PAP/HIP Protein Is an ObesogenicFactorVERONIQUE SECQ,1 CECILIA MALLMANN,2 MERITXELL GIRONELLA,1 BELEN LOPEZ,1

DANIEL CLOSA,3 ST�EPHANE GARCIA,1 LAURENCE CHRISTA,4 GIUSEPPE MONTALTO,5

NELSON DUSETTI,1 AND JUAN L. IOVANNA1*1Centre de Recherche en Carc�erologie de Marseille (CRCM), INSERM UMR 1068, CNRS UMR 7258,

Aix-Marseille University and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Marseille, France2Centre d’Investigation Clinique de Marseille, Marseille, France3Department of Experimental Pathology, IIBB-CSIC, IDIBAPS and CIBEREHD, Barcelona, Spain4Laboratory of Biochemistry, Reference Center for Inherited Metabolic Diseases, Necker Hospital, Descartes University, Paris,

France5Cattedra di Medicina Interna ed Epatologia, Department of Internal Medicine and Specialties, University of Palermo;

Consiglio Nazionale delle Ricerche, IBIM A. Monroy, Palermo, Italy

In this article we report the obesogenic role of the acute phase protein PAP/HIP. We found that the transgenic TgPAP/HIP mice developspontaneous obesity under standard nutritional conditions, with high levels of glucose, leptin, and LDL and low levels of triglycerides andHDL in blood. Accordingly, PAP/HIP-deficient mice are skinny under standard nutritional conditions. We also found that expression ofPAP/HIP is induced in intestinal epithelial cells in response to gavage with olive oil and this induction is AG490 sensitive.We demonstratedthat incubation of 3T3-L1 preadipocytes with a low concentration as 1 ng/ml of recombinant PAP/HIP results in accelerated BrdUincorporation in vitro. PAP/HIP-dependent adipocytes growth is sensitive to the MEK inhibitor U0126. Finally, patients with severe obesitypresent higher blood levels of PAP/HIP than non-obese control individuals. Altogether our data suggest that PAP/HIP could be a mediatorof fat tissue development, released by the intestine and induced by the presence of food into the gut.J. Cell. Physiol. 229: 225–231, 2014. � 2013 Wiley Periodicals, Inc.

The pancreatitis-associated protein (PAP), also known asp23, HIP, and Reg3b, is a 175-aminoacid-long polypeptidecontaining one C-type lectin originally identified as a pancreaticsecretory protein, not expressed under physiologicalconditions but strongly overexpressed during acutepancreatitis. Therefore, hereafter wewill call this gene PAP/HIP.Its expression is actually not restricted to the pancreas and ithas been showed in normal epithelial cell of the small intestine,and in certain cells of the pituitary, ovary, and uterus. PAP/HIPexpression was also linked to a variety of diseases includinginflammatory bowel disease, Alzheimer’s disease, or sometypes of cancers such as hepatocellular carcinoma, pancreaticadenocarcinoma, bladder and colorectal carcinoma, and inregenerating motor neurons (reviewed in Iovanna and Dagorn,2005; Closa et al., 2007).

PAP/HIP acts as a growth factor promoting cell proliferationin hepatocytes (Simon et al., 2003), in Schwann cells (Liveseyet al., 1997) and in intestinal epithelial cell (Moucadelet al., 2001). Widening the PAP/HIP range of functions, weshowed an anti-inflammatory role for this protein in agreementwith its strong induction during the course of inflammatorydiseases such as pancreatitis, Crohn’s disease, and ulcerativecolitis (reviewed in Closa et al., 2007). Interestingly, PAP/HIPacts as an antiapoptotic factor on pancreatic acinar cells (Ortizet al., 1998), motor neurons (Nishimune et al., 2000), andhepatocytes (Simon et al., 2003). Lastly, it was also shown anantimicrobial activity of PAP/HIP on some Gramþ intestinalbacteria (Cash et al., 2006).

