Abstract We investigated the impact of intrauterinegrowth retardation and fetal programming of hyperten-sion by maternal dexamethasone treatment on cardiacuncoupling protein (UCP) expression during develop-ment and in adulthood in the rat. Dexamethasone admin-istered via an indwelling osmotic pump (100 µg/kg bodymass per day from day 15 of gestation) decreased fetalbody mass at day 21 of gestation (by 13%; P<0.05), elic-ited significant (+24%, P<0.01) systolic hypertensionand elevated corticosterone levels (+15%; P<0.05) inadult (24-week-old) male offspring. Cardiac UCP-2 andUCP-3 protein expression was significantly upregulatedduring early postnatal development, reaching 1.7-foldand 2.7-fold the respective fetal day-21 levels by postna-tal day 7 and reaching plateaus at postnatal days 15–21(2.5-fold and 3.5-fold of respective fetal levels). CardiacUCP protein expression at fetal day 21 and the ontogenyof cardiac UCP expression during early postnatal lifewere unaffected by prenatal dexamethasone treatment.Prenatal dexamethasone treatment did not abrogate thepostnatal surge in corticosterone levels or modify plasmanon-esterified fatty acid (NEFA) levels over this period.However, UCP-2 and UCP-3 protein expression was sig-nificantly downregulated in the hearts of adult hyperten-sive male offspring of dexamethasone-treated mothers(to 27% and 65% of control values respectively). Wepropose that changes in cardiac UCP protein expressionare linked with changes in cardiac metabolic fuel selec-

tion (from glucose→fatty acids at birth and from fattyacids→glucose during hypertension).

Keywords Development · Dexamethasone · Fatty acids ·Fetal programming · Heart

Introduction

Late fetal life is associated with high rates of cardiacglucose utilization [33]. However, the transition to theearly postnatal period is associated with a switch fromcarbohydrates to fatty acids (provided in maternal milk)as the main energy source (reviewed in [12]), leading toincreased rates of cardiac lipid oxidation and suppressionof myocardial glucose utilization [21]. Studies with cul-tured neonatal cardiac myocytes have shown that fattyacids upregulate the expression of genes encoding pro-teins involved in fatty acid transport and metabolism [7,40, 41, 42]. The effects of fatty acids in this system alsoinclude stimulation of mRNA expression for uncouplingprotein 2 (UCP-2), an effect mimicked by ligands specif-ic to the fatty-acid-activated transcription factor peroxi-some proliferator activated receptor-α (PPAR-α) [43].UCP-2 mRNA expression in rat neonatal myocytes isalso upregulated by tri-iodothyronine and β-adrenergicagonists [39, 43], but not by α-adrenergic agonists.These latter observations led to the proposal that thyroidhormones and/or the sympathetic nervous system, bothof which are important for the maturational growth and function of the heart during early postnatal life [9,14, 20, 23, 25, 26, 36, 45], might be involved in the physiological upregulation of heart UCP-2 ex-pression that is observed during early postnatal develop-ment in the rat [39]. However, the greater ability of fattyacids compared to tri-iodothyronine and β-adrenergic agonists to upregulate cardiac UCP-2 mRNA expressionin neonatal myocytes prompted Van Der Lee et al. [43]to suggest that the switch from glucose to fatty acids asthe main energy source at birth might be directly respon-sible.

M.C. Sugden (✉ )Department of Diabetes and Metabolic Medicine, Medical Sciences Building, Queen Mary, University of London, Mile End Road, London, E1 4NS, UKe-mail: m.c.sugden@qmw.ac.ukFax: +44-20-89818836

M.L. Langdown · N.D. Smith · M.C. Sugden · M.J. HolnessDepartment of Diabetes and Metabolic Medicine, Division of General and Developmental Medicine, St.Bartholomew’s and the Royal London School of Medicine and Dentistry, Queen Mary, University of London, London, E1 4NS, UK

Pflügers Arch - Eur J Physiol (2001) 442:248–255DOI 10.1007/s004240100519

O R I G I N A L A RT I C L E

Maria L. Langdown · Nicholas D. Smith · Mary C.Sugden · Mark J. Holness

Excessive glucocorticoid exposure during late intrauterine development modulates the expression of cardiac uncoupling proteins in adult hypertensive male offspringReceived: 30 August 2000 / Received after revision: 19 December 2000 / Accepted: 21 December 2000 / Published online: 14 March 2001© Springer-Verlag 2001

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Under resting conditions, the adult heart derives about70% of its energy from the oxidation of lipids, and theremainder primarily from glycolysis and glucose oxida-tion [37]. Although fatty acids have been demonstratedto have a clear role in regulating UCP-2 expression inneonatal cardiac myocytes, situations associated withgreatly enhanced cardiac fatty acid utilization in adult-hood, namely fasting and experimental diabetes, do notmodulate cardiac UCP-2 mRNA expression [15, 43].This led to the suggestion that cardiac UCP-2 expressionmight already be maximal by virtue of the predilectionof the adult heart for fatty acids as fuel substrates. How-ever, cardiac UCP-3 mRNA expression in the hearts ofadult rats is upregulated both in starvation and experi-mental diabetes [15]. Taken together, these finding sug-gest either that the responses of UCP-2 and UCP-3 to up-regulation by fatty acid oxidation and/or supply differ insensitivity, or that there are different mechanisms forregulating the two UCP isoforms in the adult heart.

