attenuates streptozotocin-induced apoptotic deathof RIN-m5F cells through regulation of Bcl-2 family protein mRNA expression Satomi Onoue, Junko Hanato and Shizuo Yamada Department of Ph
Trang 1attenuates 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
Q10, an endogenous mitochondrial antioxidant, could attenuate streptozo-tocin cytotoxicity, and especially pituitary adenylate cyclase-activating polypeptide (PACAP), at a concentration of 10)7m, 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
CoQ10, coenzyme Q10; 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.
Trang 2lipotoxicity, 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 Q10 (CoQ10), as a positive control, were also evaluated and compared with those of neuropeptides because CoQ10could 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.
Trang 3ment 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 changpreincubat-ed 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 m M ; s, 10 m M ; and h, 20 m M 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.
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.
Trang 4On 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 oxidaprotec-tive 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)7m attenuated the cytotoxicity of STZ (Table 1) Of all glucagon-secretin-family peptides tested, PACAP38 at 10)7m exhibited the highest activ-ity, with approximately 34% protection against the STZ-induced LDH release (Fig 5) PACAP27 at
10)7m 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)7M )
Control (5 · 10)6M )
Trang 5PACAPs, 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 CoQ10 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 CoQ10for 48 h resulted in a significant
con-centration-dependent increase in viability of up to
25% at 5· 10)6m (Fig 5) Considering these findings
together with the effective concentration of CoQ10 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-XL) 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-XLand 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-XL, showing a 63% and 74% decrease of Bcl-2 and Bcl-XL, respectively (Fig 6B,C) Although both PACAP27 and PACAP38 were unable to attenuate down-regulation of Bcl-XL
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.
Trang 6to 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 Ca2+ 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 m M , 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-XLmRNA 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.
Trang 7insulin 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) CoQ10 also showed a
similar protective effect but only at higher
concentra-tions (> 2.5· 10)6m) 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-XL 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 Q10was 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% CO2⁄ 95% humidified air at
37C
RT-PCR analysis of mRNAs encoding neuropeptides receptors
Total RNA was isolated from RIN-m5F cells using the TRI REAGENT (Sigma), and RNA was reverse transcribed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) The resulting cDNAs were used for RT-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 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 94C for 3 min, the indicated cycles of amplification [30 s denaturing at 94C, 30 s annealing at
64C (PAC1), at 66 C (VPAC1), at 63 C (VPAC2 and
Trang 8GLP-1), at 60C (secretin) or at 67 C (glucagon), and a
1 min extension at 72C] 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
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 104cells⁄ 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 37C 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⁄ Pi for 10 min, and
BSA (2%) was added to each well and incubated at room
temperature for 10 min Cells were washed in NaCl⁄ Pi
for 5 min, then avidin-horseradish peroxidase was added
and incubated for 1 h Cells were washed twice in NaCl⁄ Pi
for 5 min, and then developed in 100 lLÆwell)1 of 0.05%
3,3¢-diaminobenzidine ⁄ 0.1 m phosphate buffer ⁄ 0.01% H2O2
for 5–7 min at room temperature
Caspase-3 activity The caspase-3 activity in the culture was measured with an Apo-ONE Homogeneous Caspase-3 ⁄ 7 Assay Kit (Pro-mega) according to the manufacturer’s instructions Briefly, cells (5· 104cells⁄ 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 37C in an atmosphere of 95% air and 5% CO2 The cells were lysed in 25 lL of 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-XL, Bcl-2 and Bax mRNA were measured with the real-time RT-PCR method using SYBR green Total RNA was extracted from the cells with TRI REAGENT (Sigma) Aliquots (1 lL) of total RNA were used for reverse transcription, employing the SuperScript 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 Taq (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-CCCAA-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-XL[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 95C for 5 s and 60 C for
34 s The dissociation stage was initiated at 95C for 15 s, followed by one cycle of 60C 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 (CT) number at which the amplification reached a significant threshold The CT 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
Trang 9We 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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