Expression of PAP/HIP is complexly regulated. In fact,several factors such as TNFa, IFNg, IL6, IL22, LPS, ROIs,intestinal flora, estrogens, thyroid hormones, CNTF,progesterone, and leptin are some of factors that activate in a

cell-specific manner the expression of PAP/HIP (reviewed inIovanna and Dagorn, 2005). Some of these factors act throughthe Jak-STAT3 intracellular cascade (Folch-Puy et al., 2006).

Mechanistically, after PAP/HIP binds its receptor PAP/HIPsignals through the MAPK signaling pathway (Ferres-Masoet al., 2009) involving p44/42, p38, and JNK MAPKs.Interestingly, we showed that PAP/HIP increased the trans-activation activity of the PAP/HIP gene by inducing the bindingon its own promoter of the nuclear factors such as C/EBPb,pCREB, pELK1, EGR1, STAT3, and ETS2 (Ferres-Maso et al.,2009).

As described above, PAP/HIP is involved in several unrelatedfunctions. In this article we report that transgenic miceoverexpressing PAP/HIP are obese whereas, on the contrary,PAP/HIP-deficient mice are slim compared to its wild-typelittermate. In addition, we observed that recombinant PAP/HIP

The authors have declared that no conflict of interest exists.

Contract grant sponsor: INSERM.

*Correspondence to: Juan L. Iovanna, 163 Av de Luminy, Campusde Luminy, 13288 Marseille, France.E-mail: juan.iovanna@inserm.fr

Manuscript Received: 16 April 2013Manuscript Accepted: 16 July 2013

Accepted manuscript online in Wiley Online Library(wileyonlinelibrary.com): 23 July 2013.DOI: 10.1002/jcp.24438

ORIGINAL RESEARCH ARTICLE 225J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology

� 2 0 1 3 W I L E Y P E R I O D I C A L S , I N C .

induces proliferation of pre-adipocytes 3T3-L1 cells, PAP/HIPexpression is induced in small intestine is response to eating,and finally, patients with severe obesity have higher serumlevels of PAP/HIP than non-obese control individuals. Inconclusion, we report a new unexpected function of the PAP/HIP protein, named obesogenic.

Materials and MethodsAnimals

PAP/HIP transgenic mice (TgPAP/HIP) development waspreviously described (Simon et al., 2003). Briefly, PAP/HIP cDNAwas under the control of albumin promoter and PAP/HIP becamesecreted into the blood. PAP/HIP�/� mice development waspreviously reported (Gironella et al., 2007).

Expression of PAP/HIP in mice intestine

Three-month-oldmale SV129micewere starved for 24 h followingby an intragastric gavage with 400ml of olive oil. An intragastricgavage with saline was given as a control. Tyrphostin AG490 waspurchased from Calbiochem (La Jolla, CA) and injected i.p. at10mg/kg 1 h before intragastric gavage. Phosphate-buffered saline(PBS) was used as control. Distal part of the small intestine wasrecovered after 300, 600, 900, 1200, and 1800 of treatment andprocessed for RNA extraction.

Serum dosages

Leptin (Leptin Mouse ELISA Kit), glucose (Glucose Assay Kit),triglycerides (TriglycerideQuantification Kit), HDL and LDL (HDLand LDL/VLDL Cholesterol Assay Kit) concentrations in mice wasmeasured in blood using commercial kits fromAbcam (Cambridge,MA) and following instruction of the manufacturer.

