Carnitine Content and Expression of Mitochondrial β-Oxidation Enzymes in Placentas of Wild-Type (OCTN2+/+) and OCTN2 Null (OCTN2−/−) Mice

Abstract

Placenta requires energy to support its rapid growth, maturation, and transport function. Fatty acids are used as energy substrates in placenta, but little is known about the role played by carnitine in this process. We have investigated the role of carnitine in the expression of the enzymes involved in fatty acid ␤-oxidation in placenta of OCTN2 Ϫ/Ϫ mice with defective carnitine transporter (OCTN2). Heterozygous (OCTN2 ϩ/Ϫ ) female mice were mated with heterozygous (OCTN2 ϩ/Ϫ ) male mice. Pregnant mice were killed and fetuses and placentas were collected. Carnitine was measured using HPLC and tandem mass spectrometry. Immunohistochemistry was used to detect enzyme expression. Enzyme activities were measured spectrophotometrically. The fetal and placental weights were similar among the three genotypes (OCTN2 ϩ/ϩ , OCTN2 ϩ/Ϫ , and OCTN2 Ϫ/Ϫ ). The levels of carnitine were markedly reduced (Ͻ20%) in homozygous OCTN2 Ϫ/Ϫ null fetuses and placentas compared with wildtype OCTN2 ϩ/ϩ controls. However, carnitine concentration in placenta was 2-to 7-fold higher than in the fetus in all three genotypes. Immunohistochemistry revealed that ␤-oxidation enzymes are expressed in trophoblast cells. Catalytic activities of these enzymes were present at comparable levels in wild-type (OCTN2 ϩ/ϩ ) and homozygous (OCTN2 Ϫ/Ϫ ) mouse placentas, with the exception of SCHAD, for which activity was significantly higher in OCTN2 Ϫ/Ϫ placentas than in OCTN2 ϩ/ϩ placentas. These data show that placental OCTN2 is obligatory for accumulation of carnitine in placenta and fetus, that fatty acid ␤-oxidation enzymes are expressed in placenta, and that reduced carnitine levels up-regulate the expression of SCHAD in placenta. Carnitine is an obligatory cofactor for the transport of long-chain fatty acids into mitochondria for subsequent ␤-oxidation (1). Carnitine deficiency is defined as a metabolic state in which the carnitine concentration in plasma and tissues falls below 10 -20% of normal values. Biologic consequences of carnitine deficiency mimic primary ␤-oxidation defects (2). OCTN2 (also known as SLC22A5) is a plasma membrane transporter that mediates the Na ϩ -coupled entry of carnitine into mammalian cells (3). Absence of OCTN2 function leads to excessive loss of carnitine in the urine due to defective reabsorption of filtered carnitine and consequently causes carnitine deficiency. Genetic defects in OCTN2 are the cause of primary carnitine deficiency in humans, a condition associated with Received November 26, 2003; accepted March 30, 2004

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

“…Deprivation of carnitine supplements from Octn2 mutant mice between 6 and 8 weeks of age markedly reduces plasma ␤-hydroxybutyrate levels and skeletal muscle glycogen stores (Knapp et al, 2008). Consistent with carnitine deprivation and altered fatty acid oxidation, Octn2 mutant mice develop enlarged fatty liver with steatosis (Shekhawat et al, 2004;Knapp et al, 2008).…”

“…The Octn2 mutation is associated with impaired L-carnitine uptake into isolated JVS mouse hepatocytes (Yokogawa et al, 1999). Heterozygous Octn2 mice are viable and fertile, but Octn2 mutant mice survive for only approximately 3 to 4 weeks without carnitine supplementation (Shekhawat et al, 2004). At 3 weeks of age, the body weight of Octn2 TRANSPORTER FUNCTION AND REGULATION mutant mice is 50% of that of age-matched wild-type mice (Shekhawat et al, 2004).…”

“…Heterozygous Octn2 mice are viable and fertile, but Octn2 mutant mice survive for only approximately 3 to 4 weeks without carnitine supplementation (Shekhawat et al, 2004). At 3 weeks of age, the body weight of Octn2 TRANSPORTER FUNCTION AND REGULATION mutant mice is 50% of that of age-matched wild-type mice (Shekhawat et al, 2004). Deprivation of carnitine supplements from Octn2 mutant mice between 6 and 8 weeks of age markedly reduces plasma ␤-hydroxybutyrate levels and skeletal muscle glycogen stores (Knapp et al, 2008).…”

Determination of Carnitine Ester Patterns During the Second Half of Pregnancy, at Delivery, and in Neonatal Cord Blood by Tandem Mass Spectrometry: Complex and Dynamic Involvement of Carnitine in the Intermediary Metabolism

“…Fetal carnitine is derived from the mother via transplacental transfer of carnitine (13). The Na ϩ -dependent high-affinity carnitine transporter OCTN2 (14,15) is expressed in human placenta (16), and placental OCTN2 is necessary for the accumulation of carnitine in placenta and fetus, and, consequently, for the oxidation of long-chain fatty acids in fetal-placental unit (17,18).…”

“…The Na ϩ -dependent high-affinity carnitine transporter OCTN2 (14,15) is expressed in human placenta (16), and placental OCTN2 is necessary for the accumulation of carnitine in placenta and fetus, and, consequently, for the oxidation of long-chain fatty acids in fetal-placental unit (17,18). Studies on OCTN2 -/-mice suggest that the placental carnitine synthesis might depend on the carnitine status of the fetus and/or the mother (13). On the basis of these observations, it is clear that further studies of maternal and neonatal carnitine status are needed, including the investigation of participation of carnitine in the ester metabolism, as a natural extension.…”