Peroxisome proliferator-activated receptor gamma modulation and lipogenic response in adipocytes of small-for-gestational age offspring

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1 Yee et al. Nutrition & Metabolism, 9:6 RESEARCH Open Aess Peroxisome proliferator-ativated reeptor gamma modulation and lipogeni response in adipoytes of small-for-gestational age offspring Jennifer K Yee, Wai-Nang Paul Lee, Mihael G Ross, Robert H Lane, Guang Han, Juan Vega and Mina Desai Abstrat Bakground: Small-for-gestational age () at birth inreases risk of development of adult obesity and insulin resistane. A model of rat offspring has been shown to exhibit inreased adipose tissue expression of a key adipogeni transription fator, peroxisome proliferator-ativated reeptor gamma (PPARγ), and inreased fatty aid de novo synthesis during the nursing period, prior to onset of obesity. PPARγ agonists have been studied for potential use in the prevention of insulin resistane. Moreover, adipoytes exhibit age-dependent differenes in lipogenesis as mediated by PPARγ. The effets of PPARγ modulators on lipogeni gene expression and de novo lipogenesis on the age-dependent hanges in adipoytes are not known. The objetives of this study were: ) to determine the adipogeni and lipogeni potential in adipoytes at postnatal day (p) and day (p), ) to determine how the PPARγ ativator- and repressor-ligands affet the lipogeni potential, and ) to determine the fatty aid metaboli response to PPARγ ativator-ligand treatment. Methods: Primary adipoyte ultures from p and p and male offspring were established from a known maternal food-restrition model of. Cell proliferation and Oil Red O (ORO) staining were quantified. Adipoytes were treated with inreasing doses of rosiglitazone or bisphenol-a diglyidyl ether (BADGE). PPARγ and SREBP protein expression were determined. De novo lipogenesis with rosiglitazone treatment at p was studied using 5% U C-gluose and gas hromatography/mass spetrometry. Results: At p and p, demonstrated inreased ell proliferation and inreased ORO staining. At p, demonstrated inreased lipogeni gene expression and inreased gluose-mediated fatty aid de novo synthesis ompared with s. In response to rosiglitazone, adipoytes further inreased gluose utilization for fatty aid synthesis. lipogeni gene expression demonstrated resistane to BADGE treatment. Conlusions: adipoytes exhibit an enhaned adipogeni and lipogeni potential in early postnatal life. By p, demonstrated resistane to PPARγ repressor-ligand treatment, and seletive response to high dose PPARγ ativator-ligand treatment in adipogeni and lipogeni gene expression. p adipoytes revealed inreased fatty aid de novo synthesis through a omplex relationship with gluose metabolism. Keywords: Adipoyte, Small-for-gestational age, Rosiglitazone, PPARγ, Stable isotope, Fatty aid, Adipogenesis Correspondene: jyee@labiomed.org Department of Pediatris, Division of Endorinology, Los Angeles Biomedial Researh Institute at Harbor-UCLA Medial Center, David Geffen Shool of Mediine at UCLA, West Carson Street, Harbor Box 6, Torrane, CA 959, USA Full list of author information is available at the end of the artile Yee et al.; liensee BioMed Central Ltd. This is an Open Aess artile distributed under the terms of the Creative Commons Attribution Liense ( whih permits unrestrited use, distribution, and reprodution in any medium, provided the original work is properly ited.

2 Yee et al. Nutrition & Metabolism, 9:6 Page of Introdution Human epidemiologial studies have shown that smallfor-gestational age () newborns demonstrate inreased risk for adult obesity and metaboli syndrome [,]. Animal studies have effetively repliated this assoiation [-5] in that maternal undernutrition during rat pregnany results in. When these pups are nursed normally, they exhibit hyperphagia [6], ath-up growth, and develop adult obesity and insulin resistane [,5]. The inreased adiposity has been attributed to programmed upregulation of the adipogeni transription fator peroxisome proliferator-ativated reeptor γ (PPARγ). newborns demonstrate inreased PPARγ expression prior to the onset of obesity [7]. The downstream lipid target of PPARγ is a lipogeni transription fator, SREBP (sterol regulatory element binding protein ), whih in turn indues expression of lipogeni enzyme fatty aid synthase (FASN) [8]. Both SREBP and FASN are upregulated in adipose tissue at the end of the nursing period (p) [7]. Hene, modulation of PPARγ may provide a therapeuti strategy in preventing adiposity in offspring. As an enhaner of adipoyte differentiation and lipid aumulation, PPARγ has been studied as a target for pharmaologial therapy. Rosiglitazone, whih is a member of the thiazolidinedione (TZD) lass of drugs, is a seletive ligand of PPARγ. It has the ability to ativate PPARγ, and hene indue adipoyte differentiation in ell ulture models and promote weight gain in rodents and humans [9,]. Reent studies have further shown that rosiglitazone may diretly indue expression of PPARγ [,]. Although rosiglitazone auses weight gain, longterm treatment leads to a smaller adipoyte phenotype, suggesting hanges in the metaboli pathways leading to fat aumulation []. While studies have