Molecular and Cellular Endocrinology 358 (2012) 63–68

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Molecular and Cellular Endocrinology

journal homepage: www.elsevier .com/locate /mce

Insulin stimulates IGFBP-2 expression in 3T3-L1 adipocytes throughthe PI3K/mTOR pathway

Zhuo Li a,1, Stéphanie Miard a, Mathieu Laplante a, Nahum Sonenberg b, Frédéric Picard a,⇑a Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Laval University, QC, Canada G1V 4G5b Department of Biochemistry, McGill University, Montréal, QC, Canada H3A 2T5

a r t i c l e i n f o a b s t r a c t

Article history:Received 2 October 2011Received in revised form 15 February 2012Accepted 23 February 2012Available online 3 March 2012

Keywords:Mouse3T3-L1mTORGene expressionChromatin immunoprecipitationC/EBPa

0303-7207/$ - see front matter � 2012 Elsevier Irelandoi:10.1016/j.mce.2012.02.022

⇑ Corresponding author. Address: Institut univerpneumologie de Québec , Y3106 Pavillon Marguerite-Foy, QC, Canada G1V 4G5. Tel.: +1 418 656 8711 ext

E-mail address: Frederic.Picard@criucpq.ulaval.ca (1 Current address: The First Teaching Hospital of Jili

130021, China.

Insulin-like growth factor binding protein 2 (IGFBP-2) has been implicated in the etiology of several dis-eases, including the metabolic syndrome. Although IGFBP-2 derives mostly from the liver, recent evi-dence in mice and humans indicate that aging and obesity are associated with altered IGFBP-2 levelsin white adipocytes. The present study was aimed at determining the mechanisms that control IGFBP-2 expression in mature adipocytes. IGFBP-2 mRNA and protein expression in serum-deprived 3T3-L1 adi-pocytes were twofold increased by acute insulin treatment. Co-treatments with the phosphatidylinositol3-kinase (PI3K) inhibitor wortmannin or the mammalian target of rapamycin (mTOR) inhibitor rapamy-cin blunted the effects of insulin. Coherently, IGFBP-2 mRNA levels were robustly increased in adipocyteslacking either TSC2 or 4E-BP1. Insulin triggered the recruitment of CAAT/enhancer binding protein a(C/EBPa) to the IGFBP-2 proximal promoter. These findings suggest that insulin upregulates IGFBP-2expression through a PI3K/mTOR/C/EBPa pathway in white adipocytes.

� 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Insulin-like growth factor (IGF) binding-protein 2 (IGFBP-2) isan abundant circulating factor that is thought to inhibit IGF-1and IGF-2 activities, but also increases their half-life (Firth andBaxter, 2002; Wheatcroft and Kearney, 2009). Altered IGFBP-2expression has been associated with several pathologies, includingdevelopmental defects in pancreas (Hill et al., 1999) and bone(DeMambro et al., 2008), as well as in cancer (Degraff et al.,2007; Miyako et al., 2009) and renal failure (Fornoni et al., 2006).In humans, a low circulating concentration of IGFBP-2 has beenlinked to obesity and insulin resistance and was suggested as amarker for the metabolic syndrome (Arafat et al., 2009; Heald etal., 2006; Li and Picard, 2010; Lukanova et al., 2002; Martin et al.,2006; Mattsson et al., 2008; Nam et al., 1997; Ruan and Lai,2010). In line with this concept, IGFBP-2 has been shown to protectagainst insulin resistance when overexpressed in mice using eithertransgenic (Hoeflich et al., 1999; Wheatcroft et al., 2007) or aden-oviral (Hedbacker et al., 2010) methods.

Whereas insulin, IGF-1, glucocorticoids, and leptin have all beenshown to influence circulating levels of IGFBP-2 (Baxter and Twigg,

d Ltd. All rights reserved.

sitaire de cardiologie et ded’Youville, 2725 Chemin Ste-3737; fax: +1 418 656 4942.F. Picard).n University, Changchun, Jilin

