Cerulein-Induced Acute Pancreatitis in PACAPKnockout Mice

Yusuke Sakurai & Norihito Shintani & Akihiro Arimori & Ken-ichi Hamagami &Naoko Higuchi & Hiroaki Inoue & Kazuya Ikeda & Atsuko Hayata & Hitoshi Hashimoto &

Akemichi Baba

Received: 30 January 2010 /Accepted: 23 May 2010 /Published online: 22 June 2010# Springer Science+Business Media, LLC 2010

Abstract In our previous study, we reported that cerulein-induced acute pancreatitis is aggravated in pancreaticβ-cell-specific pituitary adenylate cyclase-activating poly-peptide (PACAP) transgenic mice, showing that an increasein pancreatic PACAP is a risk factor for progression ofacute pancreatitis. Accordingly, in this study, we examinedthe progression of cerulein-induced acute pancreatitis inPACAP knockout (KO) mice. Unexpectedly, after cerulein,about 60% of the KO mice showed severe hypothermiabelow 30°C by 12 h and most of them died within 72 h. Incontrast, the remaining KO and wild-type mice showednormothermia with no mortality. Thus, KO mice could beclassified into two groups as hypothermic (HT-KO) andnormothermic (NT-KO) to cerulein. Only HT-KO micesubsequently showed severe mortality, although both HT-

KO and NT-KO mice exhibited similar susceptibility oflungs to cerulein toxicity, comparable to that in wild-typemice. Regarding pancreatitis, HT-KO mice showed amelio-rated pancreatic damage without any rise in serum enzymeactivities, whereas NT-KO mice exhibited a similar degreeof pancreatitis to wild-type mice. Taken together, thepresent results indicate that lack of pancreatic PACAP didnot aggravate, but rather ameliorated, cerulein-inducedpancreatitis. In addition, about half of KO mice showed anovel phenotype in which cerulein caused rapid and severehypothermia, followed by death.

Keywords Pituitary adenylate cyclase-activating polypep-tide (PACAP) . Cerulein . Acute pancreatitis . Hypothermia .

Lung injury

Introduction

Pituitary adenylate cyclase-activating polypeptide (PACAP)is a neuropeptide that belongs to the vasoactive intestinalpolypeptide/secretin/glucagon superfamily and is involvedin a wide range of physiological activities in the central andperipheral nervous tissues (Miyata et al. 1989; Vaudry et al.2009). In the pancreas, three types of receptors for PACAP,namely, VPAC1, VPAC2, and PAC1, are distributedthroughout the organ (Hannibal and Fahrenkrug 2000;Schmidt et al. 1993; Yada et al. 1994), whereas PACAP issecreted from vagus nerve and pancreatic β-cells (Hannibaland Fahrenkrug 2000; Yada et al. 1994). Several reportshave suggested that PACAP can enhance hormone secre-tion from the pancreas. For example, PACAP at extremelylow concentrations (10−14 M) stimulates insulin secretion ina glucose-dependent manner from endocrine tissue andstimulates amylase secretion from exocrine tissue (Lu et al.

Y. Sakurai :N. Shintani (*) :A. Arimori :K.-i. Hamagami :N. Higuchi :H. Inoue :K. Ikeda :A. Hayata :H. Hashimoto :A. BabaLaboratory of Molecular Neuropharmacology,Graduate School of Pharmaceutical Sciences, Osaka University,1-6 Yamadaoka, Suita,Osaka 565-0871, Japane-mail: shintani@phs.osaka-u.ac.jp

A. Hayata :H. HashimotoDepartment of Experimental Disease Model, The Osaka-Hamamatsu Joint Research Center for Child Mental Development,Graduate School of Medicine, Osaka University,2-2 Yamadaoka, Suita,Osaka 565-0871, Japan

H. HashimotoUnited Graduate School of Child Development,Osaka University, Kanazawa University andHamamatsu University School of Medicine,2-2 Yamadaoka, Suita,Osaka 565-0871, Japan