Cell treatments

3T3-L1 cells were obtained from the American Type CultureCollection. Cells were maintained in high glucose, antibiotic-freeDulbecco’s modified Eagle’s medium (DMEM) supplemented with10% fetal bovine serum. Cells were grown at 37˚C in 5% CO2.BrdU incorporation was assessed by a BrdU Cell ProliferationAssay kit (Calbiochem). 3T3-L1 pre-adipocytes were plated in96-well plates (5,000 cells/well) and 24 h later cells were treatedwith increasing concentration of recombinant PAP/HIP (Gironellaet al., 2007) for 24 h and labeled with BrdU for a 3-h period, afterwhich cells were fixed and stained with anti-BrdU antibody andvisualized in a colorimetric immunoassay. The U0126 MEKinhibitor was purchased from Sigma (Chemical Co., St Louis, MO)and used at 25mM. Spectrum absorbance was measured on a Bio-Tek Synergy HT plate reader. Cells were treated with 10 ng/ml ofPAP/HIP for 30, 60, and 90min. Protein extraction was performedon ice using total protein extraction buffer: 50mM HEPES pH 7.5,150mM NaCl, 20% SDS, 1mM EDTA, 1mM EGTA, 10% glycerol,1% Triton, 25mM NaF, 10mM ZnCl2, 50mM DTT. Before lysis,protease inhibitor cocktail at 1:200 (Sigma-Aldrich, Lyon, FranceNUPR1340), 500mM PMSF, 1mM sodium orthovanadate, and1mM b glycerophosphate were added. Protein concentration wasmeasured using a BCA Protein Assay Kit (Pierce Biotechnology,Inc., Rockford, IL). Protein samples (80mg) were denatured at95˚C and subsequently separated by SDS–PAGE gel electropho-resis. After transfer to nitrocellulose, membranewas blocking with1% BSA, samples were probed with primary anti-phospho ERK(Cell Signaling Technology, Inc., Danvers, MA) and b-tubulin(Sigma) antibodies followed by a horseradish peroxidase couplesecondary antibody. Image acquisition was made in Fusion FXimage acquisition system (Vilber Lourmat, Marne la Vallee, France)and bands were quantified ImageJ software (NIH).

qRT-PCR

RNAs from small intestine were prepared immediately afterdissection following Chirgwin’s protocol (Chirgwin et al., 1979)and reverse transcribed using Go Script (Promega France,Charbonnieres, France) according to manufacturer’s instruction.Real-time quantitative PCR was performed in a Stratagene cyclerusing Takara reagents with the forward 50-GGATCTGCAAGA-CAGACAAGATGCTTCCC-30 and reverse 50-GGAGGGAA-GGGCCAGAGAAGG-30 primers for PAP/HIP amplification. Thehousekeeping gene cyclophilin was used as control and amplifiedwith the forward 50-TCCAAAGACAGCAGAAAACTTTCG-30and reverse 50-TCTTCTTGCTGGTCTTGCCATTCC-30primers, respectively. mRNA PAP/HIP abundance in saline-treatedWT mice was taken as 1.

Immunohistochemistry of human fat tissues

Visceral tissue specimens of white human fat were obtained fromnon-obese (four samples) and obese (four samples) undergoingabdominal surgery. Formalin fixed white fat was sectioned,deparaffinized and unmasked by using heat treatment in sodiumcitrate buffer (pH 6.0 at 10mmol/L) at 95˚C for 15min beforeblocking endogenous peroxidase with 0.3% H2O2 for 15min.The sections were pre-incubated with 2% normal swine serumand were exposed to the 1:100 dilution of the polyclonal rabbitanti-human PAP/HIP for 2 h and revealed as previously published(Gironella et al., 2005).

Subjects and serum PAP/HIP levels

A group of 30 subjects (24 female and 6 males, age range 42–59years) were considered to be severe obese on the basis of theirbody mass index (BMI) value of >35. BMI was defined as weightin kg divided by height in meters (kg/m2). Thirty healthy non-obese(BMI< 30) subjects (20 female and 10 males, age range 40–60years) served as a control group. None of the subjects studied hadany clinical or laboratory (e.g., fasting blood glucose, urea andelectrolytes, liver and thyroid function tests) signs of diseaseassociated with obesity and were taking no drugs. Serum PAP/HIPlevels were determined with an ELISA kit (PANCREPAP, Dynabio,Marseille, France). This kit is a sandwich immunoenzymatic systemutilizing monoclonal antibodies that recognizes specifically PAP/HIP as previously showed (Nitta et al., 2012). Linearity was gainedbetween 0.1 and 30 ng/ml on a standard curve of this assay.

Statistical analysis

Data shown in the figures indicate the means� SE. Differencesbetween groups were compared using the non-parametricMann–Whitney U-test. Asterisks in the figures indicate statisticallysignificant differences (P< 0.05). Statistical analysis of therelationship between body weight and serum hPAP/HIP data wasconducted using the SAS version 9.3 computer program (SASInstitute Inc., Cary, NC). Spearman’s rank correlation coefficientwas applied for the analysis of significance of the correlation.