2009; Hedbacker et al., 2010), the molecular mechanisms that con-trol the transcription of the IGFBP-2 gene are not well established.In MCF-7 and T47D breast cancer cells, insulin-stimulated IGFBP-2expression can be blocked by LY294002 and rapamycin (Martinand Baxter, 2007), which are inhibitors of the phosphatidylinositol3-kinase (PI3K) and mammalian target of rapamycin (mTOR),respectively. This effect has been recently suggested to be due toincreased nuclear content of the transcription factor Sp1 and itsbinding to a proximal region within the IGFBP-2 promoter(Mireuta et al., 2010). In U251 glioma cells, induction of phospha-tase and tensin homolog (PTEN), a negative-regulator of PI3K sig-naling, also decreases IGFBP-2 protein expression (Levitt et2005). However, insulin reduces IGFBP-2 levels in C2C12 myo-blasts (Ernst and White, 1996) and in the heart (Han and Park,2006), suggesting that the modulation of IGFBP-2 expression bythe insulin signaling pathway could depend on cell types.

Compared to the liver, IGFBP-2 is expressed in low amount inwhite adipose tissue (WAT) (Gosteli-Peter et al., 1994; Han et al.,1996). IGFBP-2 can be produced and secreted by differentiated3T3-L1 adipocytes, but not by pre-adipocytes (Boney et al.,1994), indicating its adipokine nature. However, the relative con-tribution of WAT-derived IGFBP-2 to its global circulating levelsis yet unknown (Li and Picard, 2010). These observations, alongwith the association between circulating IGFBP-2 concentrationsand conditions of insulin resistance and fat accretion (Li and Picard,2010), prompted us to investigate whether IGFBP-2 transcriptionwas under the control of the insulin signaling pathway in white

64 Z. Li et al. / Molecular and Cellular Endocrinology 358 (2012) 63–68

fat cells. Here we report that insulin stimulates IGFBP-2 mRNA andprotein levels in mature 3T3-L1 adipocytes through the stimula-tion of PI3K and mTOR, and induces C/EBPa recruitment and trans-activation of the IGFBP-2 promoter. These findings suggest thatadipose-specific improvement of insulin signaling could increaseIGFBP-2 production.

A B

DC

Fig. 1. Insulin stimulates IGFBP-2 mRNA expression in adipocytes. (A) Mature 3T3-L1 adipocytes were serum (FBS)-deprived and treated for 24 h with vehicle orinsulin (10�7 M). Cells were then harvested and tested for IGFBP-2 mRNAexpression. (B) Time-course of the effects of insulin (10�7 M) on IGFBP-2 mRNAlevels in serum-deprived 3T3-L1 adipocytes. (C) Effects of a 24 h insulin treatmenton IGFBP-2 protein levels in serum-deprived 3T3-L1 adipocytes. One representativeimmunoblot is shown. The bars represent signal quantification from threeindependent immunoblots. (D) IGFBP-2 protein levels secreted in the media 24 hafter insulin treatment in serum-deprived 3T3-L1 adipocytes. Bars representmean ± S.E.M. of three independent experiments done at least in triplicate. *indicates a significant difference compared to the control group (p < 0.05).

2. Materials and methods

2.1. Cell culture

3T3L1 cells were purchased from ATCC (Manassas, USA). Tuber-ous sclerosis complex-2 (TSC2) �/� and +/+ mouse embryo fibro-blasts (MEFs), both on a p53 �/� genetic background, werekindly provided by Dr. Kwiatkowski (Zhang et al. 2003). Eukaryotictranslation initiation factor 4E binding protein 1 (4E-BP1) �/� and+/+ MEFs were described previously (Le Bacquer et al. 2007). Allcells were grown in DMEM with 10% fetal bovine serum supple-mented with 4 mM of glutamine in a 5% CO2 environment. MEFswere cultured in the above media supplemented with 1% penicil-lin/streptomycin. As described previously (Miard et al. 2009), cellswere differentiated, 2 days after confluence (D0), in the same med-ium complemented with 10 lg/mL insulin, 0.25 mM 3-isobutyl-1-methyl-xanthine and 1 lM dexamethasone. After 2 days (D2),medium was supplemented with only 10 lg/mL insulin and re-placed every other day. Differentiation was evaluated by aP2mRNA expression and lipid droplets in >80% cells. At D6, mature3T3-L1 adipocytes were serum (FBS)-deprived and treated withvehicle or insulin (10�7 M). In some experiments, cells were co-treated with 20 lM hydrogen peroxide (Miard et al. 2009), 2 mMglucosamine (Lafontaine-Lacasse et al. 2011; Wang et al. 1998),the PI3K inhibitor wortmannin (10�7 M), or the mTOR inhibitorrapamycin (10�7 M). Cells were harvested 24 h later (or otherwiseindicated in the figure legends) and tested for IGFBP-2 expressionand C/EBPa DNA binding (ChIP, see below).

