Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
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Advances in Clinical and Experimental Medicine

2010, vol. 19, nr 5, September-October, p. 563–572

Publication type: original article

Language: English

Placental Amino Acid Transport System A – an Assessment of SNAT1 and SNAT2 Expression in F1 and F2 Placentas in a Rat Model of Gestational Protein Restriction

System A łożyskowego transportu aminokwasów – ocena ekspresji SNAT1 i SNAT2 w łożyskach pokolenia F1 i F2 na szczurzym modelu ograniczenia podaży białka w ciąży

Allah Haafiz1,, Yuan-Xiang Pan2,, Donald Novak1,*

1 Department of Pediatrics, University of Florida College of Medicine, USA

2 Department of Food Science & Human Nutrition, University of Illinois at Urbana-Champaign, USA

Abstract

Background. Gestational protein restriction (GPR) can program a fetal phenotype prone to develop metabolic syndrome in successive generations. Although mechanisms are not well characterized, placental amino acid transport system A (SysA) activity is depressed in the setting of GPR.
Objectives. To determine mechanisms of GPR-induced SysA-adaptations in F1 and F2 placentas.
Material and Methods. Rats (F0) were pair-fed either a 19% normal protein diet (NPD) or an 8% low protein diet (LPD) through pregnancy and lactation. F1 placentas were studied for SNAT1 and SNAT2 mRNA and SNAT1 protein. Male and female offspring (F1) were bred to control animals and allowed to deliver at term at which time placentas were collected for the same studies. Transient transfection of HEK 293 cells was done using p-CMV-FLAG-SNAT2 or the control vector. After 36 hours of transfection, MeAIB transport, expression of SNAT2 mRNA and proteins were assessed.
Results. In F1 placentas, steady-state mRNA content of SNAT1 (140 ± 13 vs. 99 ± 11 arbitrary mRNA units; p ≤ 0.01) and SNAT2 (81 ± 6, n = 10 vs. 104 ± 9 arbitrary mRNA units; p ≤ 0.001) were higher in LPD than NPD group. An opposite but non-significant trend in mRNA expression of both isoforms was evident in F2 placentas. Despite up-regulation of mRNA in F1 placentas, SNAT1 immunoblot bands were comparable from placentalapicalmembranes (0.62 ± 0.13 vs. 0.63 ± 0.13 arbitrary units; p = 0.9), basal-membranes (0.9 ± 0.14 vs. 1 ± 0.06 arbitrary units; p = 0.6) and placental-homogenates (0.5 ± 0.16 vs. 0.7 ± 0.1 arbitrary units; p = 0.3) between LPD and NPD group. Similar results were seen in F2 placental SNAT1 protein expressions. SNAT2- mRNA over-expression by transient transfection with pLPCX-FLAG-SNAT2 construct vs. control vector in HEK 293 cells resulted in up-regulation of both SNAT2 protein and Na+ dependent MeAIB transport (1243 ± 137 vs. 390 ± 27 pmole .1mg .1 min; p ≤ 0.0008).
Conclusion. 1. Although GPR-induced SysA repression is associated with up-regulation of SNAT1 and SNAT2 mRNA in F1 placentas, the protein content is unchanged suggesting post-transcriptional regulation of SysA expression and function. Up-regulation of SNAT2 protein and transport activity following SNAT2-mRNA-over-expression noted in our tissue culture studies support this conclusion. 2. If maternal nutrition is optimized, GPR-induced SysA F1 placental abnormalities are not replicated in F2 placentas.

Streszczenie

Wprowadzenie. Ograniczenie podaży białka w ciąży (GPR) może zaprogramować u płodu fenotyp skłonności do wystąpienia zespołu metabolicznego w kolejnych pokoleniach. Chociaż mechanizmy tego zjawiska nie są dobrze poznane, wiadomo, że w warunkach GPR aktywność łożyskowego systemu A transportu aminokwasów (SysA) jest ograniczona.
Cel pracy. Ocena mechanizmów przystosowania się SysA pod wpływem GPR w łożyskach pokoleń F1 i F2.
Materiał i metody. U szczurów (F0) podczas ciąży i laktacji zastosowano dietę z 19% odpowiednią zawartością białka (NPD) i z 8% małą zawartością białka (LPD). W łożyskach F1 badano zawartość białek SNAT1 i SNAT2 mRNA i SNAT1. Potomstwo płci męskiej i żeńskiej (F1) krzyżowano ze zwierzętami z grupy kontrolnej. Młode rodziły się o czasie, wtedy pobierano łożyska do badań. Wykonano przejściową transfekcję komórek HEK 293 za pomocą p-CMV-FLAG-SNAT2 lub wektora kontrolnego. Po 36 godzinach transfekcji oceniono transport MeAIB, ekspresję SNAT2 mRNA i białka.
Wyniki. W łożyskach F1, zawartość SNAT1 w stanie równowagi mRNA (140 ± 13 vs 99 ± 11 umownych jednostek mRNA, p ≤ 0,01) i SNAT2 (81 ± 6, n = 10 vs 104 ± 9 umownych jednostek mRNA, p ≤ 0,001) była większa w grupie LPD niż w grupie NPD. Odwrotna, ale nieistotna tendencja w ekspresji mRNA obu izoform była widoczna w łożyskach F2. Mimo zwiększonej aktywacji mRNA w łożyskach F1, prążki SNAT1 błon szczytowych łożyska (0,62 ± 0,13 vs 0,63 ± 0,13 umownych jednostek, p = 0,9), błon podstawnych (0,9 ± 0,14 ± 0,06 vs 1 umownych jednostek, p = 0,6) i homogenat łożyska (0,5 ± 0,16 vs 0,7 ± 0,1 umownych jednostek p = 0,3) były porównywalne między LPD a grupy NPD. Podobne wyniki obserwowano w ekspresji białka SNAT1 w łożyskach F2. Nadekspresja SNAT2 w mRNA przez przejściową transfekcję pLPCX-FLAG-SNAT2 vs wektor kontrolny w komórkach HEK 293 powodowała zwiększoną regulację zarówno białka SNAT2, jak i Na+ zależnego transportu MeAIB (1243 ± 137 vs 390 ± 27 pmol jednostki; p ≤ 0,0008).
Wnioski. Chociaż ograniczenie SysA wywołane przez GPR jest związane z nadmierną aktywacją SNAT1 i SNAT2 w mRNA w łożyskach F1, zawartość białka nie zmienia się, co sugeruje potranskrypcyjną regulację ekspresji i czynności SysA. Nadmierna aktywacja białka SNAT1 i transport zachodzący pod wpływem nadekspresji SNAT2-mRNA wykryte w badanych tkankach potwierdza ten wniosek. Jeśli stan odżywienia matki jest optymalny, to zaburzenia SysA wywołane przez GPR w łożyskach F1 nie są replikowane w łożyskach F2.

Key words

nutrient gene interaction, fetal programming, fetal origins of adult disease, metabolic syndrome, amino acid transport system A

Słowa kluczowe

interakcja gen żywienie, programowanie płodu, płodowe pochodzenie chorób dorosłych, zespół metaboliczny, system A transportu aminokwasów

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