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Molecular and Cellular Endocrinology 264 (2007) 16–27

Prolactin-induced changes in protein expression in human pancreatic islets

L. Labriola a, G. Bomfim Ferreira a, W.R. Montor a, M.A.A. Demasi a, D.C. Pimenta c,F.H. Lojudice a, T. Genzini b, A.C. Goldberg a, F.G. Eliaschewitz b, M.C. Sogayar a,∗

a Department of Biochemistry, Chemistry Institute, University of Sao Paulo, Av. Prof. Lineu Prestes, 748,Bloco 9 Superior Sala 964, Sao Paulo 05508-900 SP, Brazil

b Albert Einstein Hospital, Sao Paulo, Brazilc Centro de Toxinologia Aplicada, Instituto Butantan, Sao Paulo, Brazil

Received 26 April 2006; received in revised form 2 October 2006; accepted 3 October 2006

bstract

Ex vivo islet cell culture prior to transplantation appears as an attractive alternative for treatment of type 1 diabetes.Previous results from our laboratory have demonstrated beneficial effects of human prolactin (rhPRL) treatment on human islet primary cultures.In order to probe into the molecular events involved in the intracellular action of rhPRL in these cells, we set out to identify proteins with altered

xpression levels upon rhPRL cell treatment, using two-dimensional (2D) gel electrophoresis and mass spectrometry (MS).An average of 300 different protein spots were detected, 14 of which were modified upon rhPRL treatment (p < 0.01), of which 12 were

uccessfully identified using MS and grouped according to their biological functions.In conclusion, our study provides, for the first time, information about proteins that could be critically involved in PRL’s action on human

ancreatic islets, and facilitate identification of new and specific targets involved in islet cell function and proliferation.2006 Published by Elsevier Ireland Ltd.

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eywords: Diabetes; Insulin; Human islets; Prolactin; Proteomics

. Introduction

Type 1 diabetes is an insulin-deficient condition resultingrom the autoimmune destruction of pancreatic beta cells. Inome cases, transplantation of isolated pancreatic islets fromadaveric organ donors is a promising possibility for treatmentf type 1 diabetes. However, this approach is severely lim-ted by the shortage of organ donors. Ex vivo islet cell culturerior to transplantation is as an attractive alternative, however,ong-term maintenance of human islets in culture has been a dif-cult task. Therefore, stimulation of islet cell proliferation andifferentiation in vitro remains a major scientific and clinicalhallenge.

The lactogenic hormones prolactin (PRL) and placental lac-ogen (PL) play an important role in the upregulation of islet
ell function during gestation (Sorenson and Brelje, 1996),eading to increased beta cell proliferation, islet cell mass,nd insulin synthesis and secretion, as well as to a decreased
∗ Corresponding author. Tel.: +55 11 3091 3820; fax: +55 11 3091 3820.E-mail address: [email protected] (M.C. Sogayar).

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303-7207/$ – see front matter © 2006 Published by Elsevier Ireland Ltd.oi:10.1016/j.mce.2006.10.004

hreshold for glucose-stimulated insulin secretion (Nielsen etl., 2001; Sorenson and Brelje, 1996; Weinhaus et al., 2000,996). Besides these stimulatory effects of PRL, a role forhis hormone in regulation of beta cell death and survivalas recently been reported (Bordin et al., 2004; Jensen et al.,005).

In rat fetuses and in pregnant mothers, the long form ofhe pancreatic prolactin receptor (PRLR) is expressed predom-nantly (Asfari et al., 1995), and, upon binding to the hormone,ctivates the receptor-associated Janus kinase (JAK2), leadingo phosphorylation, dimerization and nuclear translocation ofignal transducer and activator of transcription (STAT) pro-eins, which bind to specific consensus DNA sequences in theromoters of target genes and thereby regulate transcriptionCarter-Su and Smit, 1998). In beta cells, PRL primarily acti-ates STAT5a and STAT5b and, to a lesser extent, STAT1 andTAT3 (Galsgaard et al., 1996, 1999). Previous results from our

aboratory have demonstrated a significant beneficial effect of
ecombinant human prolactin (rhPRL) treatment on cell prolif-ration and secretory function of human islet primary cultures asell as protein expression and phosphorylation levels of JAK2
nd STAT1, 3 and 5 (Labriola et al., 2007).

mailto:[email protected]

dx.doi.org/10.1016/j.mce.2006.10.004

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L. Labriola et al. / Molecular and C

Until now, molecular mechanisms of PRL action in pancreaticslets have only been addressed in animal models and none ofhem use a proteomic approach.

Two-dimensional (2D) gel electrophoresis coupled with masspectrometry (MS) is a powerful methodology that allows theirect measurement of relative protein levels in tissues, cells andubcellular compartments and their response to specific stimulind pathological states such as cancer and diabetes (Jiang et al.,003; Li et al., 2003; Sparre et al., 2005).

In order to probe into the molecular events involved in thentracellular action of rhPRL in human pancreatic islets, weet out to identify proteins with altered expression levels uponhPRL cell treatment, using 2D gel electrophoresis and MS.n addition, in an attempt to validate the results obtained, weonfirmed, using Western blot, the protein identity and thexpression levels of some of the protein spots, upon rhPRL cellreatment.

This study provides, for the first time, information about sev-ral proteins that could be critically involved in the action ofRL on human pancreatic islets, and facilitate identification ofew and specific molecular targets involved in islet cell functionnd proliferation.