demonstrated that TZDs inrease free fatty aid uptake in adipoytes [,5], the effets of TZDs on endogenous fatty aid synthesis have not been well-studied. Conversely, bisphenol-a diglyidyl ether (BADGE), a seletive PPARγ antagonist, has shown the ability to prevent adipoyte differentiation in vitro [6]. BADGE also has the ability to interfere with rosiglitazone-mediated effets on PPARγ sine both PPARγ modulators (rosiglitazone and BADGE) bind at the ligand-binding domain [6,7]. Consistent with upregulated PPARγ, SREBP, and lipogeni target genes, our rat model of offspring exhibits inreased adipose tissue fatty aid de novo synthesis as early as weeks of age []. Thus, offspring demonstrate upregulation of the adipogenesis signaling asade and enhaned adipose tissue lipogenesis prior to development of an obese phenotype. We hypothesized that the inreased adipogeni potential of offspring is an intrinsi ellular response, whih ontributes to enhaned lipogenesis. Furthermore, we hypothesized that the enhaned lipogenesis is failitated by inreased gluose-mediated fatty aid synthesis. We addressed our hypotheses by utilizing primary adipoyte ultures from day old (p) and week old (p) offspring. We further examined the effet of PPARγ modulators, rosiglitazone (ativator-ligand) and BADGE (repressor-ligand) on the lipogeni targets downstream of PPARγ. To delineate the speifi effets of rosiglitazone treatment in the fatty aid synthesis pathways, we studied rosiglitazone-stimulated gluose utilization toward de novo fatty aid synthesis using U C-gluose as a stable isotope traer. Methods Animal studies Studies reeived approval from the Animal Care Committee at the Los Angeles Biomedial Researh Institute at Harbor-UCLA and were in aordane with the Amerian Areditation Assoiation of Laboratory Care. A desription of the maternal food-restrition rat model has been previously published []. In brief, firsttime pregnant Sprague Dawley rats (Charles River Laboratories, Hollister, CA) were housed in an animal faility with ontrolled / hour light/dark yles, and onstant temperature and humidity onditions. On day of gestation (e), dams were either given a standard laboratory ad libitum how diet (Lab Diet 5, Brentwood, Missouri) to yield normal size pups, or were 5% food-restrited to produe pups. Dams ontinued the assigned diets during pregnany and latation. After birth, on day (p), individual pups were weighed. To standardize nursing, litters were ulled to 8 pups per dam ( males and females) to inlude the offspring of median weights. offspring were ross-fostered to ad libitum-fed ontrol dams. To ontrol for rossfostering tehnique, the ontrol pups were also rossfostered to ontrol dams. Male offspring (n = 6 at eah age per group) were deapitated at postnatal day (p), or given an overdose of pentobarbital ( mg/kg intraperitoneally) at day (p), then the adipose tissue was olleted. Subutaneous adipose tissue was olleted at p due to minimal retroperitoneal adipose tissue availability at this age. Retroperitoneal adipose tissue was olleted at p beause previous data on this model already demonstrated abnormalities in this tissue []. Primary adipoyte ell ultures Pooled adipose tissue from p or p animals was mined, and digested with ollagenase type II (5U/g) in Krebs-Ringer solution for min at 7 C, filtered through μm mesh nylon filter, then entrifuged at 5 x g for 5 minutes. The ells were resuspended in

3 Yee et al. Nutrition & Metabolism, 9:6 Page of high gluose (5 mg/dl) Dulbeo s modified Eagle s medium (DMEM) (Invitrogen) with % fetal bovine serum (FBS) and % Antibioti-Antimyotis (Invitrogen), seeded into flasks and inubated at 7 C with 5% CO until onfluent. For all experiments exept for the ell proliferation assay, adipoyte differentiation was indued with dexamethasone ( μm), methylisobutylxanthine (. mm), and insulin ( μg/ml) for days. Cell proliferation assay Preadipoytes from p and p and male offspring were isolated as above, and seeded ( x ells) in DMEM supplemented with % FBS and % Antibioti- Antimyotis. After hours, -(,5-Dimethylthiazol--yl)-,5-diphenyltetrazolium bromide (MTT, from Sigma) was added to eah well, then ells were inubated at 7 C. The remaining MTT was removed, the dye rystals were solubilized in isopropanol, and the absorbane was read by spetrophotometry at 57 nm wavelength (Perkin Elmer Mulitlabel ounter VICTOR spetrophotometer). Oil red O (ORO) staining and assay Preadipoytes were seeded ( x ells) and ells were allowed to differentiate into mature adipoytes after indution as above. Cells were subsequently fixed with % paraformaldehyde, stained with.5% ORO, mounted onto slides with Vetashield mounting medium with ',6-diamidino--phenylindole (DAPI; Vetor) and images (X magnifiation) aptured (Zeiss Axioskop mirosope with Axioam HR amera). For quantifiation, adipoytes stained with ORO were dried for hour at 7 C, inubated with a fixed volume of isopropanol for minutes to elute the ORO, and absorbane measured at 57 nm. PPARγ modulation studies After differentiation, adipoytes at p and p were inubated in high gluose DMEM with rosiglitazone (Sigma) at, or μm onentration, or with BADGE (Alexis Biohemials) at, or μm onentration. Untreated ells reeived DMSO for