Maternal Plasma L-Carnitine Reduction During Pregnancy Is
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Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2019/03/27/dmd.119.086439.DC1 1521-009X/47/6/582–591$35.00 https://doi.org/10.1124/dmd.119.086439 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 47:582–591, June 2019 Copyright ª 2019 by The American Society for Pharmacology and Experimental Therapeutics Maternal Plasma L-Carnitine Reduction During Pregnancy Is Mainly Attributed to OCTN2-Mediated Placental Uptake and Does Not Result in Maternal Hepatic Fatty Acid b-Oxidation Decline s Mengru Bai, Qingquan Zeng, Yingchun Chen, Mingyang Chen, Ping Li, Zhiyuan Ma, Dongli Sun, Hui Zhou, Caihong Zheng, Su Zeng, and Huidi Jiang Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences (M.B., Y.C., M.C., P.L., Z.M., H.Z., S.Z., H.J.) and Women’s Hospital, School of Medicine (Q.Z., D.S., C.Z.), Zhejiang University, Hangzhou, People’s Republic of China Received January 18, 2019; accepted March 22, 2019 Downloaded from ABSTRACT L-Carnitine (L-Car) plays a crucial role in fatty acid b-oxidation. downregulated and the renal L-Car level was reduced, whereas the However, the plasma L-Car concentration in women markedly urinary excretion of L-Car was lower in late pregnant mice than in declines during pregnancy, but the underlying mechanism and its nonpregnant mice. Meanwhile, progesterone (pregnancy-related consequences on maternal hepatic b-oxidation have not yet been hormone) downregulated the expression of renal OCTN2 via clarified. Our results showed that the plasma L-Car level in mice at PPARa-mediated pathway, and inhibited the activity of OCTN2, but dmd.aspetjournals.org gestation day (GD) 18 was significantly lower than that in nonpreg- estradiol, corticosterone, and cortisol did not. Unexpectedly, the nant mice, and the mean fetal-to-maternal plasma L-Car ratio in GD maternal hepatic level of L-Car and b-hydroxybutyrate (an indicator 18 mice was 3.0. Carnitine/organic cation transporter 2 (OCTN2) was of mitochondrial b-oxidation), and mRNA levels of several enzymes highly expressed in mouse and human placenta and upregulated as involved in fatty acid b-oxidation in GD 18 mice were higher than gestation proceeds in human placenta, whereas expressions of that in nonpregnant mice. In conclusion, OCTN2 mediated L-Car carnitine transporter (CT) 1, CT2, and amino acid transporter B0,+ transfer across the placenta played a major role in maternal plasma were extremely low. Further study revealed that renal peroxi- L-Car reduction during pregnancy, which did not subsequently at ASPET Journals on September 26, 2021 some proliferator–activated receptor a (PPARa) and OCTN2 were result in maternal hepatic fatty acid b-oxidation decrease. Introduction concentration is only half of that in nonpregnant women, which will be returned to its original level 8 weeks after delivery (Cederblad et al., L-Carnitine (L-Car) is a hydrophilic quaternary amine that plays a 1986; Cho and Cha, 2005). It seems that pregnancy leads to a reversible crucial role in fatty acid b-oxidation. L-Car and long-chain acyl CoA form acylcarnitines by the action of carnitine palmitoyltransferase 1. secondary deficiency of L-Car (Marzo et al., 1994). However, the Acylcarnitines are then translocated across the mitochondrial membrane reasons have not yet been clarified. by the carnitine/acylcarnitine translocase. Once inside mitochondria, In humans, L-Car homoeostasis is maintained by dietary absorp- carnitine palmitoyltransferase 2 removes L-Car from acylcarnitines and tion, endogenous synthesis, and efficient renal reabsorption (Vaz and regenerates acyl-CoAs. L-Car then returns to the cytoplasm for another Wanders, 2002). About 75% of the L-Car requirement is provided by cycle while the acyl-CoAs enter b-oxidation (Ramsay et al., 2001; diet, whereas 25% is synthesized endogenously in the liver and kidney of Foster, 2004). L-Car deficiency may cause fatty acid oxidation disorder, adults. L-Car is eliminated in its original form by glomerular filtration in and lead to cardiomyopathy, muscle weakness, hypoglycemia, and fatty humans; however, most of the filtered L-Car will be reabsorbed in kidney liver (Frigeni et al., 2017). The plasma concentration of L-Car in by carnitine transporters (CTs), especially by carnitine/organic cation pregnant women is reported to be much lower than that in nonpregnant transporter (OCTN) 2 (SLC22A5 gene), a high-affinity L-Car transporter women and keeps declining as gestation proceeds (Winter et al., 1995; located in the apical side of renal tubular epithelial cells (Longo et al., Lindsay et al., 2015). At the time of delivery, the maternal plasma L-Car 2016). A defect in the OCTN2 will cause urinary L-Car wasting and subsequently will result in primary L-Car deficiency (Lahjouji et al., 2002). Besides OCTN2, CT1 (SLC22A15 gene), CT2 (SLC22A16 This work was supported by the National Natural Science Foundation of China 0,+ 0,+ [Grant 81573492, 81773804], and Zhejiang Province Natural Science Foundation gene), and amino acid transporter B (ATB ; SLC6A14 gene) can of China [Grant LZ17H300001]. also mediate L-Car uptake (Sekine et al., 1998; Nakanishi et al., 2001; https//doi.org/10.1124/dmd.119.086439. Enomoto et al., 2002). Therefore, CTs in the kidney are very important s This article has supplemental material available at dmd.aspetjournals.org. for the L-Car homeostasis. 