. Materials and methods

.1. Isolation and culture of islets

Human pancreas from adult brain-dead donors (mean age 45 ± 3 years,= 14) were harvested in accordance with Brazilian regulations and the local

nstitutional Ethical Committee. Pancreatic islets were isolated after ductalistension of the pancreas and digestion of the tissue with Liberase (Rocheiagnostics, Indianapolis, IN) according to the automated method of Ricordi et

l. (1988) with modifications (Shapiro et al., 2000). Purification was achievedsing a continuous Ficoll density gradient in a COBE 2991 cell processorGambro, Lakewood, CO). The islet preparations used in this study exhibited70 ± 4% purity as determined by dithizone staining and islet cell viability,hich was evaluated using the live/dead fluorescent method based on the incor-oration of either acridine orange by live cells (SIGMA, St. Louis, MO) orropidium iodide by dead cells (SIGMA, St. Louis, MO), being usually greaterhan 80%. Upon isolation, the islets (2 × 104 IEQ/100 cm2) were first main-ained in CMRL 1066 medium (5.6 mM glucose) (Mediatech-Cellgro, Miami,L) supplemented with 100-units/mL penicillin and 5% fetal calf serum (FCS)Cultilab, Campinas, SP, Brazil) 24–48 h. Before starting the incubation withecombinant human prolactin (rhPRL), cells were serum-starved in CMRL066 medium supplemented with 0.1% FCS for 24 h. On the following day,he cells were incubated with CMRL supplemented with 0.5% FCS and eitherhPRL (300 ng/ml) produced in our laboratory using an insect expression sys-em (Lawson et al., 1982; Sapin et al., 2001; Pereira et al., 2001) or vehicle fordays.

.2. Sample preparations

Primary cultures of human islets (passage 1–3) were washed with ice-coldBS and then lysed by scraping at 4 ◦C in a lysis buffer (10 mM Tris pH: 7.5,50 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 �g/ml aprotinin,0 �g/ml leupeptin, 5 �g/ml pepstatin, 1 mM PMSF, 25 mM NaF, 1% NP-40,.1% SDS, 0.5% sodium deoxycholate and 1 mM Na3VO4). The homogenateas centrifuged at 4 ◦C for 30 min at 12,000 × g and the supernatant fraction was

hen collected and stored at −70 ◦C. The protein content in these extracts wasuantified using a Bio-Rad kit (Bio-Rad, Richmond, CA). Aliquots containing00 �g of proteins were treated with 2D Clean-up kit (Amersham Bioscienes-GEealthcare, Buckinghamshire, UK), following the manufacturer’s instructions.he protein pellets were resuspended in rehydration solution for the isoelectric

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r Endocrinology 264 (2007) 16–27 17

ocusing (8 M urea; 2% CHAPS; 0.5% Pharmalytes 3–10 NL; 13 mM DTT) androtein concentration was determined using a Bio-Rad kit (Bio-Rad, Richmond,A). Approximately 100 �g of proteins were loaded on each strip.

.3. Two-dimensional gel electrophoresis (2DGE)

Individual 11 cm IPG strips, pH 3–10 NL, were rehydrated in 250 �L ofample, which was solubilized in a buffer containing 8 M urea; 2%CHAPS;.5% Pharmalytes 3–10 NL; 13 mM DTT and traces of bromophenol blue.n-gel sample rehydration was allowed to proceed at 20 ◦C for 15 h. The rehy-rated strips were focused on a Multiphor II system (Amersham Biosciences-GEealthcare) for about 39 kV × h at a maximum of 3.5 kV in a rapid voltage

amping mode with a maximum current per strip of 50 �A. Focused strips weretored at −70 ◦C, or directly used for the second dimension. Equilibration ofhe IPG strips after isoelectric focusing was performed in two steps: reduction0.05 M Tris–Cl pH 6.8; 6 M urea; 30% glycerol, 1% de SDS; 0.25% DTT),ollowed by alkylation (0.05 M Tris–Cl pH 8.8; 6 M urea; 30% glycerol, 1%e SDS, 4.5% iodoacetamide and traces of bromophenol blue). Each step waserformed for 10 min on a standard benchtop rocker. The second dimension wasun on a 12.5% polyacrylamide SDS gel (25 cm × 20 cm × 0.5 cm; Ettan Daltix-Amersham Biosciences-GE Healthcare, Buckinghamshire, UK). Each gelas loaded with two strips, each of which containing electrofocused proteins

rom total cell extracts corresponding to the same islet preparation, incubatedn the presence of either vehicle only or rhPRL (300 ng/ml). The gels wereun at 25 ◦C at a constant power of 5 W/gel for 30 min, followed by 17 W/gelor 3 h.

.4. Protein visualization

After 2D electrophoresis, the gels were fixed with 50% ethanol, 10% aceticcid for 30 min and washed twice with an aqueous solution of 30% ethanol for0 min and once with Milli-Q water for 30 min. The 2D gels were then stainedith colloidal Coomassie Blue G-250 (17% (NH4)2SO4; 0.1% Coomassie Bril-

iant Blue; 3% H3PO4; 33% methanol) for 16 h with gentle shaking. To removehe staining reagent excess, the gels were washed once with an aqueous solutionf 25% ethanol and then stored in 20% (NH4)2SO4 at 4 ◦C.

. Image analysis

The stained gels were imaged using a UMAX UTA100 (Hewlett Packard, GE Healthcare), calibrated densitome-er (Amersham Biosciences-GE Healthcare, Buckinghamshire,K). Raw scans were processed by the 2D gel analysis soft-are, ImageMaster Platinum 5.0 (Amersham Biosciences-GEealthcare). For between-gel comparisons, a set of parameters

smooth, saliency and minimum area) was used. To validate spotetection, the images were edited manually and streaks, specklesnd artifacts were removed. Spot patterns of different gels wereatched to each other and each spot was given a unique identi-cation number. After background subtraction, the spot volumeas normalized as a percentage of the total volume of all spotsithin the gel to minimize the effect of experimental factors onrotein spots. To find spots that differed significantly betweenreatments, the average normalized volumes (IOD) of resolvedpots were compared using quantitative, qualitative and statis-ical functions within the Image Master Platinum software. Foreliable matching, 20 different landmark proteins were manu-lly added to each gel. Significant changes between spots were
etermined using a two-tailed Student’s t-test for non-pairedbservations. Changes with a p value of less then 0.05 were con-idered statistically significant. Only matched spots presentingt least a 2-fold difference between rhPRL and vehicle-treated

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ell extracts from six different islet preparations were considereds regulated spots.