2.2. RNA extraction and qPCR

After RNA extraction (GE Healthcare, according to manufac-turer’s instructions), 1 lg of total RNA was reverse-transcribed at42 �C for 1 h with the SuperScript™ Reverse Transcriptase (Invitro-gen CA), followed by 15 min inactivation at 70 �C. Quantitative PCRwas carried out using a Rotor Gene 3000 system (Montreal Biotech,Montreal, QC, CA). Chemical detection of the PCR products wasachieved with SYBR Green Jumpstart Taq ReadyMix without MgCl2(Sigma, Oakville, ON). Relative level of gene expression was deter-mined via a standard curve composed of a mix from all the cDNA.Results were normalized to the expression level of 36B4 as ahousekeeping gene. The oligonucleotide sequences were mIGFBP-2: 50-GGCGCGGGTACCTGTGAAAAG-30, 50-TTGGGGATGTGCAGGGAGTAGAGA-30; m36B4: 50-GGCCCTGCACTCTCGCTTTCTGG-30, 50-TGGTTGCTTTGGCGGGATTAGTCG-30; mC/EBPa: 50-TTACAACAGGCCAGGTTTCC-30, 50-GGCTGGCGACATACAGTACA-30.

2.3. Western blotting

Cells were lyzed in Buffer C (20 mM Hepes pH 7.9, 420 mMNaCl, 1 mM CaCl2, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, and di-luted 1: 1000 Protease Inhibitor Cocktail (PIC)) for 20 min on iceafter two freeze/thaw cycles in liquid nitrogen. Lysates were cen-trifuged 20 min at 13,200 rpm and protein concentrations weredetermined with a Bradford assay. Proteins were then subjectedto SDS–PAGE on polyacrylamide gels and electrophoretically trans-ferred to PVDF membranes. Membranes were incubated 1 h at 4 �Cin blocking buffer (50 mM Tris.HCl pH 7, 5, 150 mM NaCl, 0.02%

Tween 20, 0.04% NP40 (wash buffer) and 5% milk), and overnightat 4 �C with IGFBP-2 (sc-25285, Santa Cruz), C/EBPa (sc-61, SantaCruz), or c-tubulin (sc-17787, Santa Cruz) primary antibodies inwash buffer containing 1% BSA. PVDF membranes were thenwashed three times in wash buffer for 7 min and incubated for1 h with anti-mouse or anti-rabbit immunoglobulin G conjugatedto horseradish peroxidase in wash buffer containing 1% BSA atroom temperature. After three washes of 7 min, the immunoreac-tive bands were detected by chemiluminescence.

2.4. ELISA assays

After incubation of cells with indicated treatment (see aboveand figure legends), fresh media was collected, centrifuged, andimmediately processed with a mouse- and rat-specific IGFBP-2ELISA kit according to manufacturer’s instructions (cat #22-MIGF-E08, Alpco Diagnostics, Salem, USA). Intra-assay CV was1.2%. Proteins were extracted from attached cells and quantifiedas described above. Data are presented as pg of secreted IGFBP-2protein per lg of cellular protein content.

2.5. Chromatin immunoprecipitation

Cross-linking of proteins and chromatin was performed for30 min in 1% formaldehyde followed by two washes with 1� PBSwith 1 mM PIC. Nuclear pellets were obtained using ChIP lysis buffer(50 mM HEPES pH 7.5, 140 mM NaCl, Triton 1%) with 1 mM PIC. Son-ication was carried out with Sonifier cell disrupter 185 (Branson) inChIP lysis buffer. Immunoprecipitations were performed using200 lL of sonicated cells and 5 lg of antibody: anti-C/EBPa (sc-61,Santa Cruz), or nonspecific rabbit IgG control (sc-2027, Santa Cruz).DNA was isolated using a PCR purification Kit (Qiagen). PCR was per-formed using primers (50-CTCGGGATGTGCATGG-30; 50-CTGGGTTCCAAGCAGCCTGG-30) that amplify a 200 bp region (�749/�549) con-taining a putative C/EBPa binding site �648/�634 bp upstream ofATG that was found through a TFSearch bioinformatics analysis