J Mol Neurosci (2011) 43:8–15DOI 10.1007/s12031-010-9396-z

2003; Onaga et al. 1997; Yada et al. 1994). To clarify theroles of PACAP in the pancreas, we generated PACAPknockout mice (PACAP-KO mice; Hashimoto et al. 2001)and transgenic mice overexpressing PACAP in pancreaticβ-cells (PACAP-βTg mice; Yamamoto et al. 2003). Inparticular, we examined the pathophysiological roles ofPACAP in pancreatic endocrine function and found that,in PACAP-βTg mice, streptozotocin-mediated destructionof pancreatic β-cells was ameliorated (Yamamoto et al.2003). In addition, overexpression of PACAP in isletsresulted in significant amelioration of hyperinsulinemiaand islet hyperplasia in a type II diabetic mouse model(Tomimoto et al. 2004). These findings have suggestedthat PACAP is involved in the pathophysiology inpancreatic β-cells.

Several lines of evidence have suggested that PACAPis also involved in the pathophysiology of pancreaticacinar tissue (Bhatia et al. 2005; Pandol et al. 2007;Saluja and Steer 1999). Pancreatitis is a disease charac-terized by inflammation, fibrosis, and general tissuedisruption especially in pancreatic acinar cells, and itsprogression and pathogenesis are mediated by severalproinflammatory cytokines and anti-inflammatory cyto-kines that are released from infiltrated monocytes andmacrophages (Bhatia et al. 2005; Pandol et al. 2007;Saluja and Steer 1999). In line with this evidence, a recentstudy showed that development of human chronicpancreatitis is associated with intrapancreatic accumula-tion of PACAP around nerves and inflammatory infil-trates, with altered patterns of PACAP-mediated cytokineresponses (Michalski et al. 2008). In addition, intravenousinfusion of PACAP aggravates the signs of acutepancreatitis (Chen et al. 2005). In accordance with theseobservations, we recently demonstrated using PACAP-βTg mice that overexpression of pancreatic PACAPaggravated cerulein-induced pancreatitis (Hamagami etal. 2009). Although these findings suggest that anincrease in pancreatic PACAP at pharmacological levelsaggravates acute pancreatitis, it is still uncertain whetherpancreatic PACAP at its physiological level is relevant inthe pathophysiology of acute pancreatitis. In the presentstudy, we examined whether the severity of cerulein-induced acute pancreatitis is affected in PACAP-KO miceto evaluate the physiological involvement of pancreaticPACAP in acute pancreatitis. The results clearly demon-strated that acute pancreatitis is not aggravated, but ratheris ameliorated, in the absence of pancreatic PACAP. Inaddition, the present study showed an unexpected resultin that about half of PACAP-KO mice appear to haveundefined central and/or peripheral mechanisms confer-ring susceptibility to cerulein-induced severe hypothermiaand subsequent death.

Materials and Methods

Mice

The generation of PACAP-KO mice on a CD1 geneticbackground has been described (Hashimoto et al. 2001).All mice were kept in a temperature-, humidity-, and light-controlled room with a 12-h light/12-h dark cycle (lights onfrom 8:00 a.m. to 8:00 p.m.) and allowed free access towater and food. All animal experiments were performed inaccordance with protocols approved by the InstitutionalAnimal Care and Use of Osaka University.

Induction of Experimental Acute Pancreatitis

After PACAP-KO and wild-type mice were fasted for 18 hwith access to water ad libitum, acute pancreatitis wasinduced. Seven consecutive hourly intraperitoneal injec-tions of saline solution (0.9% NaCl) containing cerulein (ananalog of cholecystokinin (CCK) activating its receptor(Grendell 1992), 50 µg/kg body weight; Sigma, Tokyo)were administered (10 µL/g body weight). The control micereceived an equal volume of saline solution. In thefollowing text, the experimental time indicates time afterthe first cerulein injection.