.1. Determination of molecular weight (Mr) and pI

Molecular weight values for the individual proteins on theels were interpolated from SDS-PAGE Standards (Invitrogen,arlsbald, CA), which consisted of multiple proteins with aolecular weight values ranging from 15 to 170 kDa. The pIas directly calculated by interpolation from the pI in the linearradient of the strips (pH 3–10).

.2. Protein identification by mass spectrometry

The samples were analyzed by MALDI-TOF mass spec-rometry using �-cyano-4-hydroxycinnamic acid as matrix onn Ettan Maldi-Tof/Pro instrument (Amersham Biosciences-E Healthcare), according to (Westermeier and Naven, 2002).riefly, gel spots were picked, destained, and treated withTT and IAA for reduction and alkylation of eventual disul-de bridges. The treated spots were then rehydrated with a0 ng/�L sequence grade trypsin solution (Promega, WI) in0 mM ammonium bicarbonate and the digestion was carriedut overnight at 37 ◦C. The tryptic peptides were extracted fromhe gel using an H2O/acetonitrile (ACN)/trifluoroacetic acidTFA) (1:1:0.05) solution incubated under vigorous agitation.he peptide solution was concentrated and desalted with ZipTip-18 (Millipore, Bedford, MA) prior to analysis. The samplesere pre-mixed with the matrix solution (1:1, v/v) and dupli-

ates of approximately 0.4 �L were deposited on the sampleoader and allowed to dry over the bench. Mass spectrometryas performed in the reflectron mode. All mass spectra were

alibrated with trypsin autolysis products; aminoacid sequence08–115 (MH+ = 842.51) and sequence 58–77 (MH+ = 2211.10)nd masses of known contaminants, e.g. keratin, were removed.xternal calibration was also performed in the case when

rypsin peptides were not present or clear. The obtained massist was analyzed by peptide mass fingerprinting algorithmsLDENTE (www.expasy.org/tools/aldente) (Gasteiger et al.,003) for matches with known Homo sapiens protein sequences,eposited on the SWISS-Prot and TrEBML public database.he parameters used for searches were: Monoisotopic masses, aaximum 0.5 Da mass tolerance, cysteine in carbamidomethyl

orm (fixed), methionine in oxidized form (variable), allowanceor up to one missed cleavage. The criteria used to accept pro-ein identification included the extent of sequence coverage,umber of peptides matched, score (calculated as −10 × log(p-alue), where the p-value is the probability of the observedatch being a random event, based on Swiss-Prot PMF database,

sing the Aldente software-threshold = 50). It is important toote that the majority of the proteins were identified as a singleandidate.

.3. Protein information

Information about identified proteins and putative func-ions was found at the ExPASy Molecular Biology Server

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www.expasy.org) and at the NCBI, which were accessedetween December 2004 and February 2006.

.4. Western blots

Total extracts were prepared from primary cultures of pan-reatic islets subjected to the treatments described in the “Iso-ation and culture of islets” section. Equal amounts (100 �g)f proteins from each extract were solubilized in sample buffer60 mM Tris–HCl [pH 6.8], 2% sodium dodecyl sulfate (SDS),0% glycerol, 0.01% bromophenol blue) and subjected to SDS-olyacrylamide gel (7.5 or 12%) electrophoresis (PAGE). Pro-eins were transferred to nitrocellulose membranes, which werelocked and then incubated with the following antibodies:ouse monoclonal anti-HSP27 (F-4); rabbit polyclonal anti-

ropomyosin (FL-284); mouse monoclonal anti-l-caldesmon (F-0); all of which were purchased from Santa Cruz Biotechnologync., Santa Cruz, CA, and mouse monoclonal anti-PDI (RL77)Affinity Bio Reagents, Golden, CO). The membranes were thenncubated with horseradish peroxidase-conjugated secondaryntibody (Vector Laboratories, Burlingame, CA). Enhancedhemiluminescence was performed according to the manufac-urer’s instructions (Amersham Biosciences, Buckinghamshire,K). As a loading control, the membranes were stripped and re-robed with anti-�-tubulin rabbit polyclonal antibody (kindlyrovided by Dr. Frank Solomon, Massachusetts Institute ofechnology, MS). Quantitative densitometry was carried outith a HP-Scanjet 3500 (Hewlett Packard) scanner and the

mage Quant 5.2 (Molecular Dynamics, Amersham Biosciences,uckinghamshire, UK) software. The volume density of thehemiluminescent bands (n ≥ 4 experiments performed in dupli-ate with each islet preparation) was calculated as IOD × mm2

fter background correction.

.5. Statistical analysis

The statistical differences between group means were testedy unpaired two-tailed Student’s t-test. A “p” value <0.05 wasonsidered statistically significant.