BA

Fig. 2. Insulin resistance attenuates the impact of insulin on IGFBP-2 expression inadipocytes. FBS-starved 3T3-L1 adipocytes were co-treated with insulin (10�7 M)and with either 20 lM hydrogen peroxide (A) or 2 mM glucosamine (B) to induceinsulin resistance. IGFBP-2 mRNA levels were measured 24 h later. Bars showmean ± S.E.M. of at least three experiments done in triplicate. * indicates asignificant effect (p < 0.05) of insulin, � indicates a significant effect (p < 0.05) ofH2O2 or glucosamine.

Z. Li et al. / Molecular and Cellular Endocrinology 358 (2012) 63–68 65

(mbs.cbrc.jp/research/db/TFSEARCH.html). The other putative sitesfound were �4730 to 4717; �4646 to 4633; �4574 to 4560; and�1237 to 1224 bp upstream of ATG.

2.6. Statistical analysis

Data are presented as mean ± S.E.M. Statistical differences wereanalyzed by ANOVA and Fisher’s PLSD (post hoc) when appropriatewith Statview software. A p value <0.05 was considered significant.

3. Results

In 3T3-L1 adipocytes that were FBS-starved in order to removethe impact of serum-containing insulin, IGF, and other possibleconfounders, a 24 h insulin treatment significantly increasedIGFBP-2 mRNA levels (Fig. 1A), but not those of IGFBP3 or IGF-1

A B

D E

Fig. 3. IGFBP-2 expression is under the control of the PI3K/mTOR/4E-BP1 pathway. 3T3-either the PI3K inhibitor wortmannin (A, B) or the mTOR inhibitor rapamycin (C), both aIGFBP-2 and 36B4 mRNA levels were also measured in TSC2 �/� (D) and 4E-BP1 �/� (E) aindicates a significant effect (p < 0.05) of insulin (A, B) or gene knockout (C, D).

(Supplementary Fig. 1). Expectedly, insulin treatment had no nota-ble effect on IGFBP-2 mRNA expression in differentiated 3T3-L1adipocytes incubated in the presence of FBS (data not shown).IGFBP1 was not detected in 3T3-L1 adipocytes. The stimulating ef-fect of insulin on IGFBP-2 mRNA expression was time-dependent,reaching a plateau as early as 6 h after insulin treatment(Fig. 1B). Insulin treatment in FBS-starved 3T3-L1 did not resultin an increase in intracellular IGFBP-2 protein levels (Fig. 1C), buttriggered a significant (p < 0.05) secretion of IGFBP-2 into the med-ium (Fig. 1D). These observations suggest that the insulin-stimu-lated increase in IGFBP-2 production is caused by augmentedtranscription.

Because IGFBP-2 has been linked to states of insulin sensitivity(Heald et al. 2006; Hedbacker et al. 2010; Li and Picard 2010;Wheatcroft et al. 2007), IGFBP-2 expression may be impaired whenthe insulin signaling pathway is altered. To test this hypothesis,two independent, established cellular models of insulin resistancewere assessed (Furukawa et al. 2004; Miard et al. 2009). In matureadipocytes treated with hydrogen peroxide, which reduces Aktactivity (Kim et al. 2011), the effect of insulin on IGFBP-2 mRNAlevels was significantly attenuated compared to that in controlcells (Fig. 2A). This blunted impact of insulin on IGFBP-2 expressionwas also observed in mature adipocytes pre-treated with glucosa-mine (Fig. 2B), which is known to induce insulin resistance bystimulation of the hexosamine biosynthesis pathway (Einsteinet al. 2008). Taken together, these findings indicate that insulinpositively regulates IGFBP-2 expression, and that the inductionby insulin may be impaired under conditions of insulin resistance.

