Progress 01/01/06 to 12/31/10
Outputs
OUTPUTS: We continued and concluded our proposed investigation on the role of lipoprotein lipase (LPL) in the uptake of postprandial chylomicrons by the maternal-fetal barrier. We took advantage of a mouse strain lacking LPL and over-expressing human LPL only in muscle (MCKhLPL/LPL-/-). In this strain chylomicron remnants-RE are still formed. However, neither mouse nor human LPL are expressed in placenta. MCKhLPL/LPL-/- and wild-type females were maintained on vitamin A-sufficient or -excess (10x) diet during pregnancy and sacrificed at 14.5 dpc. Placental retinoid levels were similar between MCKhLPL/LPL-/- and wild-type dams, and both showed a similar increase when dams were fed a vitamin A-excess diet. In contrast, although MCKhLPL/LPL-/- embryos were morphologically indistinguishable from wild-type, they showed reduced retinyl ester levels, with a greater difference when mothers when maintained on a vitamin A excess diet. Overall these data show that lack of LPL specifically in placenta does not affect dietary retinoid uptake in this tissue. Instead, it influences the transfer of dietary retinoid to the developing embryo. To explore the role of systemic vs. tissue specific LPL activity, we performed whole body lipase inhibition by administration of P-407, 24 hrs prior to sacrifice, followed by a gavage of 3H-ROH. Vehicle injected females were used as controls. From the data on the mice vehicle injected, we can conclude that LPL in placenta is needed for placental uptake of maternally circulating retinoids. However, the systemic inhibition of the LPL catalytic function suggests the LPL might not be the only player involved in this process. In addition, our data suggest that the systemic LPL catalytic function plays an important role in the transfer of retinoid across the maternal-fetal barrier to the embryo. Quantitative real-time PCR (QPCR) was performed on the placenta to investigate other possible molecular pathways involved in vitamin A transfer across the placenta, either in the presence or in the absence of LPL, under different maternal dietary conditions. A role for Lrp1, Ldlr and Scarb1 is shown. In contrast, Lipg and cd36 do not seem to be involved in this process. From these and other data we conclude that placenta can take up both chylomicron remnants and the whole chylomicron. Placental LPL is not the only key player that contributes to this process. However, placental LPL (mainly with its catalytic activity) is essential to allow the transfer of dietary vitamin A from the maternal bloodstream to the developing embryo, at least at 14.5 dpc. PARTICIPANTS: Dr. Loredana Quadro, Project Director, has overall responsibility for all the investigations proposed. She recently supervised and directed one postodoc and one undergraduate students working on the project and she was also responsible for communicating this work in the literature and at professional meetings. In addition she was responsible for all administrative matters associated with this project. Over the years, this project offered the opportunity for training of one undergraduate student, three graduate students and one post-doctoral fellow. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Investigating how vitamin A is delivered to and taken up by the maternal-fetal barrier to be transferred to the developing tissues is crucial to understand how the fetus can maintain vitamin A homeostasis in the face of fluctuation of the maternal dietary vitamin A intake. Addressing this question is of a great importance, especially in view of the very common practice in recent years of consuming large doses of dietary supplements and vitamins, including vitamin A. It is well know, indeed, that both vitamin A-deficiency and -excess can induce embryonic lethality and fetal malformations. Notably, 3% of all children born in the United States have a major malformation at birth, and 70% of these are of unknown etiology. These studies will expand our knowledge of maternal-fetal nutrition and dietary contribution to embryonic development and will ultimately provide new insight into appropriate dietary practices during pregnancy, ultimately reducing the incidence of fetal malformations associated with an abnormal maternal intake of micronutrients, such as vitamins.
Publications
- Y.-K. Kim and L. Quadro. Reverse-phase High Performance Liquid Chromatography (HPLC) analysis of retinol and retinyl esters in mouse serum and tissues. Methods Mol. Biol., Methods Mol Biol., 652:263-75, (2010).