. Results

.1. Global protein patterns of human pancreatic isletsxposed to recombinant human prolactin (rhPRL)

We have previously found that rhPRL stimulates both prolif-ration and insulin production and secretion in primary culturesf human pancreatic islets (Labriola et al., 2007). These resultsncouraged us to utilize the proteomic approach to identify pos-ible targets involved in the molecular mechanisms of PRLction, since the 2D gel electrophoresis strategy offers greatesolving power and permits the separation of a large quantityf proteins of wide molecular weight range and allows the iden-
ification of changes in post-translational states of proteins.
The complex structure of pancreatic islets with the presencef different cell types posed challenging problems in proteinxtraction. In order to optimize sample preparation, inclusion

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L. Labriola et al. / Molecular and Cellular Endocrinology 264 (2007) 16–27 19

Fig. 1. Human islet protein maps of human islet cells maintained in the presence or in the absence of rhPRL. Representative 2D gels of six independent experimentsanalyzing total protein extracts from primary cultures of human pancreatic islets incubated in the presence or in the absence of rhPRL (300 ng/mL). Approximately100 �g of islet proteins were separated on a 11 cm IPG strip (pH 3–10), followed by 12.5% SDS-PAGE. Proteins were detected by Colloidal Coomassie Blue (G250)s asterw cut-ofs

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taining, and image analysis of scanned gels was carried out using the Image Mhich corresponding to all of the spots that are regulated upon rhPRL treatment (

pots upon hormonal treatment.

f a combination of detergents, such as SDS and NP40, wassed to improve protein solubilization. Cleaning samples with2D Clean-Up Kit (Amersham Biosciences-GE Healthcare)as used to reduce background noise and streaking, improv-

ng isoelectric focusing and reproducibility. In order to minimizeariability between each second dimension experiment, we usedels of an adequate size so as to allow direct the loading of thetrips corresponding to electrofocused proteins from islet cellsf the same preparation treated for 4 days in the presence or inhe absence of rhPRL (300 ng/mL). The 2D map shown in Fig. 1s representative of the high resolution and good reproducibleD pattern obtained from primary cultures of human islets cul-ured in the presence or in the absence of rhPRL (n = 6 differentslets preparations).

After removal of the artifacts and speckles, 269 ± 8 and83 ± 8 spots have been detected in gels corresponding to isletells exposed to either vehicle or rhPRL, respectively. In ordero address sample similarity, the six pairs of gels were matchedsing the Image Master Platinum 5.0 (Amersham Biosciences-E Healthcare) software. Comparison of these gels demon-

trated that most abundant and visible spots were present on allels. This indicates that the protein composition of the humanancreatic islet samples was similar and that assignment of pro-ein identity based on the comparison of spots position in a 2Del is feasible.

.2. Differential protein expression profiles of primaryultures of human pancreatic islets upon rhPRL treatment

Digitalized images of the colloidal Coomassie Blue G-250tained gels were analyzed using the ImageMaster Platinum.0 software (Amersham Biosciences-GE Healthcare). The total

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Platinum Plus software. Twenty-two spots were selected and numbered, 14 off = 2-fold), the remaining 7 (black arrow-heads) corresponding to non-regulated

ensity on a gel image was used to normalize each spot vol-me in this gel image to minimize the effect of experimentalactors on spot volume. Comparative analyses were performedith the mean normalized volume (n = 6 different pairs of gels).irstly the spot volume was determined as IOD × mm2. Sec-ndly an IOD ratio was calculated as the spot volume of thepot corresponding to the rhPRL-treated sample divided by thepot volume of the spot corresponding to the vehicle sample.hirdly we established a cut-off value of 2-fold difference IOD

atio as criteria for significant differential protein expression forregulated spot.

Among the almost 300 spots detected, 14 were consistentlyegulated upon rhPRL treatment. Interestingly, all of these werepregulated (ratio rhPRL/vehicle ≥2) in all gel pairs analyzedn = 6 independent islet preparations).

Fig. 1 shows a representative 2D gel in which all excisedpots, i.e.14 spots and 8 non-regulated spots, have been num-ered and black arrowheads indicate the non-regulated ones.nlargement of some gel areas containing either regulated

Fig. 2A) or non-regulated (Fig. 2B) spots are shown in ordero allow better illustration of the type of regulation observed.uantitative data of those spots are shown in the histogram con-

aining their relative IOD ratio (rhPRL/vehicle) expressed asean ± S.E. for all gels analyzed (n = 6).In order to tackle spot identification, 22 spots (14 regu-

ated and 8 non-regulated by rhPRL treatment) were manuallyxcised from each gel (vehicle and/or rhPRL) and 19 spots,hich correspond to 16 distinct proteins, were characterized by
ALDI-TOF peptide mass fingerprinting (PMF) at 86% success
ate. A representative MALDI-TOF PMF spectrum of elonga-ion factor TU (EFTU), corresponding to spot 10, is shown inig. 3.

20 L. Labriola et al. / Molecular and Cellular Endocrinology 264 (2007) 16–27

Fig. 2. Enlargements of a representative Colloidal Coomasie Blue-stained 2D gel showing differential (A) or unmodified (B) expression of representative 2D spots.Panel C shows results of normalized relative intensity of the same spots. The results are presented as relative increase of normalized densitometric values, with theresults obtained for the control (vehicle) set as 1. The results are presented as the mean ± S.E.M. of at least six independent experiments. a vs. b: p < 0.001.

Fig. 3. Representative MALDI-TOF peptide mass spectra of spot 10 from a total of 4 independent experiments performed. Each experiment led to the identificationof the same protein. The arrows indicate the picks used to identify the protein and “T” indicates the picks corresponding to trypsin autolysis used as the internalcontrol.

L. Labriola et al. / Molecular and Cellular Endocrinology 264 (2007) 16–27 21

Table 1Known and putative functions of the identified proteins

Spot no. Protein name Accession no. IOD ratio Mw (kDa) pI Score Coverage (%)

Theor. Exp. Theor. Exp.