ResultsPAP/HIP transgenic mice are obese

Twenty TgPAP/HIP and 13 control male mice were nourishedwith standard Purina diet. Animal weight was recorded every3 weeks starting at 6 weeks old and for a period of 27 weeks,results are shown in Figure 1A,B. Control animals started witha weight of 21� 1.2 g and attained 29� 2.4 g at 15 weeksold and then they did not show more significant additionalincrease. TgPAP/HIP mice weight was higher at 6 weeks old(24� 1.0 g) and increased progressively until 18 weeks old(46.3� 4.5 g) with an additional slow increase until 27 weeks

JOURNAL OF CELLULAR PHYSIOLOGY

226 S E C Q E T A L .

old (51� 4.9 g). At the end of the experiment, animal weresacrificed and liver, kidney, testes, lungs, heart, stomach, andpancreas were dissected and weighted. To our surprise, notsignificant differences were found between these organs fromTgPAP/HIP and control mice (data not shown). However,macroscopic inspection showed that intra-peritoneal, retro-peritoneal, and peri-cardiac fat accumulation was moreabundant in TgPAP/HIP than in control mice (data not shown).

Moreover, we measured serum hPAP/HIP levels in TgPAP/HIP mice at 15 weeks old and correlated these data with theirbody weight. As shown in Figure 1D, there is a significantcorrelation (r2¼ 0.594 and P-value¼ 0.0017) between bodyweight and serum hPAP/HIP levels.

We also measured levels of glucose, triglycerides, LDL, andHDL. To our surprise, TgPAP/HIP mice showed abnormalvalues with increased glycemia (123� 19mg/dl vs. 179�8mg/dl), decreased levels of triglycerides (77� 8mg/dlvs. 57� 5mg/dl), increased levels of LDL (13� 1.5mg/dlvs. 18.5� 2.1mg/dl), and decreased HDL (58� 3mg/dl vs.44� 3.5mg/dl) indicating that high serum levels of PAP/HIPinduce a metabolic syndrome accompanied of obesity. Finally,we also measured leptin serum levels that, as expected due tothe increased fat mass in TgPAP/HIP mice, was significantlyhigher than control mice (Fig. 2).

PAP/HIP/ mice are slim

Ten PAP/HIP�/� mice and 10 wild-type counterparts were fedas described above and their weight changes followed from 6 to27 weeks old. As expected, wild-type mice progressivelygained weight from 6 to 15 weeks old, and then this gain wasless obvious until 27weeks old (Fig. 1C). In contrast, PAP/HIP�/

� mice showed a more limited increase in weight gain since at6 weeks old their weight was 20� 0.7 g and at the end of therecord their weight was 25.5� 2.4 g.

Small intestine expresses PAP/HIP mRNA as a responseto eating

Several years ago we described that starved animals decreaseddramatically the expression of PAP/HIP in the epithelia of thesmall intestine (Iovanna et al., 1993). We hypothesized thatexpression of the PAP/HIP in intestine is regulated by thenutriments present in the lumen of the gut. To test thishypothesis we treated six mice by an intragastric gavage with400ml of olive oil and measured the expression of the PAP/HIPtranscript in the mucosa of small intestine. As shown inFigure 3, expression of PAP/HIP mRNA was transientlyincreased to 5.5� 1.7-, 8.8� 1.9-, and 4.3� 1.1-fold after 60,90, and 120min of treatment, respectively. Signalization ofPAP/HIP mRNA expression is at least in part mediated by theJak-STAT3 cascade in epithelial cells (Folch-Puy et al., 2006).We therefore pretreated six mice with AG490, a potent Jakinhibitor, and measured expression of the PAP/HIP mRNAin response to the intragastric gavage. As demonstratedin Figure 3, induction of PAP/HIP transcript was almostcompletely abolished indicating a major Jak-STAT3dependence.

