Note: for non-commercial purposes only Placental amino acid transport and metabolism Dr Rohan Lewis rohan.lewis@southampton.ac.uk
Why do we need to understand placental amino acid transfer? • Amino acid transfer…. • Is required for fetal growth • Is reduced in fetal growth restriction • Is reduced prior to fetal growth restriction • Interventions could optimise amino acid transfer and fetal growth • But interventions are more likely to succeed if we understand the mechanisms
There are multiple determinants of placental function Transporters Blood flow MVM Maternal BM Fetal Structure Metabolism Diffusion distance Consumption Surface area Inter-conversion Which of these are most likely to be rate limiting for fetal growth?
Placental blood flow Amino acids must be delivered before they can be transported
Blood flow and placental transfer • Oxygen transfer is flow limited • Rapid diffusion down concentration gradients • Maternal and fetal blood flow maintain the gradients • Is amino acid transfer flow limited? • Placental uptake occurs against the gradient so is unlikely to be flow dependent • But placental transfer to the fetus occurs down gradients so could be flow dependent
Flow effects in the isolated perfused human placenta 1. Maternal artery: Perfuse 14 C-phenylalanine • Control flow rate 10, 14, 18 ml/min • 3. Fetal vein: 2. Fetal artery: Measure 14 C-phenylalanine Control flow rate 3, 6, 9 ml/min • • transfer
Study design: flow rates the perfused human placenta 20 18 Maternal flow rate 16 10, 14, 18 ml/min Flow rate (ml/min) 14 Flow rate ml/min 12 10 maternal flow fetal flow 8 Fetal flow rate 6 3, 6, 9 ml/min 4 2 0 0 50 100 150 200 250 300 Time (minutes) Time from start of tracer perfusion
Increasing maternal flow increases amino acid uptake 35 Placental uptake (pmol/min) 30 25 Plaxental uptake (umol/min) 20 15 10 5 Flow ml/min 0 10M3F 10M6F 10M9F 14M3F 14M6F 14M9F 18M3F 18M6F 18M9F Maternal 10 10 10 14 14 14 18 18 18 Fetal 3 6 9 3 6 9 3 6 9 Data is mean and SEM 2 way ANOVA Maternal flow P = 0.011, Fetal flow P = 0.41, Interaction P = 0.96
Increasing fetal flow decreases fetal vein amino acid concentration 1,8 Fetal vein concentration (pmol/l) 1,5 Fetal vein Phe concentration (umol/ml) 1,2 0,9 0,6 0,3 Flow ml/min 0 Maternal 10 10 10 14 14 14 18 18 18 Fetal 3 6 9 3 6 9 3 6 9 Data is mean and SEM 2 way ANOVA Maternal flow P = 0.54, Fetal flow P < 0.001,Interaction P = 0.22
But neither maternal or fetal flow affect placental transfer 5 Placental transfer (pmol/min) 4 Placental Phe transfer (umol/min) 3 2 1 0 Flow ml/min Maternal 10 10 10 14 14 14 18 18 18 Fetal 3 6 9 3 6 9 3 6 9 Data is mean and SEM 2 way ANOVA Maternal flow P = 0.71, Fetal flow P = 0.79,Interaction P = 0.39
Phenylalanine transfer is not flow limited • Placental uptake • Was associated with maternal but not fetal flow • Placental transfer was independent of flow • Higher uptake did not mean more transfer! • So while flow is essential for amino acid transfer it is not rate limiting • Even at half physiological flow rates
Membrane transport 20 amino acids 20+ transport proteins
Amino acid uptake Maternal blood Placental syncytiotrophoblast Fetal blood aa1 aa1 aa1 aa1 aa1 Accumulative aa1 aa2 aa2 aa2 aa2 Exchanger aa3 aa3 aa3 aa3 MVM BM Amino acids = aa
Amino acid efflux Cleal et al (2011) J. Physiol 589:987-989 Maternal blood Placental syncytiotrophoblast Fetal blood Accumulative aa1 aa1 aa2 aa3 aa2 Exchanger Exchanger aa3 aa3 aa3 aa3 aa3 aa3 aa3 Facilitated MVM BM Amino acids = aa
Amino acid transfer requires complex interactions • Amino acid transfer requires interactions • Between different classes of transporter • Between the MVM and the BM • Its more complex than shown • 20 amino acids and more than 20 transporters • Can we predict the effect of changing the level of one transporter or substrate? • Computational modelling…
Does metabolism facilitate amino acid transport? Amino acid consumption Amino acid inter-conversion