Monitoring of Body Temperature

Rectal temperature was measured using an electronicthermometer (BAT-12; Bailey Instruments, Clifton, NJ,USA). On the day before experiments, the mice werehabituated to the probe by inserting the probe into theiranus to a depth of 1.5 cm, for three times every 30 min. Insome experiments, we divided PACAP-KO mice into twogroups based on whether or not their body temperaturedropped below 30°C by 12 h after the first ceruleininjection.

Evaluation of Acute Pancreatitis

Measurement of serum enzyme activities and evaluation ofpancreatic tissue damage have been described previously(Hamagami et al. 2009). Briefly, blood samples collectedfrom tail vein were centrifuged, and the amylase and lipaseactivities in the plasma were determined with commerciallyavailable assays (Roche Diagnostics KK, Tokyo). Pan-creases and lungs rapidly removed at 12 h after the firstcerulein treatment were fixed, embedded, sectioned, andstained with hematoxylin and eosin. The stained tissuesections of the pancreas were graded in a blind fashionusing the scale system as described previously (Devière etal. 2001, Hamagami et al. 2009), where the extent of

J Mol Neurosci (2011) 43:8–15 9

damage was given a score from 0 to 4: 0 indicated theabsence of injury and 4 indicated the injury being mostsevere. The sample slides were examined for the presenceof edema, inflammation, vacuolization, and necrosis in fiverandomly chosen microscopic fields, and then summed in atotal injury score.

Myeloperoxidase Activity

Lung tissues were harvested, homogenized in 20 mMphosphate buffer (pH 7.4), and centrifuged at 10,000×g for10 min at 4°C. The resulting pellet was resuspended in50 mM phosphate buffer (pH 6.0) containing 0.5%hexadecyltrimethyl ammonium bromide (Sigma, Tokyo).Homogenates were then frozen in liquid nitrogen andthawed on four consecutive occasions before a final 40-ssonication. Samples were centrifuged at 10,000×g for10 min at 4°C, and the supernatants were collected formyeloperoxidase (MPO) assay. Supernatant (25 µL) wasmixed with 125 µL of assay buffer consisting of 1.6 mM3,3′,5,5′-tetramethylbenzidine (Sigma), 0.3 mM H2O2 (Sig-ma) diluted in 80 mM phosphate buffer (pH 5.4), and50 mM phosphate buffer (pH 6.0) containing 0.5%hexadecyltrimethyl ammonium bromide. The mixture wasincubated at 37°C for 2 min, and the chromogenic reactionwas measured as absorbance at 650 nm. An enzyme unit isdefined as the amount of enzyme that produces an increaseof 1 absorbance unit/mg wet tissue/min.

Statistical Analysis

Data are expressed as mean ± the standard error of themean. The differences between the groups were analyzedby a repeated two-way ANOVA, a two-way ANOVAwith apost hoc Tukey–Kramer test, a Student's t test, the Mann–Whitney's U test, or by the Kaplan–Meier method followedby log-rank test using StatView software (SAS Institute,Cary, NC, USA). A P value of <0.05 was consideredsignificant.

Results

PACAP-KO Mice Showed Increased MortalityAfter Cerulein Injection

We first examined the cerulein-induced increases in theserum enzyme activities in PACAP-KO and wild-type mice.As shown in Fig. 1a and b, cerulein induced a time-dependent rise in serum amylase and lipase activities inboth genotypes. No significant difference was observed inthe basal activities of serum enzyme activities between the

two genotypes. In wild-type mice, the average serumamylase and lipase activities at 12 h were fourfold and20-fold those of basal levels, respectively. In contrast, thecerulein-induced increases in these enzyme activities inPACAP-KO mice were diminished by 30% and 50%,respectively, relative to wild-type. These results are in goodaccordance with the previous finding that PACAP increasesthe release of these enzymes from pancreatic acinar cells(Lu et al. 2003; Onaga et al. 1997; Yada et al. 1994). Thepresent protocol of cerulein injection resulted in a lowmortality rate in wild-type mice (Fig. 1c). In contrast,PACAP-KO mice tended to die even within 24 h aftercerulein injection and the mortality rate rose to about 50%by 72 h after cerulein (survival rate at 72 h; 55% inPACAP-KO mice vs 90% in wild-type mice, P<0.01 bylog-rank test).