Energy transduction and redox potentials11* Fumarate hydrogenase P07954 2.0 ± 0.1 50 63 ± 5 7.0 7.4 ± 0.1 60 ± 5 13 ± 321 ATPsyntase P25705 1.2 ± 0.3 55 71 ± 3 8.3 8.6 ± 0.4 82 ± 3 18 ± 413* Aconitate hydratase Q99798 2.3 ± 0.1 82 101 ± 5 6.9 7.2 ± 0.2 65 ± 3 10 ± 3

Carbohydrate and lipid metabolism14* Piruvate kinase P14618 2.8 ± 0.4 90 76 ± 6 4.7 8.6 ± 0.1 101 ± 4 23 ± 516* Fructose aldolase P04705 2.9 ± 0.6 39 50 ± 5 8.4 8.9 ± 0.1 70 ± 5 19 ± 4

Protein synthesis, chaperones and protein folding3* GRP96/TRA1/endoplasmin P14625 2.1 ± 0.4 58 107 ± 5 8.0 4.8 ± 0.2 100 ± 3 17 ± 24* Heat shock protein 27 kDa P04792 2.1 ± 0.3 24 29 ± 5 6.0 6.1 ± 0.2 73 ± 6 28 ± 46* l-Caldesmon Q05682 2.1 ± 0.2 93 100 ± 5 5.6 6.2 ± 0.2 82 ± 4 15 ± 27 l-Caldesmon Q05682 1.6 ± 0.1 93 100 ± 5 5.6 6.3 ± 0.2 105 ± 3 18 ± 38* FUSE binding protein 2 Q92945 2.2 ± 0.4 73 103 ± 8 8.0 6.9 ± 0.1 52 ± 6 14 ± 49* FUSE binding protein 1 Q96AE4 2.1 ± 0.2 45 87 ± 12 6.3 7.3 ± 0.1 111 ± 3 24 ± 610* Elongation factor TU P49411 2.2 ± 0.9 67 61 ± 5 7.2 6.7 ± 0.2 137 ± 8 24 ± 315* Collagen-binding protein 2 P50454 3.6 ± 0.6 45 60 ± 4 8.8 9.0 ± 0.1 110 ± 4 25 ± 317 Tropomyosin 4-alpha P67936 1.3 ± 0.2 28 40 ± 3 4.7 4.9 ± 0.3 77 ± 5 19 ± 118 Protein disulfide isomerase 1 P07237 1.1 ± 0.4 55 62 ± 3 4.7 4.0 ± 0.2 75 ± 4 16 ± 319 Protein disulfide isomerase 1 P07237 0.9 ± 0.1 55 71 ± 3 4.7 4.8 ± 0.5 92 ± 5 16 ± 520 Protein disulfide isomerase 3 P30101 1.2 ± 0.2 54 78 ± 3 5.6 6.1 ± 0.3 89 ± 1 19 ± 3

Signal transduction, regulation, differentiation and apoptosis12* Voltage-dependent anion channel protein 1 P21796 2.2 ± 0.2 31 35 ± 4 8.6 8.8 ± 0.1 77 ± 3 37 ± 522 Voltage-dependent anion channel protein 1 P21796 1.2 ± 0.3 31 35 ± 3 8.6 9.0 ± 0.4 196 ± 2 45 ± 3

Non-identified proteins1* 2.9 ± 0.1 98 ± 5 4.0 ± 0.22* 2.1 ± 0.4 103 ± 5 4.2 ± 0.25 0.4 ± 0.1 78 ± 5 4.5 ± 0.2

Biological functions of identified proteins from primary cultures of human pancreatic islets incubated with either rhPRL (300 ng/mL) or vehicle for 4 days. Totalprotein extracts from human pancreatic islets were subjected to 2D gel electrophoresis. After staining, the ratios of spot volume (IOD ratio) between treated(rhPRL) and control (vehicle) cells were determined. Proteins from spots with an asterisk were regulated (cut-off = 2-fold) upon rhPRL treatment (n = 6 independentexperiments). Each protein is listed according to its major known function. Theoretical (theor.) pI and Mw values were calculated using the “Compute pI/Mw tool” att perime bservs

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he Expasy Molecular Biology Server, and the experimental (exp.) pI and Mw exxperiments. Score: −10 × log(p-value), where p-value is the probability that oearching software.

The identified proteins are categorized in Table 1 intounctional groups and include some important informationegarding accession number, relative intensity ratio (IOD ratiohPRL/vehicle), theoretical and experimental Mr and pI, scorecalculated as −10 × p value according to Aldente software) andequence coverage. All regulated spots (IOD ratio ≥2 or ≤ 0.5)ere marked with an asterisk in Table 1.The group of non-regulated spots were selected to ensure

ange in pI and molecular weight, some of which coincidedith already identified proteins demonstrating good agreementf the present map with the human islet proteome map previouslyublished (Ahmed et al., 2005). In addition, six of the nine-een identified proteins were detected, for the first time, in 2Del electrophoresis coupled to MS performed with total proteinxtracts from human pancreatic islets (i.e., fumarate hydroge-ase, caldesmon, FUSE binding protein 2, FUSE binding protein
, collagen-binding protein 2 and tropomyosin 4-alpha) and add,herefore, more information on the existent protein map.
In the majority of the spots where the theoretical Mr or pI ofhe protein did not match the experimental data, the deviation

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ould be accounted for by post-translational modifications, suchs phosphorylation and/or glycosylation of these proteins. Par-icularly, spots 6 and 7, identified as the same protein by PMF,ould be undergoing phosphorylation due to PRL treatmentince those isoforms displayed an acidic shift, but no changen the molecular weight. Unlike spots 6 and 7, spots 19 and 18,ere both identified as PDIAI 1 by PMF, present differences on

heir molecular weight. This difference could, at least in part,e the result of several phosphorylation and/or glycosylationvents, since this protein has 23 phosphorylation sites and three-glycosylation sites predicted (Blom et al., 2004, 1999). The

emaining three spots presented no reliable identification.