During the development of heart failure and pressure-overload-induced cardiac hypertrophy, the chief myocar-dial energy substrate switches back from fatty acids toglucose [6, 10, 38], with the upregulation of enzymes in-volved in glucose utilization and the downregulation ofenzymes concerned with fatty acid oxidation. The impactof the switch from fatty acid to glucose utilization oncardiac UCP-2 and UCP-3 protein expression has notbeen established. However, a key question is whetherstates associated with decreased fatty acid supply/oxida-tion are associated with suppression of cardiac UCP ex-pression. Epidemiological studies have raised the possi-bility that “early lifestyle” factors, which are not deter-mined by the individual but by the intrauterine or neona-tal environment, increase the risk of developing cardio-vascular disease and hypertension in adult life (see [3, 4,16] for recent reviews). In particular, maternal hormonalstatus has been implicated as a critical factor that mayprogramme long-term changes in cardiac function, pre-disposing to adult disease (reviewed in [16]). A rat mod-el (maternal dexamethasone treatment during the lastthird of pregnancy) has been developed that leads both tointrauterine growth retardation and hypertension in laterlife [5, 19]. Changes in cardiac UCP mRNA expressionmight be predicted to have an impact on cardiac growthand development as a result of altered energy efficiency.Indeed, ATP deficiency [15] and impaired function ofthe diabetic rat heart [1, 17, 22, 28, 29] might be causedby proton leakage following upregulation of UCP-3 ex-pression by fatty acids. It is also possible that cardiacUCP-2 and UCP-3 expression may be selectively regu-lated. Therefore, the aim of the present study was to ex-amine the effects of prenatal dexamethasone treatmenton the ontogeny of cardiac UCP-2 and UCP-3 proteinexpression during early postnatal development when theneonate is presented with a largely lipid-based diet (ma-ternal milk). We also aimed to investigate whether thisprenatal intervention, which predisposes to hypertensionin later life, leads to persistent selective changes in cardi-ac UCP-2 or UCP-3 protein expression in adulthood.

Materials and methods

Materials

General Laboratory reagents were from Roche Diagnostics(Lewes, East Sussex, UK) or from Sigma (Poole, Dorset, UK),with the following exceptions. All organic solvents were of analyt-ical grade and obtained from BDH (Poole, Dorset, UK). ECL re-agents, Hyperfilm and secondary antibodies were purchased fromAmersham Pharmacia Biotech (Little Chalfont, Bucks, UK). Anti-UCP-2 and anti-UCP-3 rabbit polyclonal IgG antibodies were pur-chased from Autogen Bioclear UK (Calne, Wiltshire, UK). Dexa-methasone (sodium phosphate) was obtained from David BullLaboratories (Warwick, UK). Bradford reagents were purchasedfrom BioRad (Hemel Hempstead, Herts., UK). Kits for the mea-surement of corticosterone and NEFA were purchased from ICNPharmaceuticals (Basingstoke, Hants., UK) and Alpha Labs (East-leigh, Hants., UK). Alzet osmotic mini-pumps were purchasedfrom Charles River (Margate, Kent, UK).

Animals

All studies were conducted in adherence to the regulation of theUnited Kingdom Animal Scientific Procedures Act (1986). Fe-male albino Wistar rats (200–220 g) were purchased from CharlesRiver. Rats were maintained at a temperature of 22±2°C and sub-jected to a 12-h light/12-h dark cycle. Rats were maintained onstandard, pelleted rodent diet purchased from Special Diet Servic-es (Witham, Essex, UK). This diet consisted of 52% carbohydrate,15% protein, 3% lipid and 30% non-digestible residue (by wt.),and contained 2.61 kcal/g metabolizable energy. In all experi-ments, rats were allowed access ad libitum to standard diet andwater. Food consumption and body weights were determined ev-ery day except during early postpartum to avoid causing the moth-er excessive stress.

Tissue and blood sampling

Rats were anaesthetized by an injection of sodium pentobarbital(60 mg/ml in 0.9% NaCl; 1 ml/kg body wt. i.p.) and, once loco-motor activity had ceased, hearts were rapidly excised and freeze-clamped using aluminium clamps pre-cooled in liquid nitrogen.Frozen hearts were stored in liquid nitrogen. Blood was sampledfrom the chest cavity of dams after the removal of the heart. Bloodwas sampled from fetuses after decapitation and from neonatalrats after decapitation following anaesthesia induction by sodiumpentobarbital injection (60 mg/ml in 0.9% NaCl; 1 ml/kg body wt.i.p.). Blood samples were centrifuged for 5 min at 12,000 g andplasma was stored at –20°C.

Dexamethasone administration

Dexamethasone was administered to pregnant rats from day 15 ofgestation by its subcutaneous infusion via a chronically implantedosmotic minipump, at a dose of 100 µg/kg body weight per day.This dosage has been used previously to elicit hypertension in theadult offspring [19]; the protocol used in the present experimentsdiffers from that used previously in that in our studies dexametha-sone was infused at a steady rate as opposed to in one daily bolusinjection. An initial priming dose (0.1 mg) of dexamethasone wasalso given by subcutaneous injection before minipump implanta-tion.