PAP/HIP protein induces adipocyte growth

3T3-L1 cells are pre-adipocytes that can be differentiatedto adipocytes under appropriate culture conditions (Zebischet al., 2012). We treated these cells by increasing

Fig. 1. Effects of PAP/HIP on body weight. Growth curves of transgenic TgPAP/HIP and control mice (A) or PAP/HIP/ and control (C)maintained on a standard chow diet for 27 weeks. B: representative picture of TgPAP/HIP and control mice. D: Body weight at 15 weeks old isplotted against hPAP/HIP values. Values are means�SE; �P< 0.05 TgPAP/HIP versus control (A) and PAP/HIP/ versus control (C).

JOURNAL OF CELLULAR PHYSIOLOGY

P A P / H I P A N D O B E S I T Y 227

concentration of recombinant PAP/HIP and estimated itsgrowth-induction capacity by measuring the BrdU incorpo-ration. As shown in Figure 4A, 0.1 ng/ml had no effect, whereas1 ng/ml or more as much as 10 or 100 ng/ml significantlystimulated BrdU incorporation. These results clearly indicatethat PAP/HIP acts as an inductor of pre-adipocyte growth.

PAP/HIP protein activates phospho-ERK and adipocytesgrowth is sensitive to the MEK inhibitor U0126

Incubation of the 3T3-L1 cells with 10 ng/ml of PAP/HIPactivates phospho-ERK after 30min (2.1� 0.2) of treatmentand 3.3� 0.8 and 3.2� 0.6 after 60 and 90min, respectively.We then evaluated the role of this pathway on the growth of3T3-L1 cells by inhibiting MEK activity by using the U0126inhibitor and found that PAP/HIP-dependent growth wascompletely inhibited as shown in Figure 4A. These resultsstrong suggest that PAP/HIP-dependent growth is mediated byMEK/pERK intracellular pathway.

PAP/HIP protein is expressed in fat tissue

Mature fat cells were strongly stained with anti-human PAP/HIP antibody. Themost prominent stainingwas observed in theperinuclear area with in some cases in the small cytoplasmicrim of the adipocytes in both obese and non-obese individuals(Fig. 5). No other cells of the adipose tissue named stromalcells, blood cells, tissue macrophages, and endothelial cellswere significantly stained. Quantification expressed as percentof positive stained cells as well as intensity of stained cells

Fig. 2. Blood concentration of glucose, triglycerides, LDL, HDL, and leptin in TgPAP/HIP mice. Blood was collected at 15 weeks old ofTgPAP/HIP and control mice and concentration of glucose, triglycerides, LDL, HDL and leptin was measured. Values are means�SE;n¼ 20 per group. �P< 0.05 TgPAP/HIP versus control.

Fig. 3. PAP/HIP mRNA is induced in intestinal epithelial cells inresponse to intragastric gavage with olive oil. Three-month-oldmale SV129 mice were starved for 24h following by an intragastricgavage with 400ml of olive oil or saline (blue bars). Distal part of thesmall intestine was recovered after 300, 600, 900, 1200, and 1800 oftreatment. A group of animals was pretreated with AG490 beforethe intragastric gavage (red bars). mRNA PAP/HIP abundance insaline-treated WT mice was taken as 1. Values are means�SE;n¼ 6 per group. �P< 0.05 of values against time 0; #P< 0.05 of valuesfrom mice treated with AG490 versus treated with PBS.

JOURNAL OF CELLULAR PHYSIOLOGY

228 S E C Q E T A L .

Fig. 4. A: Recombinant PAP/HIP promotes cell growth of the pre-adipocytes 3T3-L1. Proliferation of 3T3-L1 cells was evaluated by BrdUincorporation assay after 24h of incubation. MEK inhibitor U0126 treatment prevents the effect of PAP/HIP. Values are means�SE; n¼ 5 perpoint. �P< 0.05 of values against untreated cells. B: PAP/HIP activates pERK. 3T3-L1 cells were treated with 10ng/ml of PAP/HIP andphosphorylation of ERK was measured by immunohistochemistry.