.3. Confirmation of protein expression profiles by Westernlotting

In order to validate the data obtained from 2D gel elec-rophoresis and PMF, we confirmed the regulation profiles andhe identity of four different proteins by Western blotting. Weave chosen Heat Shock Protein 27 (HSP27) and caldesmon as

22 L. Labriola et al. / Molecular and Cellular Endocrinology 264 (2007) 16–27

Fig. 4. Protein expression levels of HSP-27, PDIAI, tropomyosin and caldesmon. Primary cultures of human pancreatic islets maintained in CMRL mediumsupplemented with 0.5% FCS were treated with rhPRL for 4 days. Cells were then lysed and HSP-27 (A), PDIAI (B), tropomyosin (C) and caldesmon (D) wereanalyzed by Western blotting with appropriate specific antibodies. The membranes were stripped and reprobed with an anti-�-tubulin antibody to confirm thatnearly equal amounts of each protein extract were loaded. The immunoblots shown are representative of a total of five independent experiments. The correspondinghistograms on the right-hand side of the figure show the relative increase of all normalized densitometric values, with the results obtained for the control (vehicle)set as 1. The results are presented as the mean ± S.E.M. of at least five independent experiments. a vs. b: p < 0.05. WB: Western blot.

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roteins belonging to the regulated-spot group and tropomyosinnd protein disulphide isomerase (PDI) as examples of the non-egulated spot group.

HSP27 was detected in primary cultures of human islets andreatment of these cells with rhPRL caused a significant increasen this protein level (3.5 ± 0.5-fold; p < 0.0001), when compar-ng rhPRL-treated cultures where compared with those treatedith vehicle only (Fig. 4A). The presence of both PDIAI 1

nd 3 was detected in human islets without any modificationn protein level induced by rhPRL treatment (0.8 ± 0.3-fold;> 0.05) (Fig. 4B). The same lack of regulation was observedhen the tropomyosin protein level was analyzed (0.8 ± 0.3-

old; p > 0.05) (Fig. 4C).When the protein level of caldesmon was studied, we real-

zed that the specific monoclonal antibody used detected notnly the predicted form of 93 kDa, but also a band of 105 kDand a prominent one, corresponding to splicing variants pre-enting Mr of approximately 64 kDa (Fig. 4D). The fact that thentibody was raised against amino acids 494–793 is in agree-ent with our results because this region is present in all the

escribed caldesmon isoforms. The 105 kDa band could be theesult of post-translation modification, particularly phosphory-ation, because it is well known that phosphoproteins cause ahift to higher molecular weights in 1D SDS-PAGE (7.5%) andhis protein has several serine and threonine residues reported toe cyclin-dependent kinases substrates (UniProtKB/Swiss-Prot)Cuomo et al., 2005). Another fact that reinforces the phospho-ylation hypothesis as one of the possible causes for this shifts the fact that this protein was detected in two spots belong-ng to a horizontal strain of protein spots (Fig. 1) and it is wellnown that phosphorylation changes in the protein charge areften indicated by a horizontal trail of protein spots on the 2Dels (Mann and Jensen, 2003; Rosen et al., 2004; Yan et al.,006).

Only the bands presenting a high molecular weight evidencedsignificant increase in their protein levels when induced by

hPRL (p < 0.05) (Fig. 4D). The caldesmon bands correspond-ng to 92 and 105 kDa showed, respectively, a 2.4 ± 0.6 and a.3 ± 0.9-fold increase upon rhPRL treatment (Fig. 4D). Theseesults are in agreement with the data obtained by 2D gel elec-rophoresis and PMF because no caldesmon spot presenting a

r of 64 kDa was found as being regulated upon treatment withhPRL.

. Discussion

Our study showed, for the first time, that changes in pro-ein expression pattern occur in primary cultures of human isletspon rhPRL treatment. Statistical analysis of the expression pro-le of the vehicle or rhPRL-treated human pancreatic islet-cellllowed us to separate a small group of 22 protein spots, 14f which were differentially expressed between these pheno-ypes. Subsequent MALDI-TOF MS allowed identification of
he majority of the proteins present in these spots, using availablerotein or translated nucleotide sequence databases. Further-ore, by using Western blotting, we were able to validate the
dentity and the regulation profiles of some of the protein spots.

hb(f


verall, the specific protein expression changes were compati-le with the functional findings reported for this model.

In fact, several studies have documented the prolactin-nduced increase in beta-cell proliferation, insulin content andecretion in rodents (Nielsen et al., 2001; Sorenson and Brelje,996; Weinhaus et al., 1996) and in human pancreatic isletsLabriola et al., 2007). Moreover, Jensen et al. (2005) haveecently shown that GH and PRL treatment lead to increasedcl-xL/Bax ratio, thus protecting beta-cells against cytotoxicytokines-induced apoptosis.

High-resolution 2D gel technology can efficiently separatehousand of proteins. Unlike transcriptome approach, proteomicnalysis offers the possibility to quantitate changes in proteinxpression and to identify post-translational protein modifica-ions, such as phosphorylation (D’Hertog et al., 2006; Larsennd Roepstorff, 2000).

We have chosen to study whole islets, since the islet cells arenown to work in concert much as an organ, with separation ofhe different cell types inducing a cellular stress response andhanges in the expression of specific proteins. Indeed, proteinhanges might originate from cell types other than beta cells,ut still they changed expression in this model and are thereforef potential interest for proper islet function.

This first proteomic examination of the effects of rhPRL onslets, by necessity, only analyzed highly abundant proteins.herefore, the changes noted are more likely to be related toell proliferation or increased insulin production than to signal-ng or regulatory mechanisms of hPRL action.