Western blotting

Cardiac samples (approx. 100 mg) were homogenized using aPolytron Tissue homogenizer (PT 10 probe; position 5, 15 s) in1 ml ice-cold extraction buffer A [20 mM Tris, 137 mM NaCl,

2.7 mM KCl, 1 mM CaCl2, 10% glycerol, 1% Igepal, 45 mM sodi-um orthovanadate (Na3VO4) 0.2 mM PMSF, 10 µg/ml leupeptin,1.5 mg/ml benzamidine, 50 µg/ml aprotinin, 50 µg/ml pepstatin A(in DMSO), pH 8.0]. Homogenates were placed on ice for 20 min,centrifuged in an Eppendorf centrifuge (10,000 g for 20 min at4°C) and the supernatants stored (–20°C) until analysis. Proteinconcentrations were determined using the Bradford method withbovine serum albumin (BSA) as standard. Samples (50–100 µg oftotal protein) were subjected to sodium dodecyl sulfate polyacryl-amide gel electrophoresis (SDS-PAGE) using a 12% resolving gel,with a 6% stacking gel. Following SDS-PAGE, resolved proteinswere transferred electrophoretically to nitrocellulose membranes,and then blocked for 2 h at room temperature with Tris-bufferedsaline (TBS) supplemented with 0.05% Tween and 5% (w/v) non-fat powdered milk. The nitrocellulose blots were incubated over-night at 4°C with polyclonal antisera raised against each of theUCPs. Anti-UCP-2 and anti-UCP-3 were used in a 1:100 dilution.Preliminary experiments established specificity by incubatingblots with and without the appropriate competing peptide antigen(2 µg/ml). The blots were washed with TBST (TBS with 0.05%Tween 20) (3×5 min) and incubated with horseradish-peroxidase-linked secondary antibody IgG anti-rabbit (1:2000 in 1% (wt./vol.)non-fat milk in TBST for 2 h at room temperature. Bound anti-body was visualized using ECL according to the manufacturer’sinstructions. The blots were then exposed to Hyperfilm and thesignals quantified by scanning densitometry and analysed withMolecular Analyst software. For each representative immunoblotpresented, the results are from a single gel exposed for a uniformduration.

Blood pressure measurements

Systolic blood pressures were determined by tail cuff plethysmog-raphy at room temperature in lightly anaesthetized (sodium pento-barbital; 10 mg/ 300 g body weight) rats. All measurements wereundertaken in the morning, and each rat had five blood pressuretraces recorded in a single session. The coefficient of variationwas 3.2%. Blood pressures were quantified using a photoelectricsensor attached to the blood pressure monitor, in conjunction withsoftware designed to determine pressures.

Hormone and metabolite measurements

NEFA concentrations were measured spectrophotometrically usinga commercial kit (Alpha Labs). Plasma corticosterone concentra-tions were measured by radioimmunoassay using a commercial kit(ICN Pharmaceuticals). The antiserum used showed <0.05% crossreactivity with cortisol and <0.01% cross reactivity with dexa-methasone.

Statistical analysis

Results are presented as the mean ± standard error (SEM), withthe numbers of rats in parentheses. Statistical analysis was per-formed by ANOVA followed by Fisher’s post-hoc tests for indi-vidual comparisons or Student’s t-test as appropriate (Statview,Abacus Concepts, Berkeley, Calif., USA).

Results

Maternal dexamethasone treatment during pregnancy in the rat leads to intrauterine growth retardation and hypertension in the adult offspring

Dexamethasone was administered via an indwelling os-motic pump at a dose of 100 µg/kg body weight per day

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during the last week of pregnancy. The efficacy of dexa-methasone treatment to suppress endogenous corticoste-rone secretion was demonstrated by significant abroga-tion of the maternal–fetal corticosterone concentrationgradient (Fig.1). Maternal dexathasone administrationdid not alter gestation length, offspring number or viabil-ity (results not shown), but led to a significant 13%(P<0.05) decrease in fetal body weight at day 21 of ges-tation (control 5.4±0.1 g; dexamethasone 4.7±0.1 g).Maternal body weight gain during the last third of preg-nancy was also significantly decreased: controls gained45±7 g body weight whereas the dexamethasone-treateddams only gained 3±1 g body weight. Fetal heart weightat day 21 of gestation was not significantly affected by maternal dexamethasone treatment (control 33.6±1.8 mg; dexamethasone 29.8±2.2 mg).

Confirming previous observations by others [19], theadult male offspring of dams treated with dexamethasonedeveloped modest but significant (P<0.01) systolic hy-pertension – a 24% increase in systolic blood pressure(as measured by tail plethysmography) at 24 weeks ofage compared with control, male offspring (Fig. 2). Inparallel with findings of hypercortisolism in adults oflow birth weight [27], plasma corticosterone concentra-tions were significantly higher (15%; P<0.05) in thelow-birth-weight adult offspring of dexamethasone-treat-ed dams than in control offspring (Fig. 2).

Effects of maternal dexamethasone treatment during pregnancy in the rat on cardiac UCP protein expression in the 21-day-old fetus

Relative levels of UCP-2 and UCP-3 protein expressionin the hearts of control (CON) rats and rats in the experi-

Fig. 1 Effect of treating pregnant rats on days 15–20 of gestationwith vehicle (CON; hatched bars) or dexamethasone (100 µg/kgbody weight per day) (DEX; solid bars) on plasma corticosteroneconcentrations in 21-day pregnant rats and fetuses. Blood was sam-pled from the chest cavity of control or dexamethasone-treated damsafter the removal of the heart. Blood was sampled from fetuses afterdecapitation. Blood samples were centrifuged for 5 min at 12,000 gand plasma was stored at –20°C. Plasma corticosterone concentra-tions were measured using a commercial kit. Further details are pro-vided in Materials and methods. Statistically significant effects ofmaternal dexamethasone treatment are indicated by *P<0.05

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mental (DEX) group (maternal dexamethasone treatmentfrom day 15 of gestation) during late fetal life (day 21)are shown in Fig. 3 together with representative immu-noblots. Both UCP-2 and UCP-3 protein expression weredetectable in the 21-day-old fetal heart. Neither UCP-2nor UCP-3 protein expression in the heart in late fetallife was significantly affected by maternal dexametha-sone treatment at a dose of 100 µg/kg body weight(Fig. 3).