Fig. 5. A: Immunohistochemical detection of PAP/HIP protein in sections of visceral fat tissue. Staining of mature adipocytes was observedin the perinuclear area and in the small cytoplasmic rim of the adipocytes in both obese and non-obese individuals. B: PAP/HIP serumconcentration in patients with severe obesity. PAP/HIP concentration was measured in serum from 30 obese patients and 30 controls using anELISA system. Data given as box and whiskers: the box extends from 25th to 75th percentile, with a horizontal line at the median, whiskersextend up to the largest and down to the smallest value. Black dots represent the extreme values.

JOURNAL OF CELLULAR PHYSIOLOGY

P A P / H I P A N D O B E S I T Y 229

showed not significant differences between samples fromobese and non-obese patients (data not shown).

Obese patients have high PAP/HIP serum level

As shown in Figure 5, there was a high significant difference(P¼ 0.0001) in serum PAP/HIP levels between patients withsevere obesity with a mean of 180.20 (21–439) and levels of thecontrol (non-obese) subjects with a mean of 54.63 (7–245). Nosignificant differences were found between males and females.

Discussion

In this article we report a new role for the acute phase proteinPAP/HIP, namely obesogenic. In fact, we describe here thatTgPAP/HIP mice develop spontaneous obesity under standardnutritional conditions, with a blood profile compatible with ametabolic syndrome and, on the contrary, that PAP/HIP-deficient mice are slim when submitted to a standard chow assingle nutrients. In addition, we found that expression of PAP/HIP is induced in epithelial intestinal cells by the presence offood into the lumen of the gut, and that recombinant PAP/HIPis able to induce growth of pre-adipocyte cells in vitro. Finally,patients with severe obesity present higher blood levels ofPAP/HIP than individuals with a BMI lower than 30. Altogetherour data suggest that PAP/HIP could be a mediator of fat tissuedevelopment, released by the intestine and induced by thepresence of food in the gut.

Ametabolic function of PAP/HIPwas suspected from severalyears by two main facts; the first one is that regulation of PAP/HIP expression is under a strict control of nutrients (Iovannaet al., 1993; Sansonetti et al., 1995) and the second one is thatPAP/HIP expression is strong activated by leptin (Waelputet al., 2000), the hormone involved in several metabolic aspectsand the main regulator of the fat tissue functions(Friedman, 1998; Friedman and Halaas, 1998). Whether or notfood-induced expression of PAP/HIP in intestine is mediated bygastric leptin (Bado et al., 1998) remains to be demonstrated;however, that is clearly showed here is that its induction is Jak-STAT3 dependent since its activation in vivo is almostcompletely AG490 sensitive (see Fig. 3).

In addition to its food-regulated expression in intestine,expression of PAP/HIP is in fact regulated as a typical acutephase protein in several other tissues, particularly in pancreasand colon in response to inflammation (Iovanna et al., 1991;Gironella et al., 2005) as well as in other diseased tissues (Konget al., 2005; Planas et al., 2010; Nitta et al., 2012). This must beparticularly taken into account since PAP/HIP could be a factorresponsible of storing sufficient quantities of energy-densetriglyceride in adipose tissue allowing survival during thefrequent periods of stress and energy demanding that aregenerally accompanied by prolonged diseases.

Among the factors activated through the Jak-STAT3-dependent pathway, including leptin, IL6, or CNTF(Febbraio, 2007), the only protein that have an adipogenicfunction is PAP/HIP, the other factors, on the contrary, arecachexiogenic. In addition, other cachexiogenic factors such asIFNg and TNFa (Tisdale, 1999) also activate PAP/HIPexpression through Jak-STAT3-independent mechanisms(Dusetti et al., 1995). It is important to be noted that, all thesecachexiogenic factors are able to activate expression of PAP/HIP, and PAP/HIP is in turn able to activate its own expression(Folch-Puy et al., 2006), suggesting an evolution conservedmechanism directed against stress- or inflammation-inducedcachexia. An important point to be noted is that at least in vitrothe molecular pathway utilized by PAP/HIP is dependent ofthe MEK/pERK intracellular signaling as demonstrated inFigure 4A,B.