A brief discussion of proteins possibly and partly responsi-le for the known morphological and functional features of thehPRL-treated islets is given below. This study did not allow uss to point a single protein or proteins responsible for the phe-otype changes induced by the hormonal treatment. Rather, its possible that phenotypical changes and lasting effects of therolactin treatment is a product of relatively small expressionhanges in the levels of several proteins.

Two protein spots involved in the tricarboxylic acid cyclefumarate hydratase and aconitate hydratase) were upregulatedfter rhPRL cell treatment, suggesting increased ATP produc-ion.

Regarding insulin secretion, it is already known that oscil-ations in the ATP/ADP ratio lead to oscillations in membraneotential modulation of the ATP-sensitive K+ (KATP)-channelLarsson et al., 1996). These variations cause oscillations inytoplasmic free Ca2+ concentration (Santos et al., 1991) thative rise to pulsatile insulin release (Bergsten et al., 1994; Gilont al., 1993). Nevertheless, ATP synthase alpha chain, which islso involved in ATP production, did not present changes in thehPRL-treated cells.

rhPRL treatment also induced the expression of two proteinsnvolved in the glycolytic pathway (piruvate kinase and fructoseldolase). The functional relevance of the glycolytic pathwayn insulin production still needs to be elucidated. However, it
as been reported that insulin and cellular ATP levels regulateoth the activity and synthesis of several glycolytic enzymesEizirik et al., 1989). The primary beta-cell displays a several-old-increased pyruvate carboxylase activity to efficiently direct

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yruvate (the major product of glycolysis in the absence of lac-ate production) toward mitochondrial tricarboxylic acid cyclend oxidative phosphorylation metabolism for efficient ATP pro-uction (Schuit et al., 1997). Therefore, an increase in pyruvateinase, which catalyzes the formation of pyruvate and ATP fromhosphoenolpyruvate, would lead to increased production ofntracellular ATP, a key metabolic stimulus-coupling factor inhe beta-cell to control insulin release (Deeney et al., 2000).urthermore, rat insulinoma INS-1 cells exposure to glucoselso induced an increase in several glycolytic enzymes, such asyruvate kinase (Roche et al., 1997).

These results are in agreement with the prolactin-inducednsulin secretion observed in human and rodent islets (Brelje etl., 2004; Labriola et al., 2007; Nielsen et al., 2001; Weinhaust al., 2000, 1996).

Three proteins (Fuse binding protein 1 and 2 (FUBP 1 and 2)nd elongation factor TU (EFTU)), involved in protein synthe-is, were upregulated in primary cultures of human islets uponhPRL treatment.

FUBP proteins are transcriptions factors that participate in theegulation of c-myc expression and loss of FUBP function arrestsellular proliferation (Bazar et al., 1995; He et al., 2000). Theyre also involved in activation of gene transcription, mediationf exon inclusion, mRNA processing and splicing (Avigan et al.,990; Barberis et al., 1995; Braddock et al., 2002; Michelotti etl., 1996).

Elongation factor TU promotes the GTP-dependent bind-ng of aminocyl-tRNA to the A-site of the ribosomes (Ogle etl., 2001; Southworth et al., 2002). Therefore, their increasedxpression levels, presented in this study, could be related withglobal increase in protein synthesis which is in agreement withell proliferation and insulin synthesis.

Formation and maintenance of the actin cytoskeleton is cru-ial in cell proliferation, and proteins with functions in thisontext were identified in our study (tropomyosin 4-alpha andaldesmon). Caldesmon, a 93 kDa protein (hCALD1), and itsplicing variants of approximately 64 kDa (hCALD 2–5), arerotein isoforms, which share conserved regions containingaldesmon’s capacity to bind to actin, tropomyosin, Ca2+-almodulin, myosin and phospholipids (Bryan, 1990). Theylay a role in the regulation of cell contractility, adhesion-ependent signaling, and cytoskeletal organization, influencingranule movement, hormone secretion, and reorganization oficrofilaments during mitosis via mitosis-specific phospho-

ylation by several complexes formed by cyclin and cyclin-ependent kinases (Bryan, 1990; Huber, 1997; Yamakita et al.,992; Yamashiro et al., 1991, 1990). Other kinases such asalmodulin-dependent kinase II and casein kinase II have alsoeen shown to phosphorylate residues within the amino-terminalyosin binding region of hCALD1 and this abolishes its inter-

ction with myosin (Sutherland et al., 1994; Wang and Yang,000).

In this work, we have found expression of both hCALD1 and
ts splicing isoforms by Western blotting, but only the hCALD
and some possible phosphorylation isoforms were regulatedy prolactin treatment. Alterations in the expression and post-ranslational modifications of this protein could explain not only

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he higher proliferation but also the higher insulin content andecretion observed in rhPRL-treated human islets.

Three proteins involved in chaperone functions and/or pro-ein folding, namely: Serpin H2 (SPH2), HSP 27 and glucose-egulated protein (GRP94) were also induced upon rhPRL treat-ent.Heat shock proteins (HSPs) function as molecular chaper-

nes assisting on protein folding, transport, translocation andegradation (Langer and Neupert, 1994), and could have pro-ective functions against exposure to several damaging stimuli,uch as heat (Langer and Neupert, 1994), cytokines (Scarim etl., 1998) and NO (Burkart et al., 2000). Furthermore, lowerxpression levels of HSP have been shown in streptozotocinnduced diabetic rats (Yamagishi et al., 2001).