- D. A. Frenz, W. Liu, A. Cvekl, Q. Xie, L. Wassef, L. Quadro, K. Niederreither, M. Maconochie, A. Shanske. Retinoid Signaling in Inner Ear Development: a Goldilocks Phenomenon. Am. J. Med. Gen. Part A, 152A: 2947-2961, (2010).
- B. Ortiz, L. Wassef, E. Shabrova, L. Cordeddu, S. Banni, L. Quadro. Hepatic retinol secretion and storage are altered by dietary conjugated linoleic acid (CLA): common and distinct actions of CLA c9,t11 and t10,c12 isomers. J. Lipid Res., 50: 2278-2289, (2009).
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The developing mammalian embryo obtains vitamin A from the maternal bloodstream. Two majors circulating forms of vitamin A are: retinol (ROH) secreted from the liver stores bound to retinol-binding protein (RBP), in the fasting state; retinyl ester (RE) within chylomicron lipoproteins of intestinal origin, in the fed state. Both forms can be used by the embryo to meet its vitamin A needs. Intestinal chylomicrons are secreted into the bloodstream and hydrolyzed by lipoprotein lipase (LPL), which is bound to the luminal surface of the vascular endothelium, into smaller chylomicron remnants-RE. Although the liver clears the majority of them from the circulation, 25% is taken up by extrahepatic tissues. It has been shown that LPL catalyzes the hydrolysis of chylomicron-RE and that the amount of postprandial RE taken up by extrahepatic tissues correlates with LPL activity. LPL is expressed in placenta and embryonic tissues. We investigated the role of lipoprotein lipase (LPL) in the uptake of postprandial chylomicrons by the maternal-fetal barrier. We took advantage of a mouse strain lacking LPL and over-expressing human LPL only in muscle (MCKhLPL/LPL-/-). In this strain chylomicron remnants-RE are still formed. However, neither mouse nor human LPL are expressed in placenta. MCKhLPL/LPL-/- and wild-type females were maintained on vitamin A-sufficient or -excess (10x) diet during pregnancy and sacrificed at 14.5 dpc. Although MCKhLPL/LPL-/- embryos were morphologically indistinguishable from wild-type, they showed reduced retinyl ester levels, with a greater difference when mothers when maintained on a vitamin A excess diet. Similar results were obtained when pregnant females on a vitamin A-sufficient diet were gavaged with 3H-ROH, 4 hrs prior to sacrifice. 3H-retinoid levels were reduced in MCKhLPL/LPL-/- embryos compared to wild-type. These data indicate that placental-specific genetic ablation of LPL affects uptake of postprandial vitamin A in this tissue and likely its transfer from the maternal bloodstream to the developing tissues. To explore the role of systemic vs. tissue specific LPL activity, we performed whole body lipase inhibition by administration of P-407, 24 hrs prior to sacrifice, followed by a gavage of 3H-ROH. We showed that the P-407 administration significantly reduces 3H-retinoid levels in wild-type embryos, suggesting that failure to form chylomicron remnants-RE through LPL action in the whole body also affects the transfer of maternal postrprandial vitamin A to the fetus. Interestingly, when a similar experiment was performed with LRAT-/-RBP-/- mice, a strain that relies heavily on dietary vitamin A to maintain normal retinoid-related functions, their embryos showed increased levels of 3H-retinoid. Overall, these data strongly indicate that LPL plays an important role in facilitating the uptake of postrandial vitamin A at the level of the maternal-fetal barrier, but they also suggest that alternative mechanisms can take place to ensure adequate transfer of dietary vitamin A from mother to fetus under extreme circumstances. Further investigations are ongoing to identify the molecular players of these alternative mechanisms. PARTICIPANTS: Dr. Loredana Quadro, Project Director, has overall responsibility for all the investigations proposed. She is responsible for communicating this work in the literature and at professional meetings. In addition she is responsible for all administrative matters associated with this project. This year the project offered the possibility to train one graduate and one undergraduate student who are currently working on this project under the supervision of Dr. Quadro. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Investigating how vitamin A is delivered to and taken up by the maternal-fetal barrier to be transferred to the developing tissues is crucial to understand how the fetus maintain vitamin A homeostasis in the face of fluctuation in the maternal dietary vitamin A intake. Addressing this question is of a great importance, especially in view of the very common practice in recent years of consuming large doses of dietary supplements and vitamins, including vitamin A. It is well know, indeed, that both vitamin A-deficiency and -excess can induce embryonic lethality and fetal malformations. These studies will expand our knowledge of maternal-fetal nutrition and dietary contribution to embryonic development and will ultimately provide new insight into appropriate dietary practices during pregnancy.