Cerulein Induces Hypothermia Followed by Deathin About Half of PACAP-KO Mice

To understand why cerulein caused high mortality inPACAP-KO mice, the change in body temperature wasmonitored after cerulein injection because single intraper-itoneal injection of CCK or its analog cerulein induceshypothermia in rats and mice (Rezayat et al. 1999; Szelényi2010). As shown in Fig. 2a, although the average bodytemperature of PACAP-KO mice was slightly decreased at12 h after cerulein, the susceptibility of PACAP-KO mice tocerulein-induced hypothermia varied extremely. At 12 hafter cerulein injection, 57% of the PACAP-KO mice (12 of21 mice) showed marked hypothermia below 30°C(Fig. 2a). The remaining ten mice showed normal bodytemperature, suggesting the mutants could be grouped intotwo types, termed hypothermic (HT-KO) and normothermic(NT-KO) mice, respectively (see also the “Materials andMethods” section). According to this grouping, only HT-KO mice showed marked and significant hypothermia time-dependently after cerulein treatment (Fig. 2b). It isremarkable that severe hypothermia below 30°C wasobserved at 6 h after cerulein, at which time pancreatitiswas not prominent. A high mortality rate was observed onlyin HT-KO mice (Fig. 2c; 20% of NT-KO mice and 83% ofHT-KO mice had died by 72 h, P<0.05 by log-rank test).These results suggested that cerulein-induced hypothermiacorrelated with the subsequent mortality in PACAP-KOmice.

Cerulein-Induced Lung Injury in PACAP-KO Mice

Cerulein produced inflammatory tissue injury not only inpancreas but in lung tissues (Bhatia et al. 1998), and suchmultiple organ dysfunctions occasionally induce individual

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death (Petrov et al. 2006). Because the mortality in HT-KOmice was possibly caused by such organ dysfunction, wenext examined the severity of the tissue damage in wild-type and PACAP-KO mice at 12 h after cerulein, at whichtime no PACAP-KO mice had died (Figs. 3 and 4).Histological examination of the lung demonstrated thatboth wild-type and NT-KO mice developed apparent tissuefibrosis (Fig. 3a–c), and HT-KO mice also exhibitedcomparable damage (Fig. 3d). For quantitative analysis of

the lung injury, we next evaluated the activity of MPO, anenzyme contained in neutrophils and monocytes involvedin tissue damage (Chooklin et al. 2009). In both genotypes,MPO activity was significantly increased by ceruleintreatment to about 2.5-fold of basal levels, and theresponses in NT-KO and HT-KO mice were comparableto that in wild-type mice (Fig. 3e). As shown in Fig. 3f,cerulein-stimulated lung MPO activity did not correlatewith hypothermia (R2 values; PACAP-KO mice=0.013,

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Figure 2 Body temperature reduction associated with mortality inPACAP-KO mice in cerulein-induced acute pancreatitis. Bodytemperature of PACAP-KO (closed symbols: hypothermic (HT-KO)represented by triangles and normothermic (NT-KO) by circles) andwild-type (open circle) mice at 12 h after the first a cerulein injectionand their b time-dependent change. Averaged body temperature ofPACAP-KO (closed square) and wild-type (open square) mice areshown as mean ± SEM (n=13–21). Statistical analysis between twogenotypes was performed using the Mann–Whitney's U test. The

dashed line represents 30°C. Time-dependent changes in bodytemperature of each mouse were examined at 0, 6, 9, and 12 h afterthe first cerulein injection. Data are expressed as mean ± SEM (n=9–13 per group). **P<0.01, repeated two-way ANOVA. c Survival rateof HT-KO (closed triangle), NT-KO (closed circle), and wild-type(open circle) mice at 0, 12, 24, 48, and 72 h after the first ceruleininjection. Data consist of five to eight mice for each group. Singlesharp indicates P<0.01, the Kaplan–Meier method followed by log-rank test