hours. Protein was extrated by soniation in RIPA buffer with phosphodiesterase inhibitor and EDTA, then the onentration was measured using the BCA Protein Assay (Piere). μg of protein was loaded on a preast BioRad BisTris gel, then transferred to nitroellulose membranes. Non-speifi binding was bloked by nonfat milk. Primary antibodies were applied overnight, membranes washed, and speies-appropriate seondary antibodies applied. Autoradiography of membranes with hemiluminesene (Piere Super Signal West Pio Chemiluminesene kit) was arried out in a darkroom. After stripping and washing of the original membrane, a primary antibody suitable for use as an internal ontrol was applied, followed by a seondary antibody and hemiluminesene. Densitometry was performed on the protein of interest and band intensities were normalized to those of the orresponding ontrol. The following are the antibodies used and their respetive soures: PPARγ (Affinity Bio Reagents, AB Cam), SREBP (Santa Cruz), FASN (Santa Cruz). Stable isotope fatty aid metabolism studies Primary adipoytes from p male RP fat were isolated and differentiated as above. For hours following differentiation, and adipoytes reeived DMSO or rosiglitazone μm (n = eah group). 5% of the gluose in the high gluose DMEM was omprised of U C-gluose as a traer. After this experimental period, ells from eah flask were olleted as pellets for fatty aid analysis and Western blotting. Fatty aid extrations Total fatty aids (from phospholipids, triglyerides, holesteryl esters and free fatty aids) were extrated from adipoyte ell pellets, using a method by Lowenstein et al. [8]. In summary, adipoytes were saponified in -proof ethanol and % KOH (w/v) in a : volume overnight on a 7 C heating blok. Samples were aidified with HCl, fatty aids were extrated three times with petroleum ether, then air dried. Fatty aids were derivatized as methyl esters using.5 N methanoli HCl, dried under a nitrogen stream, and subsequently reonstituted with hexane for gas hromatography/mass spetrometry (GC/MS) analysis. Gas hromatography/mass spetrometry GC/MS analysis was arried out using a Hewlett- Pakard model 597 seletive mass detetor onneted to a model 689 gas hromatograph. Fatty aids were analyzed as their methyl esters. Fatty aids were separated on the gas hromatograph with a Bpx7 olumn (-m length, 5-μm diameter,.5-μm film thikness) from SGE, In. (Austin, TX). The GC onditions were: helium flow rate, ml/min; initial oven temperature, 5 C; temperature was programmed to inrease at C/min to a final temperature of C. The expeted retention times under these onditions for palmitate, palmitoleate, stearate, oleate, and vaenate (the major fatty aids of interest in this study) were as follows: 6.5, 7., 9.,., and. min, respetively. Mass spetra of fatty aids were aquired using eletron impat ionization and seletive ion monitoring. Ion lusters monitored for isotopomer quantitation were mass-to-harge ratio (m/z) 7 for palmitate (6:), m/z 68 for palmitoleate (6:), m/z 98 for stearate (8:), m/z 6 for oleate (8:n-9) and vaenate (8:n-7). Distribution of the mass isotopomers was determined from the

4 Yee et al. Nutrition & Metabolism, 9:6 Page of spetral data using a method previously desribed by Lee et al. [9] that orrets for the ontribution of derivatizing agent and C natural abundane to the mass isotopomer distribution of the ompound of interest. Eah ompound of interest is omposed of the sum of isotopomer peaks within a luster. The resulting mass isotopomer distribution was expressed in molar frations (m, m, m, m, et.) orresponding to the fration of moleules that ontain,,,,... C substitutions. Fatty aid profile The overall fatty aid profile was determined by alulation of the area under the respetive gas hromatogram peaks for eah main fatty aid of interest as a perent of total fatty aids. The perent of total for eah fatty aid represents the total relative abundane (labeled plus unlabeled). Fration of new synthesis (FNS) over hours, aetyl-coa enrihment, and perent gluose ontribution to de novo synthesis The FNS represents the new fatty aids produed during the hour experimental period, and is expressed as a perentage of total for eah fatty aid. M + and M + isotopomers result from U C-gluose inorporation into palmitate in de novo lipogenesis, thereby allowing alulation of the preursor aetyl-coa enrihment from the onseutive mass isotopomer ratio m/m, and subsequent determination of the FNS. The FNS of stearate was determined from the m6/m ratio to minimize potential hain elongation effets on the m/m ratio. Perent ontribution to de novo lipogenesis (perent ontribution to de novo synthesis of palmitate, the main produt of de novo lipogenesis) from gluose was alulated based on the aetyl-coa enrihment, and 5% isotope enrihment of the medium gluose []. Medium gluose determination Gluose onentration was determined on ell ulture medium using the Gluose HK Assay Kit (Sigma) aording the manufaturer s instrutions, using spetrophotometry. Gluose ontent was alulated for the volume of medium used for eah ulture. Gluose onsumption from the medium was then determined indiretly by subtrating the gluose ontent of the posttreatment medium from the gluose ontent of the