Lastly, another interesting point is the fact that patients withsevere obesity present sustained high levels of serum PAP/HIP.There are two possibilities to explain it since fat tissue fromnon-obese and obese patients produce similar amounts of PAP/HIP; the first one is that obesity is a systemic inflammatorydisease (Stienstra et al., 2012) with high circulating levels ofseveral cytokines, and these cytokines would be able toactivate expression of PAP/HIP in almost all tissues resulting ina systemic increase of PAP/HIP levels; the other possibility isthat fat tissue produce and release leptin (Considineet al., 1996) which in turn would induce expression of PAP/HIPfrom several peripheral tissues. Obviously both possibilitiesmay occur simultaneously. Moreover, PAP/HIP is able toinduce its own expression forming a positive feedback loop.

In conclusion, in this article we report a previouslyundescribed obesogenic function of PAP/HIP protein.

Acknowledgments

We thank the cell culture platform and the mice colony facility(Cell stress, Unit�e 1068).We thank M.N. Lavaut, R. Magro, andK. Sari for technical assistance and P. Berthezene for statisticalanalysis. This work was supported by grants from the INSERMto J.L.I.

Literature Cited

Bado A, Levasseur S, Attoub S, Kermorgant S, Laigneau JP, Bortoluzzi MN, Moizo L, Lehy T,Guerre-Millo M, Le Marchand-Brustel Y, Lewin MJ. 1998. The stomach is a source ofleptin. Nature 394:790–793.

Cash HL, Whitham CV, Behrendt CL, Hooper LV. 2006. Symbiotic bacteria directexpression of an intestinal bactericidal lectin. Science 313:1126–1130.

Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. 1979. Isolation of biologically activeribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299.

Closa D, Motoo Y, Iovanna JL. 2007. Pancreatitis-associated protein: From a lectin to an anti-inflammatory cytokine. World J Gastroenterol 13:170–174.

Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, OhannesianJP, Marco CC, McKee LJ, Bauer TL, Caro JF. 1996. Serum immunoreactive-leptinconcentrations in normal-weight and obese humans. N Engl J Med 334:292–295.

Dusetti NJ, Ortiz EM,Mallo GV, Dagorn JC, Iovanna JL. 1995. Pancreatitis-associated proteinI (PAP I), an acute phase protein induced by cytokines. Identification of two functionalinterleukin-6 response elements in the rat PAP I promoter region. J Biol Chem270:22417–22421.

Febbraio MA. 2007. gp130 receptor ligands as potential therapeutic targets for obesity. J ClinInvest 117:841–849.

Ferres-Maso M, Sacilotto N, Lopez-Rodas G, Dagorn JC, Iovanna JL, Closa D, Folch-Puy E.2009. PAP1 signaling involves MAPK signal transduction. Cell Mol Life Sci 66:2195–2204.

Folch-Puy E, Granell S, Dagorn JC, Iovanna JL, Closa D. 2006. Pancreatitis-associated proteinI suppressesNF-kappa B activation through a JAK/STAT-mediatedmechanism in epithelialcells. J Immunol 176:3774–3779.

Friedman JM. 1998. Leptin, leptin receptors, and the control of body weight. Nutr Rev 56:s38–s46;discussion s54–s75.

Friedman JM, Halaas JL. 1998. Leptin and the regulation of body weight in mammals. Nature395:763–770.

Gironella M, Iovanna JL, Sans M, Gil F, Penalva M, Closa D, Miquel R, Pique JM, Panes J. 2005.Anti-inflammatory effects of pancreatitis associated protein in inflammatory boweldisease. Gut 54:1244–1253.

GironellaM, Folch-Puy E, LeGoffic A, Garcia S, Christa L, Smith A, Tebar L, Hunt SP, Bayne R,Smith AJ, Dagorn JC, Closa D, Iovanna JL. 2007. Experimental acute pancreatitis in PAP/HIP knock-out mice. Gut 56:1091–1097.

Iovanna JL, Dagorn JC. 2005. The multifunctional family of secreted proteins containing a C-type lectin-like domain linked to a short N-terminal peptide. Biochim Biophys Acta1723:8–18.