Publications
- Tepper BJ, Kim YK, Shete V, Shabrova E, Quadro L. Serum retinol-binding protein 4 (RBP4) and retinol in a cohort of borderline obese women with and without gestational diabetes. Clin Biochem. 2010. 43(3):320-3.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The developing mammalian embryo relies on maternal circulation as its source of nutrients, including vitamin A. Two majors circulating retinoids (vitamin A and its derivatives) are: retinol (ROH) secreted from the liver stores bound to retinol-binding protein (RBP), in the fast state; retinyl ester (RE) within chylomicron lipoproteins of intestinal origin, in the fed state. The embryo can use both these retinoids to meet its vitamin A needs. The overall objectives of this study are, first, to investigate the contribution of dietary vitamin A to the bulk of vitamin A delivered to the fetus. Second, to investigate the mechanisms responsible for the uptake of postprandial chylomicrons by the placenta. In particular, to establish the role of lipoprotein lipase (LPL) in this process. Once produced in the intestine, chylomicrons are secreted into the bloodstream and hydrolyzed by lipoprotein lipase (LPL) into smaller chylomicron remnants, still retaining RE. Although the liver takes up the majority of the chylomicron remnants-RE, 25% can be taken up by extrahepatic tissues. It has been shown that LPL catalyzes the hydrolysis of chylomicron-RE and that the amount of postprandial RE taken up by extrahepatic tissues correlates with LPL activity. LPL is expressed in placenta. We investigated whether LPL facilitates the uptake of postprandial vitamin A in this tissue. We showed that whole body lipase inhibition by administration of P-407, followed by gavage of 3H-ROH of wild-type females at 14.5 dpc, result in reduced 3H-retinoid levels in the embryo, likely due to a decrease transfer of postprandial vitamin A from the maternal bloodstream to the developing tissues. To explore the role of systemic vs. tissue specific LPL activity, we used a mouse strain lacking LPL and overexpressesing human LPL only in muscle (MCKhL/LPL-/-). In this strain chylomicron remnants RE are still formed. However, neither mouse nor human LPL are expressed in the placenta. MCKhL/LPL-/- mice were crossed with mice lacking RBP (RBP-/-), which do not mobilize efficiently their vitamin A stores and rely on dietary vitamin A to support normal embryogenesis. The newly generated strain (MCKhLPL/LPL-/-RBP-/-) enabled us to investigate the role played by placental LPL in facilitating the uptake of chylomicrons RE without the confounding effects of hepatic vitamin A delivered to the fetus through the retinol-RBP pathway. MCKhLPL/LPL-/-RBP-/- mice were maintained on different regimens of dietary vitamin A during pregnancy (vitamin A-sufficient, -deficient and -excess diet) and sacrificed at 14.5 dpc. MCKhLPL/LPL-/-RBP-/- embryos were morphologically similar to RBP-/- embryos bred under similar maternal dietary regimens. Also, steady state levels of maternal serum and embryonic retinoids in the MCKhLPL/LPL-/-RBP-/- strain were similar to those of the RBP-/- control strain. To date, our results suggest that systemic, but not placental-specific inhibition of LPL activity, affects uptake of postprandial vitamin A in this tissue. Further experiments are ongoing. PARTICIPANTS: Dr. Loredana Quadro, Project Director, has overall responsibility for all the investigations proposed. She supervises and directs one postodoc and one graduate student currently working on the project and she is also responsible for communicating this work in the literature and at professional meetings. In addition she is responsible for all administrative matters associated with this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The studies carried out in this proposal aim at enhancing our