Maternal protein restriction permanently programs adipocyte growth and development in adult male rat offspring

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<ul><li><p>Journal of Cellular Biochemistry 101:381388 (2007)</p><p>Maternal Protein Restriction Permanently ProgramsAdipocyte Growth and Developmentin Adult Male Rat Offspring</p><p>Ting Zhang,1,2 Haiyan Guan,1,2 Edith Arany,3 David J. Hill,2,3 and Kaiping Yang1,2*1Department of Obstetrics and Gynaecology, Childrens Health Research Institute and Lawson HealthResearch Institute, University of Western Ontario, 800 Commissioners Rd. E., London, Ontario,Canada N6A 4G52Department of Physiology and Pharmacology, Childrens Health Research Institute and Lawson HealthResearch Institute, University of Western Ontario, 800 Commissioners Rd. E., London, Ontario,Canada N6A 4G53Department of Medicine, Childrens Health Research Institute and Lawson Health Research Institute,University of Western Ontario, 800 Commissioners Rd. E., London, Ontario, Canada N6A 4G5</p><p>Abstract We previously demonstrated that maternal protein restriction (MPR) during pregnancy and lactation led tofetal growth restriction and development of increased visceral adiposity in adult male rat offspring. Here we studied therate of proliferation and differentiation of adipocyte precursors (preadipocytes) in vitro to investigate whether MPR maypermanently program adipocyte growth and development in adult male offspring. Preadipocytes were isolated fromvisceral adipose tissue of control and MPR offspring at 130 days of age, and cultured under standard conditions. The rateof proliferation was studied by [3H]-thymidine incorporation, and the rate of differentiation assessed with the use ofbiochemical and morphological markers. Although it did not affect the rate of differentiation, MPR increased the rateof preadipocyte proliferation by almost twofold. To ascertain if the increased proliferation was due to persisting in vivoinfluences or aberrations inherent in the precursor cells, we studied the rate of preadipocyte proliferation in subcultures.We found that the increased rate of proliferation of MPR preadipocytes persisted throughout the first two subcultures,indicative of an inherent abnormality. In addition, we examined the rate of preadipocyte proliferation under reducedserum conditions. We showed that MPR reduced the rate of preadipocyte proliferation to 56 and 35% of the control in thepresence of 5 and 2.5% serum, respectively. Taken together, these results demonstrate that MPR permanently programsadipocyte growth and development such that adipocyte precursors derived from MPR offspring replicate excessivelyunder standard culture conditions but exhibit markedly attenuated growth rate under reduced serum conditions. J. Cell.Biochem. 101: 381388, 2007. 2007 Wiley-Liss, Inc.</p><p>Key words: maternal nutrition; visceral adiposity; adipogenesis; preadipocytes; proliferation and differentiation</p><p>Obesity is a serious medical problem notonly because it substantially impairs quality oflife and but also because it increases the risk ofhypertension, type 2 diabetes, coronary heart</p><p>disease, sleeping disorders, and cancers [Mok-dad et al., 2003]. There is strong evidence for agenetic component to human obesity [Hofbauer,2002]. Multiple systems regulate energy home-ostasis [Montague et al., 1997; Krysiak et al.,2001] and a number of genes associated withhuman obesity have been identified [Frogueland Boutin, 2001], yet the genetic component ofthis condition cannot explain the dramaticincrease in the prevalence of obesity in recentyears.A large number of epidemiological studies</p><p>have revealed a strong statistical associationbetween poor fetal growth and the subsequentdevelopment of type 2 diabetes, hypertension,</p><p> 2007 Wiley-Liss, Inc.</p><p>Grant sponsor: Canadian Institutes of Health Research.