UCP-2 and UCP-3 protein expression are both upregulated during early postnatal life in normally developing rat hearts

The levels of protein expression of UCP-2 and UCP-3 inthe hearts of control rats during early postnatal develop-ment, as quantified by Western blot analysis and ex-pressed relative to levels of expression found in thehearts of 21-day-old fetuses, are shown in Fig. 4 togetherwith representative immunoblots. In the control group,cardiac UCP-2 protein expression was enhanced to a lev-el of approximately 1.7-fold that of the fetal level untilpostnatal day 7, and UCP-2 protein expression continuedto significantly increase between postnatal days 7 and15, but then reached a plateau (Fig. 4A). The relativelevels of UCP-3 protein expression in the hearts of con-trol rats at intervals during early postnatal development,as quantified by Western blot analysis, are shown inFig. 4B. Cardiac protein expression of UCP-3 increasedby approximately 2.7-fold between fetal day 21 andpostnatal day 7, and had reached approximately 3.6-foldof the fetal level by postnatal day 21. Thus, cardiacUCP-3 protein expression was more dramatically upreg-ulated than was UCP-2. Maternal dexamethasone treat-

ment during late pregnancy did not have a significant ef-fect on the cardiac protein expression of either UCP-2 orUCP-3 during the first 21 days of postnatal life (Fig. 4).

Corticosterone and fatty acid concentrations during early postnatal development

Circulating glucocorticoid levels rise during the thirdpostnatal week [21, 27]. In the present study, plasma cor-ticosterone concentrations increased between postnataldays 7 and 15 (see Fig. 5A), but the major increase inplasma corticosterone concentration occurred betweenpostnatal days 15 and 21. Corticosterone concentrationsdid not vary systematically between the control anddexamethasone-treated groups. Importantly, prenataldexamethasone treatment did not abrogate the postnatalsurge in corticosterone level during the third postnatalweek. Plasma NEFA levels were relatively high duringthe first two postnatal weeks, and declined during thethird postnatal week (i.e. when the pups become less re-liant on maternal milk and start to ingest chow)(Fig. 5B). Plasma NEFA levels were not significantly af-fected by prenatal dexamethasone treatment (Fig. 5B).

Fig. 2 Systolic blood pressure and plasma corticosterone concen-trations in 24-week-old male offspring of pregnant rats treatedfrom days 15–20 of gestation with vehicle (CON; hatched bars) ordexamethasone (DEX; solid bars). Systolic blood pressures weredetermined by tail cuff plethysmography at room temperature inlightly anaesthetized rats. All measurements were undertaken inthe morning, and each rat had five blood pressure traces recordedin a single session. Blood pressures were quantified using a photo-electric sensor attached to the blood pressure monitor, in conjunc-tion with software designed to determine pressures. Plasma corti-costerone concentrations were measured using a commercial kit.Further details are provided in Materials and methods. Statisticallysignificant effects of maternal dexamethasone treatment are indi-cated by *P<0.05; **P<0.01

Fig. 3A, B Uncoupling protein 2 and 3 (UCP-2 and UCP-3, re-spectively) expression in the hearts of control 21-day-old fetuses(CON; hatched bars) and 21-day-old fetuses of dams treated with dexamethasone from day 15 of gestation (DEX; solid bars).UCP-2 (A) and UCP-3 (B) protein expression was determined by Western blot analysis in homogenates of hearts obtained from21-day-old fetuses. Homogenates were subjected to SDS-PAGE,resolved proteins were transferred electrophoretically to nitrocel-lulose membranes and, after blocking with Tris-buffered saline(TBS) supplemented with 0.05% Tween and 5% (w/v) non-fatpowdered milk, were incubated overnight at 4°C with polyclonalantisera raised against each of the UCPs. The blots were washedwith TBS with 0.05% Tween 20 and incubated with horseradish-peroxidase-linked secondary antibody IgG anti-rabbit. Bound anti-body was visualized using ECL according to the manufacturer’sinstructions. The blots were then exposed to Hyperfilm and thesignals quantified by scanning densitometry and analysed withMolecular Analyst software. Further details are provided in Mate-rials and methods. Data are presented relative to the CON valuestogether with representative immunoblots. There were no statisti-cally significant effects of maternal dexamethasone treatment

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Maternal dexamethasone treatment is associated with the downregulation of cardiac UCP protein expression in adult male offspring

The protein expression of both UCP-2 and UCP-3 wassignificantly decreased in the hearts of the adult (24-week-old) male offspring of dexamethasone-treatedmothers (DEX) (Fig. 6; representative blots are shown).The downregulation of UCP-2 (to approx. 27% of thecontrol adult value, P<0.01) (Fig. 6A) was greater thanthat of UCP-3 (to 65% of the control adult value,P<0.05) (Fig. 6B). Plasma NEFA concentrations did notdiffer significantly between control and DEX male off-spring at 24 weeks of age [CON, 0.30±0.03 mM (n=9);DEX, 0.33±0.02 mM (n=8); P>0.05].