Placental amino acid metabolism Mother Placenta Fetus CONSUMPTION Reduces the size of amino acid pool available for transfer to fetus AA1 AA1 AA1 AA2 AA2 INTERCONVERSION Alters the composition of the amino acid pool available for transfer to the fetus AA = amino acid
Human placental glutamate metabolism Day et al 2013 Placenta 34, 1223-1231 Nitrogen Glutamate Carbon NH 4 + glutamine glutamine proline proline leucine α -ketoglutarate valine Intermediary isoleucine metabolites aspartate alanine aspartate ATP
Placental amino acid metabolism Day et al 2013 Placenta 34, 1223-1231 Mother Placenta Fetus CONSUMPTION Intermediary metabolites and energy ≈45% glutamate glutamate glutamate ≈45% ≈ 90% ≈ 10% glutamine glutamine glutamine INTERCONVERSION
Glutamine synthesis may drive uptake of maternal amino acids by exchange Maternal blood Placental syncytiotrophoblast Fetal blood 5 mmol/l Accumulative glutamate transporter Cycling of abundant 1. 2. amino acid glutamine 4. Exchanger Exchanger 3. 4. Lower abundance Delivery of lower essential abundance amino acids essential amino Facilitated acids to the fetus MVM BM
Metabolism summary • Glutamine synthesis may occur in order to drive amino acid uptake by exchange • Makes the system less dependent on accumulative transporters such as system A • The question we need to model is whether metabolism is a rate limiting factor
Placental structure Is this important for amino acid transfer?
Placental structure • We don’t know how placental structure may relate to amino acid transfer • Factors like surface area and diffusion distance will be important • But not as important as they are for oxygen • Modern imaging techniques may help us to investigate these questions
microCT can image large scale vascular structure
Light sheet microscopy can image up to 1 cm 3 (this image 0.2 cm 3 ) Key Red: GLUT1 Green: Capillaries
Transporters may be localised to specific regions on the villi Lat2 Key Blue: LAT2 Green: MVM
Computational Modelling An integrated approach to placental transfer
Placental function determines fetal development • We often assume that changes in placental characteristics correlate with function • e.g. more transporter = more transfer & vice versa • But placental function is complex and this assumption may not be justified • Computational modelling allows us to study complex interactions and make predictions
The simple model 3 amino acids, 3 compartments, one transporter of each type Syncytiotrophoblast Fetal artery Maternal artery aa1, aa2, aa3, aa1, aa2, aa3, aa1, aa2, aa3, 425 µM each 150 µM each 100 µM each Accumulative aa1 Exchanger Exchanger aa1, aa2, aa3 aa1, aa2, aa3 Facilitated Flow Flow diffusion aa3 All K M values 100 μ M
What can the simplified model tell us? • How changing individual factors are likely to affect placental function • Plasma amino acid concentrations • Transporter activity • Blood flow
Varying maternal amino acids ( ± 10 fold) 1.7 x 10 x 10 x -3.6 x
Increasing one amino acid alone decreases transfer of the others
Accumulative transporter V max A X X F MVM BM The V max of all other transporters
MVM Exchanger V max A X X F MVM BM The V max of all other transporters
BM facilitated transporter V max A X X F MVM BM The V max of all other transporters
Maternal and fetal flow Maternal flow* Fetal flow* *For a given level of transporter activity
Conclusions • Placental amino acid transfer requires multiple mechanisims • But many of these necessary processes may never become rate limiting • e.g. flow is essential but even at rates well below physiological is not rate limiting • Interventions to optimise fetal growth need to target rate limiting steps • Modelling will help us to identify these
Acknowledgements Southampton - Biologists Manchester • Dr Jane Cleal • Prof Colin Sibley • Emma Lofthouse • Dr Ed Johnstone • Prof Alan Jackson • Dr Ian Crocker • Prof Mark Hanson • Dr Jo Glazier • Dr Suzanne Brookes • Dr Kate Widdows • Dr Priscilla Day Southampton – Modellers • Dr Bram Sengers • Nont Panitchob • Simone Perazzollo Oxford - Maths • Prof Colin Please Gerald Kerkut Charitable Trust
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