understanding of how vitamin A delivery to the fetus is achieved and how the fetus maintain vitamin A homeostasis in the face of fluctuation in the maternal dietary vitamin A intake. Elucidating the mechanisms of uptake of dietary vitamin A by the developing tissues is of a great importance, especially in view of the very common practice in recent years of consuming large doses of dietary supplements and vitamins, including vitamin A. It is well know, indeed, that both vitamin A-deficiency and -excess can induce embryonic lethality and fetal malformations. These studies will expand our knowledge of maternal-fetal nutrition and dietary contribution to embryonic development and will ultimately provide new insight into appropriate dietary practices during pregnancy.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Vitamin A is an essential nutrient that plays a crucial role during embryonic development. To meet its requirements, the developing embryo depends on the maternal circulation, where two major forms of vitamin A can be identified. Retinol (vitamin A alcohol) bound to its specific transport protein, retinol-binding protein (RBP), is the major vitamin A form in the fasting circulation. It represents vitamin A mobilized from the liver, the main body store of vitamin A. Chylomicrons and chylomicron remnants retinyl esters represent the majority of the circulating dietary vitamin A. The overall objectives of this study are, first, to investigate the contribution of dietary vitamin A to the bulk of vitamin A delivered to the fetus. Second, to investigate the mechanisms responsible for the uptake of postprandial chylomicrons by the placenta. In particular, we will establish the role of lipoprotein lipase (LPL) in this process. During the second year of funding of this project, we
investigated the mechanisms through which mammalian developing tissues maintain adequate retinoid levels in the face of suboptimal or excessive maternal dietary vitamin A intake. Specifically, we explored the role of retinyl ester formation catalyzed by lecithin:retinol acyltransferase (LRAT) in regulating retinoid homeostasis during embryogenesis. Dams lacking both LRAT and retinol-binding protein (RBP), the sole specific carrier for retinol in serum, were maintained on diets containing different amounts of vitamin A during pregnancy. We hypothesized that the lack of both proteins would make the embryo more vulnerable to changes in maternal dietary vitamin A intake. Our data demonstrate that maternal dietary vitamin A deprivation during pregnancy generates a severe retinoid-deficient phenotype of the embryo due to the severe retinoid deficient status of the double mutant dams rather than to the lack of LRAT in the developing tissues. Moreover, in the case of excessive maternal
dietary vitamin A intake, LRAT acts together with Cyp26A1, one of the enzymes that catalyze the degradation of retinoic acid, and possibly with Stra6, the recently identified cell surface receptor for retinol-RBP, in maintaining adequate levels of retinoids in embryonic and extraembryonic tissues. In contrast, the pathway of retinoic acid synthesis does not contribute significantly to regulating retinoid homeostasis during mammalian development except under conditions of severe maternal retinoid-deficiency.
PARTICIPANTS: Dr. Loredana Quadro, Project Director, has overall responsibility for all the investigations proposed. She supervises and directs postdocs and graduate and undergraduate students working on the project and she is also responsible for communicating this work in the literature and at professional meetings. In addition she is responsible for all administrative matters associated with this project. This project offered the opportunity for training of one graduate student, two undergraduate students and one post-doctoral fellow.