original pretreatment medium. Triarboxyli aid (TCA) yle studies To determine the ontribution of the TCA yle to fatty aid synthesis, the relative ontributions from the pyruvate arboxylase (PC) and pyruvate dehydrogenase (PDH) pathways were studied through determination of glutamate fragment enrihment []. Pyruvate arboxylase (PC) ontributes toward prodution of gluoneogeni preursors through formation of oxaloaetate (OAA). PDH ativity produes aetyl-coa whih an be used for fatty aid synthesis through formation of itrate. Downstream of itrate, glutamate equilibrates with α-ketoglutarate [], and reflets different fragment enrihment patterns based on ontributions from the PC and PDH pathways. Glutamate was extrated from ml samples of medium by previously published methods using ion exhange olumns [,]. Glutamate was derivatized as its n-trifluoroaetyl-n-butylester and analyzed by GC/MS using eletron impat ionization. Two fragments were identified and the Σmn enrihment (fration of C atoms/mole) was determined for eah. The fragments were analyzed for the m isotopomer enrihment (m): m/z 5, representing the C-C fragment (pyruvate arboxylase), and m/z 98, representing the C-C5 fragment (pyruvate arboxylase + pyruvate dehydrogenase). The pyruvate arboxylase/pyruvate dehydrogenase (PC/PDH) ratio was determined by alulating (m of 5)/[(m of 98)-(m of 5)] to evaluate for differenes in gluose metabolism via pyruvate entrane into the TCA yle. When entry of gluose into the TCA yle via PC and PDH are equal, the PC/PDH ratio is approximately. A derease in the ratio suggests a relative inrease in PDH ativity, while an inrease in the ratio suggests inreased PC ativity. Statistial analysis Statistial analysis was performed using SigmaStat software. Comparisons were made by ANOVA ) within eah group ( or ) between the basal state and the treated states with inreasing dose of PPARγ modulator, and also ) between the two groups under the same treatment onditions. Fatty aid profile and stable isotope data were analyzed among treated and untreated and groups by two-way ANOVA, followed by pair-wise multiple omparisons using Tukey s test. Parametri data are presented as mean ± the standard error of the mean. Data that failed normality testing was analyzed by Kruskal-Wallis ANOVA on ranks, with pair-wise omparisons using Mann Whitney testing. The non-parametri data (individual PC and PDH data) are therefore presented as the median with the 5-75% interquartile ranges. For omparison purposes, the PC/PDH ratio is also presented as the median with the 5-75% interquartile ranges. Results Preadipoyte proliferation and adipoyte oil red O staining At both p and p, demonstrated inreased preadipoyte proliferation and inreased adipoyte lipid ontent as ompared to s. With age, preadipoyte proliferation dereased while adipoyte lipid ORO

5 Yee et al. Nutrition & Metabolism, 9:6 Page 5 of staining inreased in both and adipose tissue (Figures, ). A Adipogeni and lipogeni transription fators at p At baseline, unstimulated adipoytes demonstrated inreased PPARγ protein expression, but omparable SREBP expression as ompared to s (Figures A). With rosiglitazone, and adipoytes responded to low and high doses with marked inreases in PPARγ, and moderate, though signifiant, inreases in SREBP (Figure A). The responses to both doses were similar in eah group. Furthermore, adipoytes maintained higher expression of PPARγ, but similar SREBP expression as the adipoytes. Oil Red O (OD Units) B p p Adipogeni and lipogeni transription fators adipoyte phenotype at p At baseline, adipoytes ontinued to show upregulated PPARγ, now aompanied by inreased expression of SREBP (Figure B). With rosiglitazone treatment, adipoytes responded to both (low and high) doses, demonstrating signifiant inreases in PPARγ and SREBP expression (Figure B). In ontrast, adipoytes responded at only the higher dose of rosiglitazone treatment with further upregulation of PPARγ and SREBP expression, thereby maintaining inreased expression over s at only this dose. After observing that p adipoytes were responsive to only the higher dose of rosiglitazone, stable isotope studies were undertaken during high-dose rosiglitazone treatment. Cell Proliferation (x - OD Units) p p Figure Cell proliferation in and offspring preadipoytes. Gray bars represent s, and white bars represent. Cell proliferation dereased with age in both groups. demonstrated inreased ell proliferation ompared with s at both p and p, p <.. Figure Oil Red O (ORO) staining in and adipoytes. Gray bars represent s, and white bars represent. (A) adipoytes exhibit greater triglyeride aumulation than s at both p and p. (B) adipoytes at p demonstrate inreased lipid aumulation at X magnifiation by ORO with DAPI staining. p <.5, p <.. Fatty aid metabolism and response to μm rosiglitazone Fatty aid profile At baseline, demonstrated a similar overall fatty aid profile as ompared with s (Table ). In response to rosiglitazone,, but not adipoytes, inreased the stearate perent of total, whih inludes preexisting and newly made stearate (Table ). FNS over hours At baseline, had higher new synthesis (FNS) of palmitate, palmitoleate, stearate, and vaenate over hours (Figure ). In response to rosiglitazone (Figure ), both s and inreased the FNS of palmitate and palmitoleate. Notably, s dereased the FNS stearate with rosiglitazone, while did not respond. Overall, maintained higher FNS rates of these three fatty aids (palmitate, palmitoleate, and stearate) than s. FASN expression, whih was inreased at baseline in, also inreased in response to rosiglitazone (Figure inset). Gluose onsumption studies At baseline, adipoytes onsumed more gluose over hours (Table ) than s. In both groups, gluose onsumption was augmented by rosiglitazone