Discussion

The biological significance of UCP expression in theheart is incompletely understood. The aims of the pres-ent study were to examine the effects of prenatal dexa-methasone treatment on cardiac UCP-2 and UCP-3 pro-tein expression during early postnatal development and

Fig. 4A, B UCP-2 and UCP-3 protein expression at intervals dur-ing early postnatal development in the hearts of control offspring(open symbols) and experimental offspring of dams treated withdexamethasone from day 15 of gestation (closed symbols). UCP-2(A) and UCP-3 (B) protein expression was determined by Westernblot analysis in homogenates of hearts obtained from rats at ap-proximately weekly intervals during postnatal development(7 days, N7; 15 days, N15; and 21 days, N21) during the sucklingperiod. Further details are provided in the legend to Fig. 3 andMaterials and methods. Data are presented relative to the CONvalues in 21-day-old fetuses (F21) together with representativeimmunoblots (C Control, D dexamethasone-treated.) Protein ex-pression of both UCP-2 and UCP-3 at all intervals during postna-tal development was increased significantly (P<0.001) comparedwith corresponding values in 21-day-old fetuses. There were nostatistically significant effects of maternal dexamethasone treat-ment

Fig. 5A, B Plasma corticosterone and non-esterified fatty acid(NEFA) concentrations at intervals during early postnatal develop-ment in control (CON) offspring and the experimental (DEX) off-spring of dams treated with dexamethasone from day 15 of gesta-tion. Blood samples were obtained from control offspring (opensymbols) and experimental offspring (closed symbols) of dexa-methasone-treated dams at approximately weekly intervals duringpostnatal development (7 days, N7; 15 days, N15; and 21 days,N21) during the suckling period. Blood was sampled from neona-tal rats after decapitation following anaesthesia by injection of so-dium pentobarbital (60 mg/ml in 0.9% NaCl; 1 ml/kg body wt.i.p.). Blood samples were centrifuged for 5 min at 12,000 g andplasma was stored at –20°C. Plasma corticosterone (A) and NEFA(B) concentrations were determined using commercial kits. Fur-ther details are provided in Materials and methods

Fig. 6A, B UCP-2 and UCP-3 protein expression in the hearts ofcontrol male adult offspring and the male adult offspring of damstreated with dexamethasone from day 15 of gestation. UCP-2 (A)and UCP-3 (B) protein expression was determined by Western blotanalysis of homogenates of hearts obtained from 24-week-oldadult male offspring of control dams (CON; hatched bars) and ofdams treated with dexamethasone from day 15 of gestation (DEX;solid bars). Further details are provided in the legend to Fig. 3 andMaterials and methods. Data are presented relative to the corre-sponding CON values together with representative immunoblots.Statistically significant effects of maternal dexamethasone treat-ment are indicated by *P<0.05; **P<0.01

for the perinatal upregulation of UCP expression in thedeveloping rat heart.

In the present study we examined cardiac UCP pro-tein expression at approximately weekly intervals duringpostnatal development (7 days, 15 days and 21 days) inthe suckling period. Previous studies demonstrate thatheart UCP-2 mRNA expression, which is relatively lowduring the early postnatal days, is rapidly and signifi-cantly upregulated in the second postnatal week [39]whereas heart UCP-3 mRNA expression remains unde-tectable until the second postnatal week, at which timeexpression reaches a small yet significant peak [39]. Wedemonstrate that the upregulation of cardiac UCP-2 andUCP-3 protein expression is significant during the firstweek of postnatal development, but that the protein ex-pression of both UCP-2 and UCP-3 reaches a plateau be-tween postnatal days 15 and 21. The signals regulatingcardiac UCP-3 expression during the perinatal and neo-natal period are not known. One possibility is thatchanges in cardiac UCP protein expression reflectchanges in total mitochondrial content, which has beencalculated to increase by 20–60% between birth andadulthood in the rat heart [13]. However, the calculatedincrease in mitochondria content is less than the increasein UCP-2 and UCP-3 protein expression during the first21 days after birth (increases of approx. 2.0- and 3.4-foldrespectively) and, although the time courses of cardiacUCP-2 and UCP-3 protein expression upregulation dur-ing early post-natal life are similar, the fold changes inUCP-3 protein expression are consistently greater thanthose for UCP-2. Thus, it is unlikely that changes in mi-tochondrial content alone account for the upregulation ofUCP-2 and UCP-3 protein expression in neonatal life.During intrauterine life, the fetus is supplied with nutri-ents from the placenta, but immediately after birth thismaternal supply of substrates ceases, and the neonatal ratis presented with high-fat (70%), low-carbohydrate milk[30]. Accordingly, during the perinatal period, cardiacsubstrate metabolism shifts from predominantly non-oxi-dative glucose utilization to predominantly fatty acid ox-idation [21]. Since the exposure of neonatal cardiacmyocytes to long-chain fatty acids strongly inducesUCP-2 mRNA expression [43], it has been suggestedthat such a shift in cardiac fuel selection may cause up-regulation of the mRNA expression of UCP-2 in thepost-natal heart. Given the similarity in the time coursesof changes in UCP-2 and UCP-3 expression during earlypostnatal development (present study) and the findingthat starvation and experimental diabetes, both of whichare associated with increased rates of fatty acid oxida-tion, upregulate cardiac UCP-3 expression in adulthood[15], it is possible that fatty acids are important regula-tors of UCP-3 and UCP-2 expression during the perina-tal and neonatal periods. The observation of marked in-creases in UCP-2 and UCP-3 protein expression in theabsence of changes in plasma corticosterone concentra-tions during the first postnatal week indicates that therise in cardiac UCP protein expression is not regulatedby increasing levels of corticosterone.

