0031-3998/01/5001-0083 PEDIATRIC RESEARCH Vol. 50, No. 1, 2001 Copyright © 2001 International Pediatric Research Foundation, Inc. Printed in U.S.A. Altered Expression and Function of Mitochondrial ␤-Oxidation Enzymes in Juvenile Intrauterine-Growth-Retarded Rat Skeletal Muscle ROBERT H. LANE, DAVID E. KELLEY, VLADIMIR H. RITOV, ANNA E. TSIRKA, AND ELISA M. GRUETZMACHER Department of Pediatrics, UCLA School of Medicine, Mattel Children’s Hospital at UCLA, Los Angeles, California 90095, U.S.A. [R.H.L.]; and Departments of Internal Medicine [D.E.K., V.H.R.] and Pediatrics [R.H.L., A.E.T., E.M.G.], University of Pittsburgh School of Medicine, Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213, U.S.A. ABSTRACT Uteroplacental insufficiency and subsequent intrauterine creased in IUGR skeletal muscle mitochondria, and isocitrate growth retardation (IUGR) affects postnatal metabolism. In ju- dehydrogenase activity was unchanged. Interestingly, skeletal venile rats, IUGR alters skeletal muscle mitochondrial gene muscle triglycerides were significantly increased in IUGR skel- expression and reduces mitochondrial NADϩ/NADH ratios, both etal muscle. We conclude that uteroplacental insufficiency alters of which affect ␤-oxidation flux. We therefore hypothesized that IUGR skeletal muscle mitochondrial lipid metabolism, and we gene expression and function of mitochondrial ␤-oxidation en- speculate that the changes observed in this study play a role in zymes would be altered in juvenile IUGR skeletal muscle. To test the long-term morbidity associated with IUGR. (Pediatr Res 50: this hypothesis, mRNA levels of five key mitochondrial enzymes 83–90, 2001) (carnitine palmitoyltransferase I, trifunctional protein of ␤-oxi- dation, uncoupling protein-3, isocitrate dehydrogenase, and mi- Abbreviations tochondrial malate dehydrogenase) and intramuscular triglycer- CPTI, carnitine palmitoyltransferase I ides were quantified in 21-d-old (preweaning) IUGR and control IUGR, intrauterine growth retardation rat skeletal muscle. In isolated skeletal muscle mitochondria, AGA, appropriate for gestational age enzyme function of the trifunctional protein of ␤-oxidation and ICD, isocitrate dehydrogenase isocitrate dehydrogenase were measured because both enzymes MMD, mitochondrial malate dehydrogenase compete for mitochondrial NADϩ. Carnitine palmitoyltrans- ND-4L, NADH-ubiquinone oxidoreductase subunit 4L ferase I, the trifunctional protein of ␤-oxidation, and uncoupling HADH, trifunctional protein of ␤-oxidation protein 3 mRNA levels were significantly increased in IUGR HADHA, trifunctional protein of ␤-oxidation ␣ subunit skeletal muscle, whereas mRNA levels of isocitrate dehydroge- UCP3, uncoupling protein 3 nase and mitochondrial malate dehydrogenase were unchanged. UCP3l, uncoupling protein 3—long isoform Similarly, trifunctional protein of ␤-oxidation activity was in- UCP3s, uncoupling protein 3—short isoform Uteroplacental insufficiency causes IUGR and is causally Several investigators have demonstrated that IUGR infants related to the development of multiple adult morbidities in- expend more energy and consume more oxygen than AGA cluding insulin resistance, hypertension, coronary heart dis- infants (2–6); moreover, Bohler et al. (3) found a significant ease, and dyslipidemia (1). Although the effects of these patho- decrease in the respiratory quotient of IUGR infants, but not physiologies are most critically felt during adulthood, evidence AGA infants, over the first 8 wk of life. Similarly, Chessex et exists that metabolism may already be altered in IUGR infants. al. (6) found increased lipid oxidation in 3- to 4-wk-old IUGR infants. The molecular mechanisms of how the transient in utero event of uteroplacental insufficiency affects IUGR post- Received September 18, 2000; accepted February 23, 2001. Correspondence: Robert Lane, M.D., Mattel Children’s Hospital @ UCLA, B2-375 natal metabolism are unknown. MDCC, 10833 Le Conte Avenue, Los Angeles, CA, 90095, U.S.A.; e-mail: Skeletal muscle mitochondrial metabolism contributes sig- [email protected] nificantly to energy expenditure, oxygen consumption, and This research was supported, in part, by National Institute of Child Health and Human Development grants P30HD-28836-05 (R.H.L.), 1KO8BD01225-01 (R.H.L.), and 5P30- lipid oxidation. We have previously demonstrated that utero- DK46204-05 (R.H.L.), and the Magee-Womens Hospital Twenty-Five Club. placental insufficiency leads to altered skeletal muscle mito- 83 84 LANE ET AL. chondrial gene expression and metabolism in juvenile IUGR counterparts (7). Fasting serum triglyceride levels in 21-d-old rats (7). IUGR rat skeletal muscle is characterized by reduced (preweaning) control and IUGR rats are 90 Ϯ 10 and 125 Ϯ 24 mitochondrial NADϩ/NADH