Impacts
The long term objective of this proposal is to enhance our understanding of how vitamin A delivery to the fetus is achieved and how the fetus maintain vitamin A homeostasis in the face of fluctuation in the maternal dietary vitamin A intake. Consumption of large doses of dietary supplements and vitamins, including vitamin A, has become a very common practice in recent years, generating the necessity to investigate the effects of high doses of vitamin A intake at different stages of the lifecycle, including pregnancy and development. These studies will expand our knowledge of maternal-fetal nutrition and dietary contribution to embryonic development and will ultimately provide new insight into appropriate dietary practices during pregnancy.
Publications
- Y-K. Kim, L. Wassef, L. Hamberger, R. Piantedosi, K. Palczewski, W. S. Blaner and L. Quadro. (2007). Retinyl ester formation by lecithin:retinol acyltransferase (LRAT) is a key regulator of retinoid homeostasis in mouse embryogenesis. J. Biol. Chem., Epub ahead of print.
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Progress 01/01/06 to 01/01/07
Outputs
The role and mechanisms of utilization of dietary vitamin A during pregnancy and development are the main focuses of our study. To meet its high retinoids (vitamin A and its derivatives) requirements, the developing embryo relies on the maternal circulation, where two major forms of vitamin A are found. Retinol (vitamin A alcohol) bound to its specific transport protein, retinol-binding protein (RBP), is the major form in the fasting circulation. RBP transports vitamin A mobilized from the liver, the main body store of retinoids. Chylomicron retinyl ester represents the majority of the circulating dietary vitamin A. Indeed, once ingested, retinoids are packaged into chylomicrons within the intestine and secreted into the lymphatic system to reach the general circulation and, hence, the target tissues. To investigate the role and the functions of retinyl ester during development, we used mice lacking RBP (RBP-/-) and mice lacking both RBP and lecithin:retinol
acyltransferase, LRAT (LRAT-/-RBP-/-), which is the enzyme that primarily catalyzes retinol esterification. Firstly, we have shown that LRAT is expressed throughout development, in mouse embryo, yolk sac and placenta although at different levels depending on the developmental stage. These data suggest that the ability to generate vitamin A stores is important for the developing tissues. Secondly, we have shown that, although able to breed and generate live progeny when maintained on a vitamin A-sufficient diet, LRAT-/-RBP-/- dams show a significantly increased number of resorptions (39% of the total number of implantations) compared to wild-type or RBP-/- dams. Furthermore, dietary vitamin A deprivation of LRAT-/-RBP-/- dams during pregnancy affects embryonic development and further increases the number of resorptions. The embryonic malformations of the LRAT-/-RBP-/- embryos from dams on a vitamin A-deficient diet are more severe than those reported for RBP-/- embryos from RBP-/- dams
maintained on similar dietary regimen. Indeed, while RBP-/- embryos displayed small eyes and cardiac insufficiency (peripheral edema), LRAT-/-RBP-/- embryos showed eyes malformed or completely undetectable, peripheral edema and abnormal mid-facial regions and forelimbs. The elevated number of resorptions and the embryonic defects are likely due to the dramatically reduced circulating retinoids levels in the bloodstream of LRAT-/-RBP-/- dams maintained on a vitamin A-deficient diet. Investigations are ongoing to verify whether lack of LRAT specifically in the developing tissues may also contribute to this phenotype. We are also exploring the effects of excessive (not toxic) dietary vitamin A intake on embryonic development by using RBP-/- mice, which rely on postprandial vitamin A to support vitamin A-dependent functions, including embryonic development. We have shown that excess of maternal dietary vitamin A intake during pregnancy does not affect the development of RBP-/- mice.
However, an elevation in the number of resorptions seems to occur. Experiments are ongoing to verify whether the amount of vitamin A circulating in the maternal blood and transferred to the embryo is affected by this dietary regimen.
Impacts
This project aims at unraveling the contribution of dietary vitamin A to the bulk of vitamin A delivered to the fetus. These studies will ultimately expand our knowledge of maternal-fetal nutrition and dietary contribution to embryonic development. They will also provide new insight into appropriate dietary practices during pregnancy.
Publications
- No publications reported this period
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