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Figure 1 Serum amylase and lipase activities and survival rate inPACAP-KO mice during cerulein-induced acute pancreatitis. Theactivities of a serum amylase and b lipase were measured for PACAP-KO (closed circle) and wild-type (open circle) mice at 0, 6, 9, and12 h after the first cerulein injection. Data are expressed as mean ±

SEM (n=10–11 per group). *P<0.05, repeated two-way ANOVA. cSurvival rate of PACAP-KO (closed circle) and wild-type (opencircle) mice at 0, 12, 24, 48, and 72 h after the first cerulein injection.Data consists of 22–29 mice for each group. Double sharps indicateP<0.01, the Kaplan–Meier method followed by log-rank test

J Mol Neurosci (2011) 43:8–15 11

wild-type mice=0.010). These results suggested thatcerulein-induced lung injury did not induce the death ofHT-KO mice.

Cerulein-Induced Pancreatic Injury in PACAP-KO Mice

As reported previously (Hamagami et al. 2009), the presentprotocol of cerulein treatment significantly induced exper-imental pancreatitis which was clearly monitored byhistological examination. Thus, the cerulein treatmentincreased the histological scores in all groups as shown inFig. 4e. HT-KO mice showed ameliorated injury comparedwith NT-KO mice, which exhibited comparable damage towild-type mice. In accordance with these observations, NT-KO mice showed a similar magnitude of response in bothserum amylase and lipase activities, which are additionalmarkers for pancreatic injury, compared with wild-typemice (Fig. 5a, b). Interestingly, this increase was almostcompletely ablated in HT-KO mice, suggesting a clearcorrelation between hypothermia and attenuated proteaseresponse in PACAP-KO mice. These data indicated only inPACAP-KO mice a higher positive correlation betweenbody temperature and pancreatic phenotype at 12 h aftercerulein treatment (R2 values in total injury score: PACAP-KO mice=0.63, wild-type mice=0.0028; those in serum

amylase levels at 12 h: PACAP-KO mice=0.70, wild-typemice=0.055). Taken together, these data suggest thatcerulein-induced hypothermia was highly correlated withthe ameliorated development of pancreatitis in PACAP-KOmice.

Discussion

Previously, we found that overexpression of PACAP inpancreatic β-cells aggravated the pathology of cerulein-induced acute pancreatitis (Hamagami et al. 2009). Simi-larly, exogenous application of PACAP at pharmacologicaldoses also aggravated the cerulein-induced pancreatitis inrats (Chen et al. 2005). Taken together, these previousresults suggested that marked increases in pancreaticPACAP aggravate the pathology of pancreatitis. Thepresent study was aimed at determining the possible effectsof a deficiency in pancreatic PACAP on the development ofpancreatitis, by using PACAP-KO mice. The resultssuggested that the susceptibility of PACAP-KO mice tocerulein was significantly variable between mice, at leastduring the first 12 h after cerulein injection where about50% of PACAP-KO mice tested exhibited normal pancre-atitis characterized by an increase in serum amylase and