6 Yee et al. Nutrition & Metabolism, 9:6 Page 6 of A p p p p PPAR? (fold hange) 6 5 Rosiglitazone ( M) SREBP (fold hange) Rosiglitazone ( M) Rosiglitazone ( M) Rosiglitazone ( M) B p p p p PPAR? (fold hange) 6 5 SREBP (fold hange) Rosiglitazone ( M) Rosiglitazone ( M) Rosiglitazone ( M) Rosiglitazone ( M) Figure PPARγ and SREBP expression by Western blotting in p and p adipoytes at baseline and with rosiglitazone treatment. maintained higher expression of PPARγ than s at baseline and stimulated states. (A) p and adipoytes responded to rosiglitazone at both doses. (B) p adipoytes responded only to high dose rosiglitazone. p <.5 ompared to untreated ells within the same group. p <.5 ompared to s treated with the same dose of rosiglitazone. treatment (Table ), but inreased more in than in s (+interation). Aetyl-CoA enrihment At baseline, demonstrated inreased aetyl-coa enrihment as well as inreased perentage of U C- Table Profile of major fatty aids, at baseline and with rosiglitazone Fatty Aids Perent of Total (%) Fatty Aid Baseline Rosiglitazone Baseline Rosiglitazone Palmitate 6: 6. ± ±. 7.5 ± ±. Palmitoleate 6:n-7. ±..9 ±.. ±.. ±. Stearate 8:.6 ±.9 9. ±.9.8 ±.6. ±. Oleate 8:n ±.7 7. ±.. ±.. ±. Vaenate 8:n-7. ±.5. ±.. ±.. ±.9 p <.5 ompared with reeiving the same treatment. p <.5 ompared to the untreated state within the same group. The fatty aid perentages of total are presented as the mean ± the standard error of the mean (SEM). With rosiglitazone treatment, s demonstrated inreased stearate abundane. gluose ontribution to palmitate de novo synthesis (Figure 5). With rosiglitazone treatment, these measures were enhaned in both groups, with maintaining higher levels than s. Greater inreases were observed in (+interation) than in s. TCA yle ativities To eluidate potential TCA yle ontributions to the inreased aetyl-coa enrihment, glutamate enrihment was studied to determine the relative pyruvate arboxylase and pyruvate dehydrogenase ativities. demonstrated inreased entry of U C-gluose into the TCA yle in based on the higher Σmn enrihment (fration of C atoms/mole) in both the glutamate C- and C-5 fragments (Table A). demonstrated a lower PC/PDH ratio as ompared with s (Table B), suggesting higher PDH ativity relative to PC ativity, possibly leading to inreased aetyl-coa prodution for lipogenesis. In response to rosiglitazone, both groups inreased enrihment (Σmn) in the C- and C-5 glutamate fragments

7 Yee et al. Nutrition & Metabolism, 9:6 Page 7 of FNS Fatty Aid (%) FASN FASN expression (fold hange) +Rosi +Rosi +Rosi +Rosi PalmitatePalmitoleate Stearate Oleate Vaenate Figure Fration of new synthesis (FNS) of fatty aids and FASN expression in s and at baseline and with μm rosiglitazone treatment. The data is presented as the mean perentage or mean fold-hange in expression ± the standard error of the mean. demonstrated inreased FNS palmitate and palmitoleate at baseline and with rosiglitazone when ompared to s, with a similar pattern in FASN protein expression (inset). p <.5 ompared to untreated state within the same group, p <.5 ompared to s reeiving the same treatment. (Table A). s exhibited a derease in the PC/PDH ratio while inreased it to a level similar to s (Table B), with a signifiant interation between and rosiglitazone. The inrease of the PC/PDH ratio suggested that shifted its gluose entry to be more balaned between PC and PDH, but this hange ould not be attributed to signifiant hanges in either the PC or PDH enrihment beyond the hanges expeted based on the Σmn. BADGE treatment At p, both and adipoytes showed moderate dereases in PPARγ and SREBP expression at low dose BADGE, then further dereases with high dose BADGE. As before, adipoytes ontinued to exhibit higher expression of PPARγ (Figure 6A). At p, and adipoytes differed in their response to BADGE treatment. While adipoytes responded with downregulation of PPARγ and SREBP, adipoytes were ompletely unresponsive Table Gluose onsumption from ell ulture medium in hours at baseline or with rosiglitazone Gluose Consumption (mg/ml of ulture medium) Baseline.99 ±.. ±. Rosiglitazone Treatment. ±..5 ±. i p <.5 ompared with reeiving the same treatment. p <.5 ompared to the untreated state within the same group. i interation with rosiglitazone. (Figure 6B). Due to the lak of response, the fatty aid metaboli studies were not repeated with BADGE. Disussion This study demonstrates that adipoytes are intrinsially programmed by maternal malnutrition as evident in the prinipal findings of: () enhaned adipogenesis through upregulated PPARγ expression, and () enhaned lipogenesis through upregulated SREBP and FASN expression, with inreased gluose-mediated fatty