Iovanna JL, Keim V, Michel R, Dagorn JC. 1991. Pancreatic gene expression is altered duringacute experimental pancreatitis in the rat. Am J Physiol 261:G485–G489.

Iovanna JL, Keim V, Bosshard A, Orelle B, Frigerio JM, Dusetti N, Dagorn JC. 1993. PAP, apancreatic secretory protein induced during acute pancreatitis, is expressed in ratintestine. Am J Physiol 265:G611–G618.

Kong SW, Bodyak N, Yue P, Liu Z, Brown J, Izumo S, Kang PM. 2005. Genetic expressionprofiles during physiological and pathological cardiac hypertrophy and heart failure in rats.Physiol Genomics 21:34–42.

Livesey FJ, O’Brien JA, Li M, Smith AG, Murphy LJ, Hunt SP. 1997. A Schwann cell mitogenaccompanying regeneration of motor neurons. Nature 390:614–618.

Moucadel V, Soubeyran P, Vasseur S, Dusetti NJ, Dagorn JC, Iovanna JL. 2001. Cdx1promotes cellular growth of epithelial intestinal cells through induction of the secretoryprotein PAP I. Eur J Cell Biol 80:156–163.

Nishimune H, Vasseur S, Wiese S, Birling MC, Holtmann B, Sendtner M, Iovanna JL,Henderson CE. 2000. Reg-2 is a motoneuron neurotrophic factor and a signallingintermediate in the CNTF survival pathway. Nat Cell Biol 2:906–914.

Nitta Y, Konishi H, Makino T, Tanaka T, Kawashima H, Iovanna JL, Nakatani T, Kiyama H.2012. Urinary levels of hepatocarcinoma-intestine-pancreas/pancreatitis-associatedprotein as a diagnostic biomarker in patients with bladder cancer. BMC Urol 12:24.

Ortiz EM, Dusetti NJ, Vasseur S, Malka D, Bodeker H, Dagorn JC, Iovanna JL. 1998. Thepancreatitis-associated protein is induced by free radicals in AR4-2J cells and confers cellresistance to apoptosis. Gastroenterology 114:808–816.

JOURNAL OF CELLULAR PHYSIOLOGY

230 S E C Q E T A L .

Planas R, Carrillo J, Sanchez A, de Villa MC, Nunez F, Verdaguer J, James RF, Pujol-Borrell R,Vives-Pi M. 2010. Gene expression profiles for the human pancreas and purified islets intype 1 diabetes: New findings at clinical onset and in long-standing diabetes. Clin ExpImmunol 159:23–44.

Sansonetti A, Romeo H, Berthezene P, Scacchi P, Dusetti N, Keim V, Dagorn JC, Iovanna JL.1995. Developmental, nutritional, and hormonal regulation of the pancreatitis-associatedprotein I and III gene expression in the rat small intestine. Scand J Gastroenterol 30:664–669.

Simon MT, Pauloin A, Normand G, Lieu HT, Mouly H, Pivert G, Carnot F, Tralhao JG,Brechot C, Christa L. 2003. HIP/PAP stimulates liver regeneration after partial

hepatectomy and combines mitogenic and anti-apoptotic functions through the PKAsignaling pathway. FASEB J 17:1441–1450.

Stienstra R, Tack CJ, Kanneganti TD, Joosten LA, Netea MG. 2012. The inflammasome putsobesity in the danger zone. Cell Metab 15:10–18.

Tisdale MJ. 1999. Wasting in cancer. J Nutr 129:243S–246S.WaelputW, Verhee A, Broekaert D, Eyckerman S, Vandekerckhove J, Beattie JH, TavernierJ. 2000. Identification and expression analysis of leptin-regulated immediate early responseand late target genes. Biochem J 348:55–61.

Zebisch K, Voigt V, Wabitsch M, Brandsch M. 2012. Protocol for effective differentiation of3T3-L1 cells to adipocytes. Anal Biochem 425:88–90.

JOURNAL OF CELLULAR PHYSIOLOGY

P A P / H I P A N D O B E S I T Y 231