ratios and decreased ND-4L mg/dL, respectively (19). mRNA levels (7). ND-4L is a necessary component of the We quantified mRNA levels of CPTI (muscle isoform), mitochondrial respiratory chain complex I. These finding are HADHA, MMD, ICD, UCP3 in whole skeletal muscle, and intriguing because NADϩ is believed to be rapidly recycled enzyme function of HADH and ICD in skeletal muscle mito- between complex I of the respiratory chain and the mitochon- chondria from 21-d-old sham-operated control and IUGR rats drial HADH (Fig. 1) (8). (preweaning). We also measured skeletal muscle triglyceride HADH is an inner mitochondrial membrane protein that levels in both IUGR and sham-operated animals because al- contributes three of the four reactions necessary for mitochon- tered intramuscular triglycerides have been associated with drial long-chain fatty acid ␤-oxidation, including the reduction dysregulation of skeletal muscle mitochondrial metabolism of NADϩ to NADH (9). HADH competes directly for NADϩ (20). with the Krebs cycle dehydrogenases ICD and MMD. As a result, this intramitochondrial competition is a potential point METHODS of flux control connecting the Krebs cycle and ␤-oxidation, which may be particularly important in skeletal muscle (10, Animals. All procedures were approved by the Institutional 11). Animal Care and Use Committee of the Magee-Womens Re- CPTI and UCP3 also affect mitochondrial ␤-oxidation flux search Institute, Pittsburgh, PA, U.S.A. (12, 13). CPTI catalyzes the exchange of acyl-carnitines across Timed pregnant Sprague Dawley rats (Taconic Farms Inc., the mitochondrial outer membrane. UCP3 catalyzes mitochon- Germantown, NY, U.S.A.) were housed in individual cages drial proton leak and is preferentially expressed in skeletal and were exposed to 12-h light-dark cycles. All animals were muscle (14). Increased substrate flux through skeletal muscle fed routine Purina rat chow ad libitum. The animals were mitochondrial ␤-oxidation is associated with increased UCP3 allowed at least2dofacclimatization before experimental mRNA levels (15). handling. We therefore hypothesized that induction of IUGR in rats by On d 19 of gestation (term is 21.5 d), the maternal rats were maternal uteroplacental insufficiency would increase gene ex- anesthetized with intraperitoneal xylazine (8 mg/kg) and ket- pression and function of HADH as well as gene expression of amine (40 mg/kg), and both inferior uterine arteries were CPTI and UCP3, while not affecting the Krebs cycle dehydro- ligated (IUGR) (n ϭ 12 litters). Sham surgery was performed genases ICD and MMD. To test this hypothesis, we used a upon control animals who underwent identical anesthetic and well-characterized rat model of uteroplacental insufficiency surgical procedures except for the uterine artery ligation (n ϭ and IUGR that is induced by ligating both uterine arteries of 12 litters). Maternal rats recovered within a few hours and had the pregnant rat. Fetal and neonatal rats in this model are ad libitum access to food and water. The rats were allowed to significantly lighter than controls that undergo identical anes- delivery spontaneously, and the litter size was randomly culled thesia and sham surgery, and litter size does not differ between to six at birth in both control and IUGR litters by veterinary control and IUGR groups (7, 16). Like the human, the IUGR staff who were blinded to the group and the purpose of the rat fetus is characterized by hypoxia, acidosis, hypoglycemia, study. Maternal rats were allowed ad libitum access to food and hypoinsulinemia (16, 17). These values normalize in the and water, and pups remained with their dams until d 21 of life. perinatal period (18). At d 21 of life, both male and female To perform studies upon d 21 skeletal muscle, pups were IUGR rats weigh significantly less than sham-operated control separated from their dams for 4 h (to minimize individual Figure 1. Illustration of mitochondria and enzymes significant in this study. In bold are the enzymes whose mRNA and/or enzyme function were measured in d 21 sham-operated control or IUGR skeletal muscle. CPTI and HADH are enzymes that play significant roles in determining the rate of ␤-oxidation; ICD and MMD are Krebs cycle NADϩ dehydrogenases that compete with HADH for NADϩ; UCP3 is the skeletal muscle UCP isoform whose expression correlates with fat metabolism and proton leak. I, II, III, and IV identify complexes I, II, III, and IV, respectively. Between complexes I and II, complexes III and IV, and after complex IV are representations of F0 F1 ATP synthase. F1 ATP synthase production of ATP is powered by proton flow through the F0 channel. IUGR ALTERS SKELETAL MUSCLE ␤-OXIDATION 85 hormonal variations associated with feeding) and sedated with retinal RNA. Reactions