunable UV detector with a detection wavelength of 254 nm and square-law detector, connected to Waters masslynx software. An Acquity UPLC BEH C18 column (particle: size 1.7 lm, column size: F 2.1 · 50 mm; Waters) was used. The pure peptides showed the expected molar ratio of the constituent amino acids in amino acid analysis with a L-8500 amino acid analyzer (Hit- achi, Tokyo, Japan). Molecular mass was confirmed with a MALDI-TOF mass spectrometer (Kratos, Manchester, UK). Streptozotocin (STZ) was purchased from Sigma (St Louis, MO, USA). Water-soluble formulation of coenzyme Q 10 was provided from Yokohama Oils and Fats Industry Corpora- tion (Yokohama, Japan). Cell cultures RIN-m5F is a clonal rat insulinoma pancreas-cell line, derived from the RIN-m rat islet cells [43]. RIN-m5F cells were obtained from the American Type Culture Collection (Rockville, MD, USA). RIN-m5F cells were cultured in RPMI-1640 medium (Sigma) supplemented with 5% (v ⁄ v) newborn calf serum (Gibco-BRL, Grand Island, NY, USA), 5% (v ⁄ v) horse serum (Gibco-BRL) and 1% (v ⁄ v) kanamycin sulfate (Invitrogen, Tokyo, Japan). The cul- tures were maintained in 5% CO 2 ⁄ 95% humidified air at 37 °C. RT-PCR analysis of mRNAs encoding neuropeptides receptors Total RNA was isolated from RIN-m5F cells using the TRI REAGENTÔ (Sigma), and RNA was reverse transcribed using SuperScriptÔ First-Strand Synthesis System for RT- PCR (Invitrogen). The resulting cDNAs were used for PCR with specific primers based on rat cDNA: 5¢- and 3¢-primers for PAC1 (GenBank accession no. Z23279 for basic, Z23273 for hip, Z23274 for hop1, Z23275 for hop2, and Z23272 for hiphop1) were 5¢-TTTCA TCGGC ATCAT CATCA TCATC CTT-3 ¢ (sense) and 5¢-CCTTC CAGCT CCTCC ATTTC CTCTT-3¢ (antisense); those for VPAC1 (M86835) were 5¢-GCCCC CATCC TCCTC TCCAT C-3¢ (sense) and 5¢-CCGCC TGCAC CTCAC CATTG-3¢ (antisense); those for VPAC2 (U09631) were 5¢-ATGGA TAGCA ACTCG CCTTT CTTTAG-3¢ (sense) and 5¢-GGAAG GAACC AACAC ATAAC TCAAA CAG-3¢ (antisense); those for GLP-1 (CV871385) [44] were 5¢-AGTAG TGTGC TCCAA GGGCA T-3¢ (sense) and 5¢-AAGAA AGTGC GTACC CCACC G-3¢ (antisense); those for secretin [27] were 5¢- GGG TTC TCC AGC CAT TTT TG-3¢ (sense) and 5¢- GTCCC AGCAC CAGTA TTTTC TAGA-3¢ (antisense); and those for glucagon [45] were 5¢-GTCCG CATCA TTCAT CTTCT T-3¢ (sense) and 5¢-CTGCC TGCAC TCATA AGCTG A-3¢ (antisense). PCR for respective recep- tors and b-actin was performed for 35 cycles. After an initial denaturation at 94 °C for 3 min, the indicated cycles of amplification [30 s denaturing at 94 °C, 30 s annealing at 64 °C (PAC1), at 66 °C (VPAC1), at 63 °C (VPAC2 and PACAP attenuates streptozotocin-induced apoptotic death S. Onoue et al. 5548 FEBS Journal 275 (2008) 5542–5551 ª 2008 The Authors Journal compilation ª 2008 FEBS GLP-1), at 60 °C (secretin) or at 67 °C (glucagon), and a 1 min extension at 72 °C] were performed in a DNA Thermal Cycler (Perkin-Elmer, Norwalk, CT, USA), iCycler (Bio- Rad, Hercules, CA, USA), Program Temp Control System PC708 (ASTEC, Hukuoka, Japan), and the GeneAmp Ò PCR system 9700 (Applied Biosystems, Tokyo, Japan). The size of each PCR product was expected to be 290 bp for the basic PAC1 receptor, 374 bp for a PAC1 receptor with a sin- gle cassette insert (hip, hop1), 371 bp for a PAC1 hop2 recep- tor, 458 bp for a double insert (hiphop1 or hiphop2), 299 bp for VPAC1, 326 bp for VPAC2, 190 bp for GLP-1, 73 bp for secretin and 407 bp for glucagon. The amplified PCR prod- ucts were separated by electrophoresis (2% agarose gel in Tris-acetic acid-EDTA buffer) and visualized with ethidium bromide staining. LDH assay The RIN-m5F cells were seeded at 10 4 cells ⁄ well in 96-well plates (AGC Techno Glass, Chiba, Japan) at least 72 h before the experiment and cultured in serum-containing RPMI 1640. STZ at the indicated concentrations was added to each well and, after 2 h of incubation, STZ-containing medium was carefully replaced with freshly prepared med- ium containing neuropeptides. The extent of cell death was assessed by measuring the activity of LDH released from the dead cells. The level of LDH activity in the culture medium was determined using a commercially available kit (LDH-Cytotoxic test; Wako, Osaka, Japan), according to the manufacturer’s instructions. TUNEL staining RIN-m5F cells were treated for 48 h in the absence or pres- ence of conditioned medium, and then fixed in 10% neutral buffered formalin for 30 min at room temperature. The TUNEL method