0,+ 0,+ ABBREVIATIONS: ATB , amino acid transporter B ; CORT, corticosterone; CT, carnitine transporter; d3-L-Car, d3-L-carnitine; DMEM, Dulbecco’s modified Eagle’s medium; E2, estradiol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GD, gestation day; hOCTN, human carnitine/organic cation transporter; L-Car, L-carnitine; LD, lactation day; MDCK, Madin-Darby canine kidney; OCTN, carnitine/organic cation transporter; P4, progesterone; PPAR, peroxisome proliferator–activated receptor; qRT-PCR, quantitative real-time polymerase chain reaction; TC, total cholesterol; TG, triglyceride; WY14643, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid. 582 The Mechanism of Maternal Plasma L-Carnitine Reduction 583 A series of physiologic changes, including increased maternal fat, Specific pathogen-free female nonpregnant and pregnant (gestation day [GD] blood volume, cardiac output, and blood flow to the kidneys, occur in 14) ICR mice, 8–10 weeks of age, were obtained from Beijing Vital River pregnant women (Costantine, 2014). In addition, the plasma concentra- Laboratory Animal Technology Co., Ltd. (certificate number SCXK 2016-0011; ’ tions of several hormones are gradually increased during pregnancy. The Beijing, People s Republic of China). The animals were housed in cages at 6 ° 6 plasma levels of estradiol (E2) and progesterone (P4) in pregnant women controlled temperature (22.0 1 C) and humidity (50 10)% with a 12-hour light/dark cycle and free access to food and water throughout the study. Mice during the third trimester will reach tens to hundreds folds of the original consumed ad libitum a commercial standard mouse diet (Laboratory Rodent Diet levels (La Marca et al., 2005; Zhang et al., 2015). And the plasma 5001; LabDiet, St. Louis, MO). No dietary manipulations were otherwise concentrations of glucocorticoids (cortisol in humans and corticosterone conducted, and all dietary conditions were the same for the control and treated [CORT] in rodents) are also gradually increased during pregnancy groups. (Soldin et al., 2005; Jung et al., 2011). Blood, livers, kidneys, hearts, and muscles of the nonpregnant (n = 10) and It is noteworthy that the placenta mediates maternofetal transport of a pregnant (GD 18, n = 10) mice were collected after cervical dislocation, and the variety of nutrients, including L-Car. L-Car is pivotal for the fetal fetal blood samples were collected immediately after fetuses were sacrificed by organism, but the fetus has a limited capacity for L-Car biosynthesis decapitation. The plasma/serum was collected immediately after centrifugation at 2 ° (Hahn, 1981; Shenai and Borum, 1984); thus, the fetus obtains almost all 8000g for 10 minutes and stored at 40 C until analysis. Placentas of the pregnant mice at GD 15 (n = 8) and GD 18 (n = 8) were collected, and the tissues of the required L-Car from the maternal circulation. And the L-Car level were frozen in liquid nitrogen immediately after dissection and stored at 280°C in umbilical cord plasma is reported to be higher than that in the mother until analysis. (Talián et al., 2007; Keller et al., 2009). We hypothesized that it might Other nonpregnant mice (n = 10, two mice per cage) and pregnant mice Downloaded from be one of the reasons for the reduction of maternal plasma L-Car (n = 5, one mouse per cage) were used in the urine excretion study. On GD concentration during pregnancy. It has been reported that OCTN2, 16–18 and lactation day (LD) 8–10, urine samples within 24 hours from which is located in the apical membrane of syncytiotrophoblasts of pregnant or postpartum mice were collected. On another successive 3-day the human placenta, mediates the placental transport of L-Car (Lahjouji period (days 1–3), urine samples were collected within 24 hours from et al., 2004; Grube et al., 2005); however, it is not clear whether other nonpregnant mice. CTs are involved in the process. The concentrations of L-Car in plasma, tissues, and urine were determined by It was known that plasma lipid levels, including those of triglyceride the liquid chromatography-tandem mass spectroscopy method described pre- dmd.aspetjournals.org (TG) and total cholesterol (TC), were markedly higher in pregnant viously (Wang et al., 2019). The mRNA and protein levels of related genes in the liver, kidney, and placenta were measured by quantitative real-time polymerase chain women than those in nonpregnant women, and were increased as reaction (qRT-PCR) and Western blot. The concentrations of b-hydroxybutyrate in gestation proceeds (Alemu et al., 2018; Wang et al., 2018).