To determine the key contributors to the impact of insulin onIGFBP-2 expression, 3T3-L1 adipocytes were first co-treated withthe PI3K inhibitor wortmannin. Compared with cells incubatedwith insulin only, adipocytes co-treated with wortmannin showeda robust reduction in IGFBP-2 secreted levels (Fig. 3A). Wortman-nin also completely blocked insulin-stimulated IGFBP-2 mRNA

C

L1 adipocytes were serum-deprived, and co-treated with insulin (10�7 M) and witht 10�7 M. Secreted (A) and mRNA (B, C) levels of IGFBP-2 were measured 24 h later.dipocytes. Bars show mean ± S.E.M. of at least three experiments done in triplicate. *

66 Z. Li et al. / Molecular and Cellular Endocrinology 358 (2012) 63–68

expression (Fig. 3B). Moreover, IGFBP-2 expression in insulin-trea-ted adipocytes was also blunted by rapamycin, an inhibitor ofmTOR (Fig. 3C). To further pinpoint the downstream effectors ofinsulin, MEFs bearing genetic deletion of mTOR-inhibiting proteinTSC2 (TSC2 �/�) were differentiated into adipocytes. Compared totheir wild-type controls, these cells had a fourfold increased IGFBP-2 expression despite lower 36B4 mRNA levels (Fig. 3D). A similartest in 4E-BP1 knockout adipocytes resulted in a 140-fold increasein IGFBP-2 mRNA levels (Fig. 3E). In both TSC2 �/� and 4E-BP1 �/� adipocytes, insulin treatment did not further induced IGFBP-2expression (data not shown). These results demonstrate that insu-lin stimulates IGFBP-2 expression through the PI3K/mTOR/4E-BP1pathway.

4E-BP1 �/� adipocytes were shown to have increased expres-sion levels of the transcription factor CCAAT/enhancer binding pro-tein a (C/EBPa) as part of a stimulated adipogenic program (LeBacquer et al. 2007). Acute treatment of differentiated 3T3-L1 adi-pocytes with insulin had no impact on C/EBPa mRNA (Fig. 4A) orprotein (Fig. 4B) levels. The contribution of C/EBPa to IGFBP-2expression was then tested through a bioinformatics search(TFSearch) of C/EBPa binding sites on the mouse IGFBP-2 pro-moter. Five putative regions within a 5 kb frame upstream of thestart codon were detected (Fig. 4C). As assessed by chromatinimmunoprecipitation, insulin treatment of FBS-starved 3T3-L1 adi-pocytes induced the binding of C/EBPa to the most proximal(�640 bp) region of the IGFBP-2 promoter (Fig. 4D), but not theother putative sites (not shown). These findings suggest that insu-lin increases IGFBP-2 mRNA expression in part by stimulating site-specific C/EBPa transcriptional activity.

C

D

A B

Fig. 4. Insulin promotes C/EBPa recruitment on the IGFBP-2 promoter. Serum-deprived 3T3-L1 adipocytes were treated with insulin (10�7 M) or vehicle. 24 hlater, mRNA (A) and protein (B) levels of C/EBPa were quantified. One represen-tative immunoblot is shown. The bars represent signal quantification from threeindependent immunoblots. (C) Schematic representation of the 5 putative C/EBPabinding sites (black bars) on the mouse IGFBP-2 promoter as found through aTFSearch bioinformatics analysis (�4730 to 4717; �4646 to 4633; �4574 to 4560;�1237 to 1224, �648 to 634 bp upstream of ATG). (D) ChIP assay using cells treatedwith insulin (10�7 M) or vehicle for 24 h. The PCR was performed with primers thatamplified the most proximal site shown in C. Data shown are representative of atleast three experiments done in triplicate.

4. Discussion

The modulation of IGFBP-2 levels has been involved in the eti-ology of several diseases, including cancer and the metabolic syn-drome. However, the molecular mechanisms controlling IGFBP-2expression are not well established. The present study demon-strates that, in differentiated 3T3-L1 adipocytes, insulin stimulatesIGFBP-2 gene transcription, and increases its secretion. Our find-ings are consistent with those of previous studies performed in car-cinoma cells, in which the insulin signaling pathway also regulatesIGFBP-2 transcription (Martin and Baxter 2007). Inhibition of PI3Kand mTOR blunted the effects of insulin on IGFBP-2, whereas ge-netic deletion of TSC2 or 4E-BP-1 robustly increased IGFBP-2mRNA levels.