SPH2 is a protein belonging to the HSP47 family presentxclusively in the ER, which plays a vital role in procollagenrocessing (Nandan et al., 1990). It is reported to be involveds a chaperone in the biosynthetic pathway of collagen in sev-ral tissues and it has also been implicated in tumor progressionhrough the endogenous processing of collagen XVIII in tumorells (Hah et al., 2002; Ollins et al., 2002; Rocnik et al., 2002).he presence of HSP27 was identified in several human tissues,uch as brain, lung, pancreas and endometrium, where it plays anmportant role in the implantation, decidualization and placen-ation processes during pregnancy (Ahmed et al., 2005; Carpert al., 1990; Ciocca et al., 1996; Strausberg et al., 2002; Yu etl., 1997).

In this work, we have identified the presence and the rhPRL-nduced levels of HSP27 by 2D gel electrophoresis–MS andalidated these results by Western blot. Even though the molec-lar mechanisms involved in HSP27 regulation in human isletsave not been well established, there are evidences indicatinghat synthesis of this protein is induced by environmental stressnd its expression is also regulated by estrogen and proges-erone in mammary tumors (Ciocca et al., 1996). Furthermore,t has been shown, in a model of mumps virus-infected cells,hat the induction of HSP27 by stress requires Stat-1, in addi-ion to the activated heat shock factor-1 (HSF-1) (Yokota et al.,003). Additionally, it was demonstrated that activation of HSF-induces the expression of heat shock proteins and attenuates

tress-induced cell death (Dodge et al., 2006; Kiang and Tsokos,998; Morimoto and Santoro, 1998). In fact, preliminary west-rn blot results of our laboratory have shown rhPRL-inducedrotein levels of HSF-1 in human islets (data not shown). Theseesults, together with the presence of STAT1 in human isletsescribed by data from our laboratory (Labriola et al., 2007)re in agreement with the findings of Yokota and collaboratorsnd could represent a link between rhPRL treatment and HSP27nduction. Further experiments are required in order to validatehis hypothesis.

TRA1 is an abundant calcium-binding, endoplasmic retic-lum glycoprotein believed to function in the translocation ofascent proteins across the endoplasmic reticulum membrane
Koch et al., 1986; Mazzarella and Green, 1987) and the foldingf denatured proteins as well as multimer assembly (Nigam etl., 1994). TRA1 could be modulating protein biosynthesis andxport in the endoplasmic reticulum of human pancreatic islet

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ells. Since protein quality control and clearance mechanismsre particularly important in pancreatic beta cells, whose func-ion is to synthesize and secrete biologically active insulin, thenduction of TRA1 expression levels found in the rhPRL-treateduman pancreatic islets could be related to this specific cellu-ar function. In addition, the rhPRL-induced expression of theseroteins could also be involved at least in cell survival, sincerinivasan et al. (2005) have recently highlighted a new mech-nism whereby signaling through the insulin/IGF pathway inancreatic islet beta-cell mediates ER stress-induced apoptosisnd may provide a means to enhance survival.

We also identified two proteins of the protein disulphide iso-erase family (PDIAI 1 and 3), which were found in more

han one spot, but their expression levels were not altered uponormonal treatment. These proteins are specifically involved inhe re-arrangement of both intra-chain and inter-chain disulfideonds in proteins to form native protein structures.

Voltage-dependent anion channel (VDAC) proteins formores in the outer mitochondrial membrane, serving as theajor permeability pathway for metabolite flux between cyto-

lasm and the mitochondria (Hodge and Colombini, 1997).ro-apoptotic proteins BAX and BAK bind to VDAC chan-els and induce apoptosis by releasing cytochrome c, whereashe anti-apototic protein bclxL closes the VDAC channels andnhibits apoptosis (Shimizu et al., 1999). Furthermore, Jensent al. (2005), using rat insulinoma INS-1E cells have recentlyeported that PRL could be involved in increasing cell survival,y inducing an increase in the ratio of bclxL/BAX. If rhPRLould also promote this increase in human islets, we couldypothesize that in the case of rhPRL-treated cells, there wille more bclxL available to interact with the VDAC and, thusontribute to apoptosis inhibition.

In our study, VDAC1 protein was identified in at least twopots (spot 12 and 22). Interestingly, only one of them waspregulated upon rhPRL treatment, the other one remainingnchanged but maintaining constant a higher level of expres-ion than protein spot 12. As VDAC is a protein, which containseveral phosphorylation sites, it is reasonable to state, based onhe pI of the spots (Yan et al., 2006), that spot 12 might presenthigher extent of phosphorylation than spot 22.

Application of proteomics is a rapidly growing research areahat encompasses both genetic and environmental factors. Ourtudy using a proteomic approach shows, for the first time inrimary cultures of human islets, that rhPRL treatment mightrogram islets protein expression toward cell proliferation andorrect function. Whether the lasting consequences induced byhe rhPRL treatment in human islets (i.e. increased beta-cellumber and insulin content and secretion) can be directly relatedo the changes in protein expression described here is a possi-ility but has not been proven.

Even though the data presented do not allow distinctionetween primary and secondary changes induced by rhPRL,ither in time or in importance, or between active and inactive
soforms of the proteins, we believe that they provide impor-ant insights for the understanding of the molecular mechanismsnvolved in the beneficial effects of prolactin action in humanslets. We anticipate that such knowledge could provide useful
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nsights for the design of novel and rational preventive and/orurative strategies for T1DM, both in relation to beta cell growth,ifferentiation and to inhibition of cell death.

cknowledgements

We are especially grateful the excellent technical assistancerovided by Patricia Barros dos Santos, Zizi de Mendonca,ebora Costa and Sandra Regina Souza. We sincerely thankrof. Dr. Chantal Mathieu and Dr. Lut Overbergh for criticaleading of the manuscript.

This work was supported by grants from FAPESP, CNPq,INEP and PRP-USP.

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Prolactin-induced changes in protein expression in human pancreatic islets

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