Effects of parental folate deficiency on folate content, DNA methylation and expressions of folate transporters and IGF proteins in the postnatal rat liver

Abstract

본 연구는 임신전 수컷과 암컷 쥐의 엽산 영양상태에 따른 생후 3주령 새끼 간의 엽산 함량, DNA methylation, folate transporters (FRα • PCFT • RFC)과 IGF proteins (IGF-1 • IGF-2 • IGF-1R)의 발현과 혈장 호모시스테인 농도에 미치는 영향을 알아보고자 하였다. Sprague-Dawley종 수컷 흰쥐와 암컷 흰쥐에게 4주간 실험 식이를 공급하였으며 [0mg/kg of folic acid: 아빠 결핍군 (PD)과 엄마 결핍군 (MD) 그리고 8mg/kg of folic acid: 아빠 보충군 (PS)과 엄마 보충군 (MS)], 5주째에 교배를 시켜 각각 PSxMS, PSxMD, PDxMS과 PDxMD군으로 나누웠다. 출생후 3주령새끼 쥐의 간에서 엽산 함량, global DNA methylation, 그리고 folate transporters (FRα • PCFT • RFC)와 IGF proteins (IGF-1 • IGF-2 • IGF-1R)의 발현을 측정하였다. 또한 새끼의 혈장에서 호모시스테인 농도를 측정하였다. 생후 3주령 새끼 간의 엽산 함량의 경우 PDxMD와 PSxMD군이 PSxMS군보다 유의적으로 낮았으며 (P<0.0001), 혈장 호모시스테인 농도는 PSxMS군보다 PSxMD과 PDxMD군에서 유의적으로 낮았다 (P<0.0001). 이 결과에 엄마 엽산 결핍상태는 생후 새끼 간조직의 엽산함량에 직접적인 영향을 미치는 것으로 나타났다. 따라서 혈장 호모시스테인 농도에도 영향을 미친다. 또한, 생후 3주령 새끼 간조직의 global DNA methylation은 PSxMS군 보다 나머지 세 군 모두에서 유의적으로 감소하였다 (P<0.0001). 이는 아빠 엽산 결핍 또는 엄마 결핍이 부모 모두의 엽산 결핍만큼 간조직의 global DNA methylation에 중요하게 작용하는것으로 보인다. 간조직의 FRα 발현은 PDxMS이 PDxMD군 만큼 감소하였다 (P<0.05). 그러나 PCFT 발현은 네 군간의 차이가 없었다. 간의 IGF-1과 IGF-1R 발현은 PSxMS군에 비하여 PDxMS군에서 감소하였으며 (P<0.01, P<0.05), PDxMD군에서 가장 많이 감소하였다. IGF-2 발현은 PSxMS군 비교하였을 때 PDxMD군에서 가장 많이 감소하였다 (P<0.01). 이 결과는 엄마의 엽산 보충 상태에도 불구하고, 아빠 엽산 결핍이 FRα, IGF-1 및 IGF-1R 발현에 영향을 줄 수 있다는 것을 의미할 수 있다. 3주령 새끼 간의 엽산 함량은 간조직의 global DNA methylation (P<0.01) 그리고 FRα (P<0.05), PCFT (P<0.05), IGF-1 (P<0.05), IGF-2 (P<0.01), IGF-1R (P<0.01) 발현과 양의 상관 관계를 보였으며 간조직의 엽산 함량은 혈장 호모시스테인 농도와 음의 상관 관계가 있었다.;This study investigated the effect of parental folate deficiency on the hepatic folate content, global DNA methylation, protein expressions of folate receptors (FRα, PCFT and RFC), and insulin-like growth factor proteins (IGF-1, IGF-2 and IGF-1R) in the liver, and plasma homocysteine levels in the 3-week-old pups. Sprague-Dawley rats were mated after a 4-week diet intervention: 0mg/kg of folic acid (paternal folate-deficient, PD and maternal folate-deficient, MD), or 8mg/kg of folic acid (paternal folate-supplemented, PS and maternal-folate-supplemented, MS), and were grouped as PSxMS, PSxMD, PDxMS, and PDxMD. Pups were sacrificed at day 21 of lactation. Folate content, DNA methylation, and expressions of FRα, PCFT and RFC, IGF-1, IGF-2 and IGF-1R proteins were analyzed in the liver, while homocysteine concentration was analyzed in the blood plasma of the pups. The hepatic folate content was markedly reduced in the PDxMD and PSxMD groups as compared to the PSxMS group (P<0.0001). The homocysteine levels were significantly lower in the PSxMS and PDxMS groups, while elevated levels were found in the PSxMD, and PDxMD groups (P<0.0001). The result indicates that regardless of paternal folate status, maternal folate deficiency directly influences the liver folate status, and consequently affects homocysteine levels in the 3-week old pups. The hepatic DNA methylation was significantly lower in the PDxMD, PDxMS and PSxMD groups than in the PSxMS group (P<0.0001), indicating that a deficiency of either one of the parents may result to a reduced global DNA methylation in the pup liver as similar to a deficiency in both. The expression of hepatic FRα were significantly lowest in the PDxMD and PDxMS groups, followed by the PSxMD group, as compared with the PSxMS group (P<0.05). On the other hand, the PCFT expression was not significant among the four groups. The respective hepatic IGF-1 and IGF-1R expressions were lower in the PDxMS group and decreased the most in the PDxMD group, as compared to the PSxMS group (P<0.05; P<0.01), while IGF-2 expression decreased the most in the PDxMD group, as compared to the PSxMS group (P<0.01). These results may imply that paternal folate deficiency, despite adequate maternal folate status, can influence the protein expressions of FRα, IGF-1 and IGF-1R. Moreover, significant positive correlations were found between hepatic folate content and global DNA methylation (P<0.01), and protein expressions of FRα (P<0.05), PCFT (P<0.05), IGF-1 (P<0.05), IGF-2 (P<0.01) and IGF-1R (P<0.01), whereas an inverse correlation was found between hepatic folate content and plasma homocysteine level in the 3-week-old rat pup.