aid synthesis. Importantly, adipoytes are overall responsive to PPARγ ativator-ligand, but are seletively responsive at p to the higher dose of PPARγ ativator-ligand, with pathway-speifi effets on fatty aid synthesis. In ontrast, adipoytes at p are unresponsive to PPARγ inhibitor-ligand. Together, these results indiate that the adipoyte phenotype is programmed in utero, as demonstrated by enhaned preadipoyte proliferation and lipid aumulation at p. Due to resistane to treatment at p, therapeuti strategies to prevent the obesogeni adipoyte phenotype should be onsidered during the nursing period prior to onset of the resistane. Consistent with previous studies [,5], preadipoyte proliferation delined and adipoyte fat aumulation amplified with age in both the and offspring. Notably, despite growth-restrition at birth, the adipose tissue exhibited inreased preadipoyte proliferation with enhaned fat aumulation, onsistent with an obese phenotype. The upregulated transription fators (PPARγ, SREBP) together with inreased gluose-

8 Yee et al. Nutrition & Metabolism, 9:6 Page 8 of A Aetyl-CoA Enr/Aetyl Unit (%) B U C-Gluose to DNL (%) i i Figure 5 Aetyl-CoA enrihment and U C-gluose ontribution to de novo lipogenesis (DNL). Gray bars represent s, and white bars represent. Solid bars represent adipoytes untreated at baseline, and hathed bars represent adipoytes treated with μm rosiglitazone. (A) adipoytes exhibited inreased aetyl- CoA enrihment, and therefore, (B) inreased U C-gluose ontribution to de novo synthesis of palmitate. There was a signifiant interation between and rosiglitazone for eah of these measures. p <.5 ompared to untreated state within the same group, p <.5 ompared to s reeiving the same treatment, i interation with rosiglitazone. mediated fatty aid synthesis likely failitate enhaned adipogenesis and lipogenesis in offspring. Moreover, these hanges are evident and persistent at postnatal age p in offspring. Inreased risk for obesity in has been well-desribed, with humans exhibiting inreased viseral adiposity [6] or inreased linial features of abdominal adiposity suh as waist irumferene []. Although obesity may not manifest until adulthood, our data in nursing pups demonstrating inreased hyperplasia and hypertrophy along with enhaned fatty aid de novo synthesis indiates the early presene of abnormalities that may ontribute toward the propensity for future fat aumulation. The differential response to rosiglitazone revealed pathway-speifi and age-speifi hanges in lipogenesis between s and. Expression of adipogeni and lipogeni fators was stimulated in both groups at p. At p, however, s demonstrated inreased responsiveness with dose inrease, while responded from its basally inreased expression to only high-dose rosiglitazone. The seletive response of to the highdose rosiglitazone indiates a deline in responsiveness by the end of the nursing period. Nonetheless, the dereased responsiveness of at p indiated hanges leading toward an obese phenotype as evident by the fatty aid synthesis data. Further studies are needed in offspring from p to p to determine the fators in the metaboli milieu that may ultimately lead to p rosiglitazone resistane. In s and, rosiglitazone treatment led to inreased FNS of palmitate and palmitoleate, with a similar pattern of inrease in FASN expression. However, the two groups demonstrated a differential response in 8-arbon stearate synthesis. The group inreased its perent of total of stearate in response to rosiglitazone, despite dereasing the FNS stearate, suggesting dereased stearate turnover as a normal response to rosiglitazone. In ontrast, adipoytes did not derease the FNS stearate in response to rosiglitazone, and maintained the new synthesis rate, suggesting abnormal resistane to modulation in the 8-arbon fatty aid synthesis pathway. The differene in FNS stearate versus palmitate response to rosiglitazone supports possibilities of differential regulation and/or ompartmentalization of the 6-arbon versus 8-arbon fatty aid synthesis pathways []. Sine the FASN expression refleted the FNS palmitate response to rosiglitazone in both groups, the stearate responses to rosiglitazone are likely mediated by a posttranslational mehanism. Whether these differenes in fatty aid metabolism after rosiglitazone treatment impat adipoyte funtion in is not known from our study, but would be of interest in the setting of longterm treatment. Rosiglitazone improves blood gluose ontrol in humans with diabetes through inreased gluose uptake by musle and adipose tissue [7]. Therefore, we used the stable isotope U C-gluose to determine the relationship between the fatty aid synthesis pathways and gluose metabolism in the adipoytes. In fat, our data demonstrate that adipoytes use more gluose toward fatty aid synthesis (inreased aetyl-coa enrihment). Moreover, demonstrated an augmentation of response to rosiglitazone (+interation) possibly through a programmed inrease in sensitivity to gluose abundane. Correspondingly, demonstrated an inreased perent ontribution of U C-gluose to de novo synthesis with rosiglitazone treatment. These findings led to investigations into the PC and PDH-mediated gluose entry pathways into the TCA yle.