</p><p>*Correspondence to: Dr. Kaiping Yang, Childrens HealthResearch Institute, Room A5-132, Victoria ResearchLaboratories- Westminster Campus, 800 CommissionersRoad East, London, Ont., Canada N6A 4G5.E-mail: kyang@uwo.ca</p><p>Received 16 August 2006; Accepted 30 September 2006</p><p>DOI 10.1002/jcb.21176</p></li><li><p>and obesity, visceral obesity in particular[Osmond and Barker, 2000]. These observa-tions were made initially by Barker and collea-gues in England, but have now been reproducedin a large number of populations worldwide[Byrne and Phillips, 2000]. These findings haveled to the fetal origins hypothesis, whichstates that an adverse intrauterine environ-ment programs or imprints the development offetal tissues, permanently determining physio-logical responses, and ultimately producingdysfunction and disease [Purdy and Metzger,1996]. However, the molecular mechanismsthat underpin this relationship remain elusive.Since the world-wide maternal malnutrition</p><p>is themost common cause for poor early growth,and amino acids play a critical role in fetalgrowth [Petry et al., 2001], thematernal proteinrestriction (MPR) rat model has become one ofthe most extensively studied models of fetalgrowth restriction [Ozanne, 2001]. In thismodel, rat dams are subjected to a low proteindiet (8% protein) instead of control diet (20%protein) throughout pregnancy and lactation.As a consequence, the resulting offspringexhibit low-birth weight and become diabetic,insulin resistant, and hypertensive [Haleset al., 1996; Petry et al., 1997]. It has beenreported thatMPRhad a long-term influence onthe structure and function of certain organs,such as pancreas [Snoeck et al., 1990; Dahriet al., 1991; Petrick et al., 1999; Holness et al.,2000; Joanette et al., 2004], liver [Ozanne et al.,1996a; Burns et al., 1997] and muscle [Ozanneet al., 1996b].Recently we have demonstrated that protein</p><p>restriction during pregnancy and lactationleads to fetal growth restriction and develop-ment of increased visceral adiposity in adultmale rat offspring [Guan et al., 2005]. Further-more, we have also obtained evidence suggest-ing that visceral adiposity in our rat model ischaracterized by adipocyte hyperplasia becausethere were no apparent differences in the sizeof adipocytes between control and MPR off-spring. This contention is consistent with theDNAmicroarray results showing that levels of anumber of genes involved in cellular prolifera-tion and differentiation were up-regulated inMPR adipose tissue while the expression ofgenes involved in apoptosis did not change[Guan et al., 2005]. Since adipocyte hyperplasiaresults from the recruitment of new adipocytesfrom precursor cells in adipose tissue and</p><p>involves the proliferation and differentiationof preadipocytes, the present study wasdesigned to test the hypothesis that preadipo-cytes from MPR offspring possess a greaterpotential for proliferation and differentiation.</p><p>MATERIALS AND METHODS</p><p>Experimental Animals and Dietary Manipulations</p><p>The MPR rat model was established asdescribed previously [Guan et al., 2005]. Inbrief, virgin female Wistar rats (Charles RiverLaboratories,Wilmington,MA)weighting 240260 g were housed individually andmaintainedat 228C on a 12:12-h light-dark cycle. They weremated, and day 0 of gestation was set as theday on which vaginal plugs were expelled.The pregnant dams were fed either a diet con-taining 20% protein (control diet) or an isoca-loric diet containing 8% protein (low-proteindiet) throughout pregnancy and lactation. At3 days of age, litters were randomly reduced to8 pups, thus ensuring a standard litter size permother. At 21 days of age, all offspring wereweaned onto a 20% protein diet. At 130 days ofage, male offspring were sacrificed and theirvisceral fat pads (composed of mesenteric,omental, and retroperitoneal fat masses) wereisolated. For simplicity, the two groups ofoffspring will be termed control and MPR rats.