was used to detect DNA fragmentation in the cell nuclei. The TUNEL method used was an adapta- tion of that of Gavrieli et al. [46]. All cells were pre-incu- bated in 50 UÆwell )1 of terminal deoxynucleotidyl transferase (TdT) buffer (Promega, Madison, WI, USA) for 10 min at room temperature and then the buffer was removed. A 100 lL aliquot of reaction mixture containing 5.0 U TdT and 0.4 mm biotin-14-dATP in TdT buffer was added to each well and incubated for 1 h at 37 °C. This mixture was removed and 100 lLÆwell )1 of standard saline citrate was added and incubated for 15 min at room tem- perature. Cells were washed in NaCl ⁄ P i for 10 min, and BSA (2%) was added to each well and incubated at room temperature for 10 min. Cells were washed in NaCl ⁄ P i for 5 min, then avidin-horseradish peroxidase was added and incubated for 1 h. Cells were washed twice in NaCl ⁄ P i for 5 min, and then developed in 100 lLÆwell )1 of 0.05% 3,3¢-diaminobenzidine ⁄ 0.1 m phosphate buffer ⁄ 0.01% H 2 O 2 for 5–7 min at room temperature. Caspase-3 activity The caspase-3 activity in the culture was measured with an Apo-ONEÔ Homogeneous Caspase-3 ⁄ 7 Assay Kit (Pro- mega) according to the manufacturer’s instructions. Briefly, cells (5 · 10 4 cells ⁄ well) in 96-well plates (AGC Techno Glass) were rinsed twice with serum-free RPMI 1640. The cultures were incubated with or without the indicated stim- ulants in RPMI 1640 (25 lL) at 37 °C in an atmosphere of 95% air and 5% CO 2 . The cells were lysed in 25 lLof Homogeneous Caspase-3 ⁄ 7 buffer containing the caspase-3 substrate Z-DEVD-rhodamine 110, and the cell lysates were incubated for 18 h at room temperature. After incubation, the fluorescence (excitation, 485 nm; emission, 535 nm) of cell lysates (25 lL) was measured with a Multilabel Counter (Perkin-Elmer). Real-time PCR The levels of Noxa, Bcl-X L , Bcl-2 and Bax mRNA were measured with the real-time RT-PCR method using SYBR Ò green. Total RNA was extracted from the cells with TRI REAGENTÔ (Sigma). Aliquots (1 lL) of total RNA were used for reverse transcription, employing the SuperScriptÔ First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time PCR was performed with a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex TaqÔ (Takara Bio Inc., Shiga, Japan). The specific primers based on rat cDNA: 5¢- and 3¢-primers for Noxa [47] were 5¢-GAACG CGCCA TTGAA CCCAA- 3¢ (sense) and 5¢-CTTTG TCTCC AATTC TCCGG-3¢ (anti- sense); those for Bcl-2 [48] were 5¢-AGGAT TGTGG CCTTC TTTGA GT-3¢ (sense) and 5¢-GCCGGTTCAGG TACTCAGTCAT-3¢ (antisense); those for Bcl-X L [49] were 5¢-GCCCA TCTCT ATTAT AAAAA T-3¢ (sense) and 5¢-CACAG TGCCC CGCCA AAGG AG-3¢ (antisense); and those for Bax [48] were 5¢-GGTTG CCCTC TTCTA CTTTG CT-3¢ (sense) and 5¢-TGAGC CCATC TTCTT CCAGA-3¢ (anitsense). The reaction was performed at 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. The dissociation stage was initiated at 95 °C for 15 s, followed by one cycle of 60 °C for 1 min and 95 °C for 15 s. The fluorescence of the STBR green dye was determined as function of PCR cycle number, giving the threshold cycle (C T ) number at which the amplification reached a significant threshold. The C T values were used to quantify the PCR product, and relative quantitative method was used for data analysis. All values were normalized to the b-actin gene. Statistical analysis For statistical comparisons, a one-way analysis of variance with the pairwise comparison by Fisher’s least significant difference procedure was used. P < 0.05 was considered statistically significant for all analyses. S. Onoue et al. PACAP attenuates streptozotocin-induced apoptotic death FEBS Journal 275 (2008) 5542–5551 ª 2008 The Authors Journal compilation ª 2008 FEBS 5549 Acknowledgements We are grateful to Mr Kazuki Kuriyama for his excel- lent technical assistance throughout the study. This work was supported in part by a Grant-in-Aid for Young Scientists (B) (No. 20790103; S. Onoue) from the Ministry of Education, Culture, Sports, Science and Technology, and by a grant from the Smoking Research Foundation Japan. 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Pituitary adenylate cyclase-activating polypeptide attenuates streptozotocin-induced apoptotic death of RIN-m5F cells through regulation of Bcl-2 family protein. attenuate down -regulation of Bcl-X L mRNA, they promoted expression of Bcl-2 mRNA significantly. According to these novel findings, the regulation of Bcl-2, Noxa