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Figure 3 Cerulein-induced lung tissue damage in PACAP-KO mice.a–d Representative micrographs of hematoxylin- and eosin-stainedlung sections are shown in a saline- and b cerulein-treated wild-typemice. Those of normothermic PACAP-KO (NT-KO, c) and hypother-mic PACAP-KO (HT-KO, d) mice are also presented. Bars indicate100 µm. e–f Myeloperoxidase (MPO) activity in lungs of e saline- orcerulein-treated mice and the f correlation between lung MPO activityand body temperature at 12 h after the first cerulein injection inPACAP-KO (closed circles) and wild-type (open circles) mice. Data

were expressed as mean ± SEM (n=3–8 per group). *P<0.05 vs salinegroups in each genotype, one-way ANOVA followed by Tukey–Kramer test. f Correlation lines were calculated between the bodytemperature (x-axis) and MPO activity (y-axis) at 12 h after ceruleininjection in 12 PACAP-KO and five wild-type mice. The dashed linerepresents 30°C. All data were obtained using lungs harvested fromeach group at 12 h after cerulein or saline administration, as describedin the “Materials and Methods” section

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lipase activities followed by significant pancreatic inflam-mation, but the remaining 50% showed attenuatedresponses to cerulein. Furthermore, about 50% of thePACAP-KO mice died within 72 h after cerulein. We,therefore, focused on the unexpected cerulein-induced

death in these PACAP-KO mice in terms of the severityof pancreatic and lung injuries.

To address the heterogeneity of PACAP-KO mice in theresponse cerulein, PACAP-KO mice were classified intotwo groups: one group exhibited marked hypothermia even

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Figure 4 Cerulein-induced pancreatic tissue damage in PACAP-KOmice. a–d Representative micrographs of hematoxylin- and eosin-stained pancreatic sections are shown in a saline- and b cerulein-treated wild-type mice. Those of normothermic PACAP-KO (NT-KO,c) and hypothermic PACAP-KO (HT-KO, d) mice are also presented.At 12 h after cerulein or saline administration, the pancreas from wild-type mice and PACAP-KO mice were harvested, processed, andstained as described in the “Materials and Methods” section. Barindicates 100 µm. e, f Total histological scores of injury e in cerulein-

induced pancreatitis in PACAP-KO and wild-type mice and the fcorrelation between the injury score and the body temperature at 12 hafter the first cerulein injection in PACAP-KO (closed circles) andwild-type (open circles) mice. Data were expressed as mean ± SEM(n=5–7 per group). **P<0.01 vs cerulein-treated PACAP-KO micewith normothermia, by Student's t test. f Correlation lines werecalculated between the body temperature (x-axis) and pancreatic injuryscore (y-axis) at 12 h after cerulein injection in ten PACAP-KO andseven wild-type mice. The dashed line represents 30°C

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Figure 5 Cerulein-induced increase in serum protease activity inPACAP-KO mice. a, b Time-dependent changes in serum amylaseand lipase activities are presented in wild-type (open circles) andPACAP-KO mice with normothermia (closed circles) and hypother-mia (closed triangles). The activities of a serum amylase and b lipasein each group were measured at 0, 6, 9, and 12 h after the first cerulein

injection. Data are expressed as mean ± SEM (n=5–10 per group).*P<0.05, **P<0.01, repeated two-way ANOVA. c Correlation lineswere calculated between the body temperature (x-axis) and serumamylase activity (y-axis) at 12 h after cerulein injection in 11 PACAP-KO and eight wild-type mice. The dashed line represents 30°C

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at 6 h after cerulein at which time no sign of pancreatitiswas observed (HT-KO) and the other group did not showany significant hypothermia (NT-KO). In addition, wefound a strong correlation between mortality and hypother-mic response to cerulein in HT-KO mice. The time courseof the change in body temperature and mortality clearlyshowed that marked hypothermia preceded and wasprerequisite for subsequent death. Concerning the severityof cerulein-induced pancreatitis and lung injury, HT-KOmice were more unsusceptible than NT-KO mice. Clinical-ly, acute pancreatitis-associated multiple organ dysfunctionand systemic inflammatory response syndromes are themain cause of death in patients with acute pancreatitis(Bhatia 2005). The results of animal studies also suggestedthat lung injury observed in pancreatitis may be related toindividual death (Bhatia 2005). However, the presentfindings are not in agreement with this, and instead leadus to conclude that the severe hypothermia and subsequentdeath in HT-KO mice were not due to inflammatoryresponses in the pancreas and lung.