</p><p>Isolation and Culture of Preadipocytes</p><p>Preadipocytes were isolated from control andMPR rats as described previously [Aoki et al.,2004]. Briefly, the visceral fat pads weredissected from visible blood vessels and con-nective tissue, weighed, finely minced anddigested in digestion buffer (3 ml/g tissue)consisting of Dulbeccos modified Eagles med-ium (DMEM) (Invitrogen Life Technologies,Burlington, Ontario, Canada), 0.5 mg/ml col-lagenase class IV (Sigma, Oakville, Ontario,Canada) and 1.5% bovine serum albumin (BSA)(Sigma) for 45 min at 378C, under mildcontrolled agitation. The resultant digest mate-rial was filtered through 250 mm nylon meshand centrifuged at 600g for 5 min to separatethe floating adipocytes. The cell pellet wasresuspended, washed with dulbeccos phos-phate-buffered saline (DPBS) containing 10%newborn calf serum (NCS) (Invitrogen LifeTechnologies), filtered through 25 mm nylonmesh and then centrifuged. The pelletedpreadipocytes were resuspended in standard</p><p>382 Zhang et al.</p></li><li><p>culturemedium (DMEM/F-12medium (Invitro-gen Life Technologies) supplemented with 10%fetal bovine serum (FBS) (Sigma), 50 U/mlpenicillin and 50 mg/ml streptomycin (Invitro-gen Life Technologies)). The cell number wasdetermined with a hemocytometer. Preadipo-cytes were seeded in 24-well plates and 100-mmdishes and cultured in ahumidified incubator at378C, in the presence of 5% CO2. For simplicity,preadipocytes derived from control and MPRrats will be termed control and MPR preadipo-cytes.For subculture, primary preadipocytes were</p><p>seeded in 100-mm dishes and cultured instandard culture medium for 34 days untilthey reached 6070% confluence. Cells werethen washed, detached by incubation withTrypsin-EDTA and centrifuged at 600g for5 min. The resulting pellets were used forsubculture. The first two subcultures were usedfor proliferation study.</p><p>Proliferation Assay-[3H]-ThymidineIncorporation</p><p>Proliferation capacity of preadipocytes wasassessed by measuring the rate of [3H]-thymi-dine incorporation. Primary preadipocyteswereseeded in 24-well plates at a density of 1 104cells/cm2 (day 0) and incubated in standardculture medium for 24 h. Subsequently, themedium was changed to DMEM/F-12 contain-ing with 10, 5, or 2.5% FBS. [3H]-thymidine(0.5 mCi/well) (75.2 Ci/mmol, PerkinElmer Lifeand Analytical Sciences, Woodbridge, Ontario,Canada) was added at 24-h intervals untilday 4. At days 2, 3 and 4, the medium wasremoved, and cells were washed twice with ice-cold phosphate-buffered saline (PBS), oncewith5%trichloroacetic acid (TCA) andoncewith95%ethanol. Cells were then solubilized by theaddition of 200 ml of 0.5 M NaOH. Thesolubilized cell lysate (100 ml) was added to4 ml of scintillation fluid and the incorporationof [3H]-thymidine into DNA was determined byscintillation counting. Protein concentration inthe cellular lysate was quantified by theBradford technique. The incorporation ofthymidine was normalized by protein content.Preadipocytes in the first and second sub-</p><p>cultures were plated in 24-well plates at adensity of 7,500 cells/cm2, and incubated instandard culture medium for 24 h. Thereafter,medium was changed, and cells were pulselabeled with [3H]-thymidine (0.5 mCi/well) for</p><p>24 h. The rate of [3H]-thymidine incorporationwas determined as described above.