The recruitment of the transcription factor C/EBPa to a proximalsite on the IGFBP-2 promoter upon insulin treatment suggests thatthis mechanism could contribute, at least in part, to the stimulat-ing effect of insulin on IGFBP-2 mRNA levels. Interestingly, in sim-ilarity with our findings, a promoter site containing the proximal633 bp of the human IGFBP-2 promoter was shown to be activatedby IGF-1 and synthetic analogues (Elminger et al. 2001). Theimportance of C/EBPa to IGFBP-2 expression is supported by thefact that IGFBP-2 can be quantified only in mature adipocytes(Boney et al. 1994), at which stage C/EBPa is also expressed(Farmer 2006). Although not the scope of the present study, it re-mains to be tested whether chronic activation of C/EBPa in matureadipocytes results in an increase in IGFBP-2 expression (Olofssonet al. 2008). Indeed, long-term transactivation of the IGFBP-2 pro-moter could be triggered by different transcription factors as a re-sult of their relative abundance (e.g. Sp1 or C/EBPa in cancer andadipose cells). Interestingly, Sp1 and C/EBPa DNA bindings sitesare found in different promoter regions located at �200 and�640 bp, respectively, which could suggest additional effects.

Our findings indicate that methods to restore positive regula-tors of the insulin signaling pathway could be associated with

enhanced IGFBP-2 expression. This concept may only apply to spe-cific conditions that are reflective components of experimental FBSstarvation (in which for instance negative regulators are lacking orcompensatory survival pathways are turned on), since insulin hadno effect on IGFBP-2 expression in normal adipocytes incubatedwith FBS-containing media. Interestingly, treatment of mice withleptin, at doses that stimulate glucose metabolism without affect-ing body weight and food intake, strongly upregulates IGFBP-2 lev-els in blood (Hedbacker et al. 2010). Notably, this effect is seenwithout significant long-term changes in insulinemia. We havealso found in rats that improved insulin sensitivity associated withcaloric restriction triggers an important increase in IGFBP-2expression in liver (Boivin & Picard, unpublished data). In addition,overexpression of IGFBP-2 in mice not only ameliorates insulinresistance (Hoeflich et al. 1999; Wheatcroft et al. 2007) but canalso correct diabetes (Hedbacker et al. 2010). Thus, more studiesare required to dissociate the respective impact of insulin sensitiv-ity and IGFBP-2 on each other.

Modulation of the mTOR pathway has recently been suggestedas an important contributor of lipid biosynthesis (Laplante andSabatini 2009), notably by activating the transcription factorSREBP1 (Duvel et al. 2010; Peterson et al. 2011; Porstmann et al.2008). Previous work performed in mice also indicated the adipo-genic effect of this pathway (Um et al. 2004). It is therefore para-doxical that increased mTOR activity induced the expression ofIGFBP-2, which has been shown to exert anti-lipogenic effects inadipocytes, especially by blunting the stimulating impact of IGF-1 on adipogenesis (Wheatcroft et al. 2007). Interestingly, animals

Z. Li et al. / Molecular and Cellular Endocrinology 358 (2012) 63–68 67

genetically modified to have lower body fat have been shownnumerous times to live longer (Bluher 2008), a condition observedin mice with a deficiency or downregulation of the mTOR pathway(Selman et al. 2009) or in mice heterozygous for the IGF-1 receptor(Holzenberger et al. 2003). Although it remains to be assessedexperimentally, IGFBP-2 could thus, apart from its insulin-sensitiz-ing effects, positively influence lifespan in part by modulating theimpact of IGF-1 on adipose mass.

In conclusion, the present findings demonstrate that, in adipo-cytes, insulin upregulates IGFBP-2 transcription through a PI3K/mTOR cascade, possibly in part by promoting the recruitment ofC/EBPa on the IGFBP-2 promoter. Other studies are required tofully understand the coordinated pathways that could driveIGFBP-2 expression and secretion after insulin stimulation/sensiti-zation in physiological settings.

Conflict of interest

The authors have no conflict of interest with the present work.

Acknowledgements

We thank Dr. David Kwiatkowski and Dr. Katherine Cianflonefor their generous help on providing MEFs cell lines, and LouiseBoivin for expert technical assistance. F. Picard holds a FRSQ Junior2 Investigator Award. This work was supported by operating grantsfrom the CIHR (MOP-66967 and CCI-85677).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.mce.2012.02.022.

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