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to investigate whether this prenatal intervention, whichpredisposes to hypertension in later life, leads to persis-tent selective changes in cardiac UCP-2 or UCP-3 pro-tein expression in adulthood. The present study demon-strates that UCP-2 and UCP-3 protein expression is detectable in the rat heart during late fetal life, is up-regulated and highly expressed in the rat heart duringearly postnatal development and adulthood. Low-dose(100 µg/kg body weight per day) maternal dexametha-sone treatment during the last third of pregnancy in therat, which induces intrauterine growth retardation in as-sociation with the development of hypertension in theoffspring in adulthood [5, 19], did not modify cardiacUCP-2 or UCP-3 protein expression during late fetal life(day 21 gestation, term = 23 days) or during the first3 weeks of postnatal life. In contrast, the study dem-onstrates that protein expression of both UCP-2 andUCP-3 in the adult heart is significantly downregulatedin the hypertensive offspring of rat dams treated withdexamethasone compared with control offspring. Ourdata are consistent with the concept that changes inUCP expression in the heart may be linked with changesin cardiac metabolic fuel selection (from glucose to fat-ty acids at birth and from fatty acids to glucose duringhypertension), but that UCP-2 and UCP-3 protein ex-pression may be differentially responsive to such meta-bolic signals.

Maternal glucocorticoid levels are considerably high-er than fetal concentrations, the fetus being normallyprotected from higher maternal levels of glucocorticoidsby feto-placental 11-β-hydroxysteroid dehydrogenasetype-2 (11-β-HSD2), which inactivates glucocorticoids(reviewed in [34]). The synthetic glucocorticoid dexa-methasone is a poor substrate for 11-β-HSD2; therefore,dexamethasone administration during the last third ofpregnancy exposes the fetus to excessive levels of gluco-corticoids. In the present experiments we used a dexa-methasone dose that provides a concentration in the low-er range of doses used in previous animal studies and isconsidered comparable to the doses used to promote lungmaturation in humans [8]. Suppression of maternal corti-costerone concentrations by exogenous dexamethasoneadministration was clearly evident. Average litter sizeand the duration of pregnancy were unaltered, but the fe-tal body weight at day 21 of gestation was reduced andthe male offspring exhibited significantly elevated bloodpressures and elevated plasma corticosterone concentra-tions at 24 weeks of age. The glucocorticoid type II re-ceptor has been identified in the rat heart during the peri-natal period [18], suggesting that mechanisms are inplace whereby glucocorticoids can modulate cardiacfunction at birth. However, although glucocorticoids nor-mally increase during late pregnancy and modify fetaltissue structure and function in preparation for birth [11],the present finding that low-dose maternal dexametha-sone treatment during the last third of pregnancy doesnot modify the profiles of cardiac UCP-2 and UCP-3protein expression in late-fetal life suggests that the pre-natal glucocorticoid surge is not primarily responsible

References

1. Allison TB, Bruttig SP, Crass MF 3rd, Eliot RS, Shipp JC(1976) Reduced high-energy phosphate levels in rat hearts. I.Effects of alloxan diabetes. Am J Physiol 230:1744–1750

2. Barger PM, Kelly DP (1999) Fatty acid utilization in the hy-pertrophied and failing heart: molecular regulatory mecha-nisms. Am J Med Sci 318:36–42

3. Barker DJP (1999) Fetal origins of cardiovascular disease.Ann Med 31 [Suppl. 1]:3–6

4. Barker DJP (2000) In utero programming of cardiovasculardisease. Theriogenology 53:555–574

5. Benediktsson R, Lindsay RS, Noble J, Seckl JR, Edwards CR(1993) Glucocorticoid exposure in utero: new model for adulthypertension. Lancet 341:339–341

6. Bishop SP, Altschuld RA (1970) Increased glycolytic metabo-lism in cardiac hypertrophy and congestive failure. Am JPhysiol 218:153–159

7. Brandt JM, Djouadi F, Kelly DP (1998) Fatty acids activatetranscription of the muscle carnitine palmitoyltransferase Igene in cardiac myocytes via the peroxisome proliferator-acti-vated receptor alpha. J Biol Chem 273:23786–23792

8. Carlos RQ, Seidler FJ, Slotkin TA (1992) Fetal dexametha-sone exposure alters macromolecular characteristics of ratbrain development: a critical period for regionally selective al-terations? Teratology 46:45–59

9. Chizzonite RA, Zak R (1984) Regulation of myosin isoen-zyme composition in fetal and neonatal rat ventricle by endog-enous thyroid hormones. J Biol Chem 259:12628–12632

10. Christe ME, Rodgers RL (1994) Altered glucose and fatty acidoxidation in hearts of the spontaneously hypertensive rat. J Mol Cell Cardiol 26:1371–1375

11. Fowden AL, Li J, Forhead AJ (1998) Glucocorticoids and thepreparation for life after birth: are there long-term conse-quences of the life insurance? Proc Nutr Soc 57:113–122

12. Girard J, Ferre P, Pegorier JP, Duee PH (1992) Adaptations ofglucose and fatty acid metabolism during perinatal period andsuckling-weaning transition. Physiol Rev 72:507–562