</p><p>Adipocyte Differentiation</p><p>Primary preadipocytes were seeded in 24-well plates at a density of 5 104 cells/cm2 andcultured in standard culture medium until theyreached confluence. Two days after confluence(day 0), cells were induced to differentiate bythe addition of a differentiation cocktail con-taining 1 mM dexamethasone (Dex) (Alpharma,Boucherville, Quebec,Canada), 0.5mMmethyl-3-isobutylxanthine (IBMX) (Sigma), and 5 mg/ml insulin (Eli Lilly Canada Inc., Toronto,Ontario, Canada) and 10% FBS in DMEM.After 48 h, mediumwas replaced with standardculture medium plus insulin only, and cellswere then fed every 2 days. Differentiatedprimary preadipocytes were harvested atdays 3, 6, and 9 of differentiation. The rate ofdifferentiation was scored by analysis of theexpression of differentiation marker genes(peroxisome proliferator-activated receptor g(PPARg) and lipoprotein lipase (LPL)), and bylipid accumulation (oil red O staining).</p><p>Real-time Quantitative RT-PCR (qRT-PCR)</p><p>To determine if MPR altered the rate ofpreadipocyte differentiation, levels of PPARgmRNA and LPL mRNA were assessed by atwo-step real-time quantitative RT-PCR (qRT-PCR),asdescribedpreviously [Guanetal., 2005].Briefly,1mgof totalRNAwasreverse transcribedin a volume of 20 ml with the High CapacityComplementaryDeoxybribonucleicAcid (cDNA)Archive Kit (Applied Biosystems, Foster City,CA), following the manufacturers instructions.For every RT reaction, one RNA sample was setup without reverse transcriptase enzyme toprovide a negative control against possiblegenomic DNA contamination. Gene-specific pri-mers were designed by using Primer Expresssoftware (Applied Biosystems), and the optimalconcentrations for each gene were determinedempirically. All primers were purchased fromSigma Genosys: PPARg, 50-TTGGCCATATTTA-TAGCTGTCATTATT-30 and 50-TGTCCTCGAT-GGGCTTCA-30; LPL, 50-GGGTCGCCTGGTC-GAAGT-30 and 50-AAAGTGCCTCCATTGGGA-TAAA-30; 28S, 50-GAATCCGCTAGGAGTGTG-TAACAA-30 and 50-GCTCCAGCGCCATCCAT-30. The SYBRGreen I assay was performed withthe SYBP Green PCR Master Mix (AppliedBiosystems) and a modified universal thermal</p><p>Effects of MPR on Adipogenesis 383</p></li><li><p>cycling condition (2 min at 508C and 10 min at958C, following by 40 cycles of 10 s each at 95, 60,and 728C) with the standard disassociation/melting parameters (15 s each at 95, 60, and958C) on the ABI Prism 7900HT SequenceDetection System (Applied Biosystems). Thespecificity of the SYBR Green I assay wasverified by performing a melting curve analysisand by subsequent sequencing of the PCRproducts.Levels of 28S rRNA (housekeeping gene) and</p><p>target mRNAs in each RNA sample werequantified by the relative standard curvemethod (Applied Biosystems). Briefly, standardcurves for 28S rRNA and each target gene weregenerated by performing a dilution series of amixed cDNA pool. For each RNA sample, theamount of target mRNA relative to that of 28SrRNA was obtained. For each target gene, foldchanges in the MPR group compared with thecontrol were then calculated, and expressed asmeanSEM.</p><p>Oil Red O Staining</p><p>Adipocyte monolayers (at days 3, 6, and 9)were washed with DPBS, fixed for 1 h with 4%paraformaldehyde at room temperature andincubated in 60% isopropanol for 5 min. Oil redO (3g/L) (Sigma) in99% isopropanolwasdilutedwith water, filtered and added to the fixed cellmonolayers for 5 min and then nuclei werestained with hematoxylin for 30 s. Cell mono-layers were then washed with water and thestained triglyceride droplets were visualizedand photographed.</p><p>Statistical Analyses</p><p>Results are presented as meanSEM of fourto eight independent experiments (i.e., indivi-dual rats), as indicated.Datawereanalyzed byastandard Student t-test. Significance was set atP&lt; 0.05. Calculations were performed usingSPSS software version 9.0 (Chicago, IL).</p><p>RESULTS</p><p>Effects of MPR on DNA Synthesis in Preadipocytesin Primary Culture</p><p>To determine if preadipocytes fromMPR ratsexhibited a greater potential for proliferation,[3H]-thymidine incorporation was used to mea-sure the rate of DNA synthesis in preadipocytesisolated from control and MPR visceral adiposetissue under standard culture conditions at</p><p>several time p...</p></li></ul>

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