9 Yee et al. Nutrition & Metabolism, 9:6 Page 9 of Table Relative PC and PDH ativities determined from adipoyte exposure to U C-gluose A. Σmn C- fragment (mean ± SEM) Σmn C-5 fragment (mean ± SEM) Baseline.56 ±..7 ±. C.6 ±..8 ±. C Rosi Treatment.7 ±..96 ±. C.8 ±.. ±. C B. PC (m 5) (median with 5-75%) PDH (m 98 m 5) (median with 5-75%) PC/PDH (median with 5-75%) Baseline.5 (.5-.7).8 (.7-.8).9 (.9-.).7 (.6-.8).66 (.6-.75).9 (.9-.) C Rosi Treatment.9 (.8-.8).5 (.5-.6). (.-.. (.-.). (.-.9).7 (.-.8) i C p <.5 ompared with reeiving the same treatment. p <.5 ompared to the untreated state within the same group. i interation between and rosiglitazone. A) The Σmn representing the average fration of C atoms/moleule for the C- fragment and C-5 fragments are shown for ultures with and without rosiglitazone treatment. The data are presented as frations of enrihment ± the SEM. Enrihment inreased with rosiglitazone treatment in both s and, while maintained higher levels than s regardless of treatment. B) The PC and PDH pathway ontributions are represented based on glutamate fragment enrihments. These data are presented as the median with the 5-75% interquartile ranges. At baseline, demonstrated a lower PC/PDH ratio than s. With rosiglitazone, s dereased the PC/PDH ratio, while inreased it. A p p p p PPAR? (fold hange) BADGE ( M) SREBP (fold hange) BADGE ( M) BADGE ( M) B p p p p PPAR? (fold hange) BADGE ( M) BADGE ( M) SREBP (fold hange) BADGE ( M) BADGE ( M) Figure 6 PPARγ and SREBP expression by Western blotting with BADGE treatment. (A) p, (B) p. At p, was resistant to both doses of BADGE. p <.5 ompared to untreated ells within the same group. p <.5 ompared to s reeiving the same dose of BADGE.

10 Yee et al. Nutrition & Metabolism, 9:6 Page of Analysis of the PC/PDH ratio was performed to determine how entry of gluose into the TCA yle ontributed to the inreased aetyl-coa enrihment in. adipoytes at baseline exhibited a lower PC/PDH ratio than s, suggesting that adipoytes demonstrate enhaned gluose entry into the TCA yle to produe aetyl-coa for fatty aid synthesis via PDH. Rosiglitazone treatment led to a derease in the PC/PDH ratio in s, supporting enhaned lipogenesis, while adipoytes unexpetedly demonstrated an inreased ratio. Changes in the separate PC and PDH enrihments were therefore examined relatively to hanges in the Σmn. The Σmn was inreased in at baseline and further inreased with rosiglitazone in both glutamate fragments, with orresponding trends in PC and PDH m enrihment. However, the PC enrihment inreased by 9% with rosiglitazone treatment, while the PDH enrihment inreased by only 9%, resulting in a net inrease in the PC/PDH ratio. An alternative explanation for the inreased aetyl-coa enrihment in our study ould be inreased reyling of labeled palmitate, through oxidation and reassembly of labeled aetyl units in de novo synthesis. Additional fators may also be ontributing to our findings, sine the turnover of glutamate is low, and the perentage of gluose ontribution to palmitate omprised only up to 6-%. The pathway of gluose utilization to produe palmitate is likely not diret [8], involving other pathways before these arbons are inorporated into fatty aids. However, this data illustrates the omplex relationship that exists between fatty aid synthesis and gluose metabolism, whih involve multiple metaboli pathways. adipoytes responded to BADGE at both p and p, with further derease in expression of both PPARγ and SREBP at the high dose. In ontrast, adipoytes demonstrated BADGE resistane at both doses of rosiglitazone, suggesting that stimulatory fators that are present annot be overome by BADGE at the experimental doses. It is unknown whether even a higher dose may have been effetive. Despite the urrent obesity epidemi, effetive strategies for the prevention or treatment of obesity and related disorders are very limited. adipoytes, previously adapted to the in utero environment of low energy resoures, suddenly experiene a relative nutrient exess with normal postnatal nutrition [9]. As a result, adipoytes of offspring demonstrate enhaned ell proliferation, and lipogenesis, whih may allow offspring to ath-up in growth. The inreased adipogenesis however, leads to adult obesity and insulin resistane. TZDs are used to improve insulin sensitivity, but they have also been studied for their potential to