Already in 1983, Homma and Malik reported thatcerulein is a vasodilator (Homma and Malik 1983), andseveral reports suggest that hypothermia induced byintraperitoneal injection of CCK or cerulein is related tonot only vasodilation but also fall in metabolic rate andblood pressure and afferent or efferent nervous pathwaysalteration (Szelényi 2010). Thus, the altered response ofthese central and/or peripheral functions toward ceruleinmay contribute to the severe hypothermia in HT-KO mice.However, CCK and cerulein generally caused small andshort-lived drop (approximately by 1°C to 3°C no morethan 2 h) in their core temperature in certain strains ofanimals (Szelényi 2010), and relevant doses of cerulein didnot cause significant hypothermia in CD1 mice in thisstudy. Therefore, the cerulein-induced marked hypothermiain HT-KO mice is an unexpected finding. Recently, wereported that hypothermic response to ethanol was attenuat-ed in PACAP-KO mice (Tanaka et al. 2010) and is related tothe changes in serotonergic neurotransmission. In addition,the heat production is inadequate in PACAP-KO mice,which is due to insufficient norepinephrine stimulation onbrown adipose tissue under prolonged mild cold stress(Gray et al. 2002). These findings suggest that thermalregulation in PACAP-KO mice is not intact. However, it isnoted that about 40% of the KO (NT-KO) mice did notshow any hypothermia in response to cerulein. Therefore,unknown factors or mechanisms are apparently involved inthe phenotypic heterogeneity of PACAP-KO mice in thehypothermic response to cerulein.

Concerning the severity of pancreatitis in PACAP-KOmice, cerulein induced a typical response in NT-KO miceand an attenuated response in HT-KO mice. We considerthat the attenuated response in HT-KO mice is due to the

severe hypothermia in this group, and this is supported bythe following findings. Early induction of moderatehypothermia suppressed pancreatic inflammatory cell infil-tration in rats with cerulein-induced pancreatitis (Fujimotoet al. 2008), and hypothermic pretreatment inhibited thepancreatic expression of proinflammatory cytokine IL-6 inrats with sodium taurocholate-induced pancreatitis (Wang etal. 2005). These results suggest that induction of hypother-mia generally ameliorated experimental pancreatitis.

On the other hand, the strong tolerance to cerulein-induced increases in serum enzyme activity in HT-KOmice, but not in NT-KO mice, may be related to a failure inenzyme secretion from acinar tissues. The secretion ofpancreatic enzymes such as amylase and lipase is regulatedunder metabolic states of acinar cells, and intracellular Ca2+

and cAMP are signals involved in these secretory process-es. PACAP induced zymogen activation in pancreaticacinar cells (Lu et al. 2003; Onaga et al. 1997), andinduction of hypothermia suppressed the increase in serumamylase activity in cerulein-induced pancreatitis (Fujimotoet al. 2008). Along the lines of these discussions, it is likelythat PACAP deficiency and hypothermia in HT-KO miceare responsible for the defect in cerulein-induced enzymesecretion in HT-KO mice.

In summary, in contrast to the previous finding that a risein pancreatic PACAP content resulted in aggravation ofcerulein-induced pancreatitis, the present study revealedthat PACAP deficiency in pancreatic tissue did notmarkedly affect the pathology of pancreatitis. In addition,about half of the population of PACAP-KO mice showedan unexpected response to cerulein, severe hypothermiafollowed by death, the mechanisms of which are not yetelucidated.

Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research (A) and (B) and for Young Scientists (B)from the Japan Society for the Promotion of Science (JSPS). Thiswork was also supported in part by grants from the Japan-FranceIntegrated Action Program (SAKURA) funded by JSPS and theMinistère des Affaires Etrangères in France (MAE), the UeharaMemorial Foundation, Senri Life Science Foundation, and TaishoPharmaceutical Co., Ltd.

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