13. Glatz JF, Veerkamp JH (1982) Postnatal development of palm-itate oxidation and mitochondrial enzyme activities in rat car-diac and skeletal muscle. Biochim Biophys Acta 711:327–335

14. Gustafson TA, Bahl JJ, Markham BE, Roeske WR, Morkin E(1987) Hormonal regulation of myosin heavy chain and alpha-actin gene expression in cultured fetal rat heart myocytes. J Biol Chem 262:13316–13322

15. Hidaka S, Kakuma T, Yoshimatsu H, Sakino H, Fukuchi S,Sakata T (1999) Streptozotocin treatment upregulates uncou-pling protein 3 expression in the rat heart. Diabetes 48:430–435

16. Holness MJ, Langdown ML, Sugden MC (2000) Early-lifeprogramming of susceptibility to dysregulation of glucose me-tabolism and the development of Type 2 diabetes mellitus.Biochem J 349:657–665

17. Jenkins RL, McDaniel HG, Digerness S, Parrish SW, Ong RL(1988) Adenine nucleotide metabolism in hearts of diabeticrats. Comparison to diaphragm, liver, and kidney. Diabetes 37:629–636

18. Katz SE, Penefsky ZJ, McGinnis MY (1988) Cytosolic gluco-corticoid receptors in the developing rat heart. J Mol Cell Car-diol 20:323–328

19. Levitt NS, Lindsay RS, Holmes MC, Seckl JR (1996) Dexa-methasone in the last week of pregnancy attenuates hippocam-pal glucocorticoid receptor gene expression and elevates bloodpressure in the adult offspring in the rat. Neuroendocrinology64:412–418

20. Lloyd TR, Marvin WJ Jr (1990) Sympathetic innervation im-proves the contractile performance of neonatal cardiac ventric-ular myocytes in culture. J Mol Cell Cardiol 22:333–342

21. Makinde AO, Kantor PF, Lopaschuk GD (1998) Maturation offatty acid and carbohydrate metabolism in the newborn heart.Mol Cell Biochem 188:49–56

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Situations associated with an elevated fatty acid sup-ply (namely starvation and experimental diabetes) upreg-ulate cardiac UCP-3 expression in adulthood [15]. Incontrast, UCP-2 mRNA expression in the adult heart isrefractory to starvation and diabetes, even though fattyacids promote UCP-2 mRNA expression in neonatal car-diac myocytes [43], leading to the suggestion that themaximal stimulation of cardiac UCP-2 expression by fat-ty acids is already achieved in adulthood. A key ques-tion, therefore, is whether states associated with de-creased fatty acid supply/oxidation are associated withthe suppression of cardiac UCP protein expression. Theheart becomes increasingly dependent upon glucose,rather than lipid, to meet its metabolic demands duringpressure-overload hypertrophy [2, 6, 10, 32, 38]. Adult(24-week-old) male offspring of dexamethasone-treateddams are hypertensive and exhibit left ventricular hyper-trophy, as assessed by an increase in left ventricle plusseptum-to-body weight ratio (control, 0.0016±0.0001;dexamethasone, 0.0020±0.0001; P<0.01). In the presentexperiments UCP-2 and UCP-3 protein expression wassuppressed in the hearts of hypertensive adult offspringof dexamethasone-treated dams. This suggests that cardi-ac UCP-2 expression in adulthood responds predomi-nantly to decreased rather than increased rates of fattyacid oxidation. The downregulation of cardiac UCP-3protein expression in the hypertensive offspring of dexa-methasone-treated dams compared with age-matchedcontrols is also consistent with a metabolic switch fromthe use of fatty acids to that of glucose, even though cir-culating fatty acid supply is unchanged. Thus our datasuggest that changes in cardiac UCP-3 protein expres-sion are elicited by changes in fatty acid utilization, rath-er than simply reflecting changes in fatty acid supply perse.

Both UCP-2 and UCP-3 are thought to be active inuncoupling electron transport from oxidative phosphory-lation [31, 44]; thus, increased UCP protein expressionwould be predicted to reduce cardiac energy efficiency.In addition, it has been hypothesized that mitochondrialuncoupling in the heart reduces the formation of reactiveoxygen species, which may initiate mitochondrial dam-age when rates of fatty acid oxidation are high [24, 35].It is tempting to speculate that the suppression of cardiacUCP protein expression in the adult hypertensive off-spring of dexamethasone-treated dams arises because therequirement to suppress the formation of reactive oxygenspecies is reduced by lower rates of fatty acid oxidation.Furthermore, the differential specificity of the effect ofexcessive exposure to glucocorticoids in early life todownregulate UCP protein expression in adulthood, withmore marked downregulation of UCP-2 than UCP-3,suggests that UCP-2 expression is sensitive to changes infatty acid supply/oxidation in a lower concentrationrange compared to UCP-3.

Acknowledgements This study was supported in part by projectgrants from the British Heart Foundation (PG98/044 andPG99/99197) to M.C.S. and M.J.H. M.L.L. was a recipient of aBritish Heart Foundation Studentship (FS/97079).