prevent insulin resistane [,]. Adipose tissue is the major site of ation of TZDs. Further studies are needed to define the relationship between gluose utilization for fatty aid synthesis and insulin sensitivity, in order to determine if TZDs may be effiaious in preventing insulin resistane in. Studies are also needed to determine how the 6- arbon versus 8-arbon fatty aid synthesis pathways ontribute to adipoyte phenotype in, and whether differenes in response of these pathways to rosiglitazone may interfere with its potential to prevent insulin resistane. If gluose-mediated lipogenesis ontributes to insulin sensitivity in TZD treatment, then risks of further adipogenesis during TZD therapy must be weighed against potential benefits of gluose ontrol. Conlusions adipoytes at p and p exhibit enhaned adipogenesis and lipogenesis. p adipoytes respond to both ativator-ligand and repressor-ligand treatment. However, p adipoytes demonstrate pathway-speifi responses to PPARγ ativator-ligand treatment in fatty aid synthesis. p adipoytes are also resistant to PPARγ repressor-ligand treatment. PPARγ ativatorligand treatment enhanes gluose-mediated lipogenesis. The omplex relationship between fatty aid synthesis and gluose metaboli pathways should be further investigated in adipoytes. Abbreviations : Small-for-gestational age; PPARγ: Peroxisome proliferator-ativated reeptor gamma; BADGE: Bisphenol-A diglyidyl ether; TZD: Thiazolidinedione; ORO: Oil Red O; GC/MS: Gas hromatography/mass spetrometry; FNS: Frational synthesis (or fration of new synthesis); TCA: Triarboxyli aid; PC: Pyruvate arboxylase; PDH: Pyruvate dehydrogenase; SREBP: Sterol regulatory element binding protein ; FASN: Fatty aid synthase; DMEM: Dulbeo s modified Eagle s medium; FBS: Fetal bovine serum; MTT: -(,5-Dimethylthiazol--yl)-,5- diphenyltetrazolium bromide; Rosi: Rosiglitazone. Competing interests The authors have no ompeting interests to delare. Authors ontributions JKY partiipated in the study design, exeution, analysis and interpretation of the fatty aid/stable isotope studies, and also drafted the manusript. WPL partiipated in the fatty aid/stable isotope data analysis and interpretation, and ontributed to manusript preparation. MGR partiipated in manusript preparation. RHL partiipated in manusript preparation. GH established the adipoyte ultures, performed the MTT assay, ORO staining, and Western blotting, and ontributed to manusript preparation. JV performed Western blotting and ontributed to manusript preparation. MD partiipated in study design, data analysis and interpretation, and drafting of the manusript. All authors read and approved the final manusript. Aknowledgements We appreiate Linda Day, Stay Behare, and Catherine S. Mao for their assistane in animal are. We thank Shu Lim and Paulin Wahjudi for tehnial assistane in glutamate analysis. J.K.Y. is supported by grant K DK8 from the NIDDK and the Clinial Sholar Award from the Pediatri Endorine Soiety. M.D. is supported by grant RDK8756 from the NIDDK. The GC/ MS analyses were performed at the LABioMed at Harbor-UCLA Biomedial Mass Spetrometry Faility, whih is supported by the UCLA CTSI through grant UL-RR76. Author details Department of Pediatris, Division of Endorinology, Los Angeles Biomedial Researh Institute at Harbor-UCLA Medial Center, David Geffen Shool of

11 Yee et al. Nutrition & Metabolism, 9:6 Page of Mediine at UCLA, West Carson Street, Harbor Box 6, Torrane, CA 959, USA. Department of Obstetris and Gyneology, Los Angeles Biomedial Researh Institute at Harbor-UCLA Medial Center, David Geffen Shool of Mediine at UCLA, West Carson Street, Harbor Box 6, Torrane, CA 959, USA. Division of Neonatology, University of Utah Shool of Mediine, Williams Building, P.O. Box 5889, Salt Lake City, UT 858, USA. Reeived: April Aepted: June Published: June Referenes. Ravelli AC, van Der Meulen JH, Osmond C, Barker DJ, Bleker OP: Obesity at the age of 5 y in men and women exposed to famine prenatally. Am J Clin Nutr 999, 7: Ravelli GP, Stein ZA, Susser MW: Obesity in young men after famine exposure in utero and early infany. N Engl J Med 976, 95:9 5.. Yee JK, Lee WN, Han G, Ross MG, Desai M: Organ-speifi alterations in fatty aid de novo synthesis and desaturation in a rat model of programmed obesity. Lipids Health Dis, :7.. 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