34. Seckl JR, Cleasby M, Nyirenda MJ (2000) Glucocorticoids,11beta-hydroxysteroid dehydrogenase, and fetal programming.Kidney Int 57:1412–1417

35. Skulachev VP (1998) Uncoupling: new approaches to an oldproblem of bioenergetics. Biochim Biophys Acta 1363:100–124

36. Steinberg SF, Drugge ED, Bilezikian JP, Robinson RB (1985)Acquisition by innervated cardiac myocytes of a pertussis tox-in- specific regulatory protein linked to the alpha 1-receptor.Science 230:186–188

37. Taegtmeyer H (1994) Energy metabolism of the heart: frombasic concepts to clinical applications. Curr Prob Cardiol 19:59–113

38. Taegtmeyer H, Overturf ML (1988) Effects of moderate hy-pertension on cardiac function and metabolism in the rabbit.Hypertension 11:416–426

39. Teshima Y, Saikawa T, Yonemochi H, Hidaka S, YoshimatsuH, Sakata T (1999) Alteration of heart uncoupling protein-2 mRNA regulated by sympathetic nerve and triiodothyronineduring postnatal period in rats. Biochim Biophys Acta 1448:409–415

40. Van Bilsen M, De Vries JE, Van Der Vusse GJ (1997) Long-term effects of fatty acids on cell viability and gene expressionof neonatal cardiac myocytes. Prostaglandins Leukot EssentFatty Acids 57:39–45

41. Van Bilsen M, Van Der Lee KAJM, Van Der Vusse GJ (1999)Long-chain fatty acids and signal transduction in the cardiacmuscle cell. In Doevendans PA, Reneman RS, Van Bilsen M (eds) Cardiovascular specific gene expression. Kluwer,Dordrecht, The Netherlands, pp 257–268

42. Van Der Lee KAJM, Vork MM, De Vries JE, Willemsen PH,Glatz JF, Reneman RS, Van Der Vusse GJ, Van Bilsen M(2000) Long-chain fatty acid-induced changes in gene expres-sion in neonatal cardiac myocytes. J Lipid Res 41:41–47

43. Van Der Lee KAJM, Willemsen PH, Van Der Vusse GJ, VanBilsen M (2000) Effects of fatty acids on uncoupling protein-2expression in the rat heart. FASEB J 14:495–502

44. Voehringer DW, Hirschberg DL, Xiao J, Lu Q, Roederer M,Lock CB, Herzenberg LA, Steinman L (2000) Gene microar-ray identification of redox and mitochondrial elements thatcontrol resistance or sensitivity to apoptosis. Proc Natl AcadSci USA 97:2680–2685

45. Zhang JF, Robinson RB, Siegelbaum SA (1992) Sympatheticneurons mediate developmental change in cardiac sodiumchannel gating through long-term neurotransmitter action.Neuron 9:97–103

255

22. Miller TB Jr. (1979) Cardiac performance of isolated perfusedhearts from alloxan diabetic rats. Am J Physiol Heart CircPhysiol 236:H808–H812

23. Nadal-Ginard B, Mahdavi V (1989) Molecular basis of cardiacperformance. Plasticity of the myocardium generated throughprotein isoform switches. J Clin Invest 84:1693–1700

24. Negre-Salvayre A, Hirtz C, Carrera G, Cazenave R, Troly M,Salvayre R, Penicaud L, Casteilla L (1997) A role for uncou-pling protein-2 as a regulator of mitochondrial hydrogen per-oxide generation. FASEB J 11:809–815

25. Ogawa S, Barnett JV, Sen L, Galper JB, Smith TW, Marsh JD(1992) Direct contact between sympathetic neurons and ratcardiac myocytes in vitro increases expression of functionalcalcium channels. J Clin Invest 89:1085–1093

26. Orlowski J, Lingrel JB (1990) Thyroid and glucocorticoid hor-mones regulate the expression of multiple Na,K-ATPase genesin cultured neonatal rat cardiac myocytes. J Biol Chem 265:3462–3470

27. Phillips DIW, Walker BR, Reynolds RM, Flanagan DE, WoodPJ, Osmond C, Barker DJP, Whorwood CB (2000) Low birthweight predicts elevated plasma cortisol concentrations inadults from 3 populations. Hypertension 35:1301–1306

28. Pieper GM, Murray WJ (1987) In vivo and in vitro interven-tion with L-carnitine prevents abnormal energy metabolism inisolated diabetic rat heart: chemical and phosphorus-31 NMRevidence. Biochem Med Metab Biol 38:111–120

29. Pieper GM, Salhany JM, Murray WJ, Wu ST, Eliot RS (1984)Lipid-mediated impairment of normal energy metabolism inthe isolated perfused diabetic rat heart studied by phosphorus-31 NMR and chemical extraction. Biochim Biophys Acta803:229–240

30. Prip-Buus C, Thumelin S, Chatelain F, Pegorier JP, Girard J(1995) Hormonal and nutritional control of liver fatty acid oxi-dation and ketogenesis during development. Biochem SocTrans 23:500–506

31. Ricquier D, Bouillaud F (2000) The uncoupling protein homo-logues: UCP1, UCP2, UCP3, StUCP and AtUCP. Biochem J345:161–179

32. Sack MN, Rader TA, Park S, Bastin J, McCune SA, Kelly DP(1996) Fatty acid oxidation enzyme gene expression is down-regulated in the failing heart. Circulation 94:2837–2842

33. Santalucia T, Camps M, Castello A, Munoz P, Nuel A, TestarX, Palacin M, Zorzano A (1992) Developmental regulation ofGLUT-1 (erythroid/Hep G2) and GLUT-4 (muscle/fat) glucosetransporter expression in rat heart, skeletal muscle, and brownadipose tissue. Endocrinology 130:837–846