고함수탄소 식이에 의한 G-6-PDH와 FAS의 생합성 증가기전에 관한 연구

Abstract

[한글]

백서를 굶기면 간장 세포막의 insulin수용체가 급격히 증가하며, 고함수탄소 식이를 재투여하여 혈액내 insulin이 증가하면 insulin과 수용체가 결합하여 그 복합체가 세포내로 신속히 이동되어 세포내의 insulin농도가 증가하고, 이 증가된 insulin이 핵막의 insulin수용체와 결합하면 핵으로부터 mRNA efflux를 증가시켜 증가된 mRNA가 lipogenic enzyme의 생합성을 증가시키는 요인이라고 보고한 바 있으며 (Whang등, 1982 : Yoon등, 1983),그 중에서도 glucose-6-phosphate dehydrogenase (G-6-PDH)를 합성 하는 mRNA가 증가되어

G-6-PDH 합성 증가가 일어나는 기전을 일부 밝힌 바 있다(Kim등, 1985). 또한 insulin과 포도당은 간장세포질 내 fatty acid synthetase(FAS) mRNA함량을 증가시켜 FAS 생합성이 증가된다는 기전일부를 밝힌 바 있다(Suh등, 1986).

본 연구는 장시간 굶긴 백서에 고함수탄소 및 insulin처리와 동시에, 핵으로부터 RNA운반을 억제하는 colchicine, 또는 RNA합성 억제물질인 actinomycin D를 처리한 후 각 동물의 간장세포로 부터 mRNA를 정제 분리하여 cell free system에서 단백질을 합성시킨 후,

G-6-PDH와 FAS를 정량하고, 이들 두 효소를 합성하는 mRNA의 핵으로 부터 원형질내로 운반과 생합성과의 상호관계를 관찰하여, 이들 효소합성 증가기전을 밟히려고 본 실험을 착수하여 다음과 같은 결과를 얻었다.

2일간 굶긴 백서에 ((14)**C)-orotate를 복강내 주입과 동시에 insulin. colchicine 및 actinomycin D등을 각각 처리한 각 군의 간장 세포질내 polysomal RNA를 정제 분리한 후, radioactivity를 측정한 결과 대조군에서 440±9.09dpm, colchicine 단독 처리군은 445

±12.9dpm으로 두 군간에 큰 차가 없었으나, insulin 처리군은 대조군에 비하여 polysomal RNA는 2.16배 증가하였다. Insulin과 colchicine을 동시에 처리한 군에서는 insulin단독 처리군에 비하여 핵으로부터 원형질내로 RNA운반이 35.91% 억제되었다. 또한 insulin

과 actinomycin D를 동시에 처리한 군에서는 insulin처리군에 비하여 65.01% 억제하였다.

각 군의 polysomal RNA로부터 oligo(dT)-cellulose chromatography를 시행하여 순수 분리된 mRNA의 radioactivity를 측정한 결과, 식염수 또는 colchicine단독 처리군에서는 1,400.79±14.60dpm과 1,420.99±31.58dpm로 두 군간에 큰 차가 없었으나, insulin처리군은 대조군에 비하여 1.8배가 증가하였다. Insulin과 colchicine 동시 처리군에서는 insulin처리군에 비하여 mRNA운반은 42.59%가 억제되었으며, insulin과 actinomycin D 동시 처리군은 63.72%가 억제되었다.

각 군의 백서 간장에서 정제 분리한 mRNA를 가지고 소정의 방법에 따라 nuclease를 처리한 가토 reticulocyte lysate system을 이용하여 시험관 내에서 단백질을 합성시키고,G-6-PDH 또는 FAS에 대한 항혈청으로 침전시킨 후, SDS-polyacrylamide gel electrophore

sis(PAGE)를 시행하고, gel slice를 만들어서 radioactivity를 측정 비교한 바, G-6-PDH합성은 대조군, colchicine단독 투여군 및 insulin과 actinomycin D 동시 처리군에서 radioactivity를 관찰 할 수 없었으나, insulin처 리군에서는 1,027 dpm으로 polysomal mRNA

중 G-6-PDH를 합성하는 mRNA 함량이 급격히 증가한 사실을 관찰하였다.

FAS는 대조군과 colchicine단독 처리군에서 radioactivity가 없었으나, insulin처리군은 2300dpm, insulin과 colchicine, actinomycin D와 insulin처리군에서는 insulin처리군에 비하여 54%, 71%가 각각 억제되었다.

이상과 같은 실험결과를 종합하면 insulin은 세포질내 총 polysomal RNA함량보다도 특히 polysomal mRNA함량 증가를 자극하였으며, colchicine은 핵으로부터 세포질내로 총 polysomal RNA운반보다 polysomal mRNA운반을 약간 억제하는 경향을 보였으나 통계학적으로

의미가 없었다. 또한 insulin은 G-6-PDH를 합성하는 polysomal mRNA함량보다 FAS를 합성하는 mRNA함량을 양적으로 증가시켰다.

백서간장 세포에서 insulin은 lipogenic enzyme인 G-6-PDH와 FAS의 mRNA 생합성 및 핵으로부터 efflux를 자극하여 세포질내에서 lipogenic enzymes생합성을 증가시키는 것으로 사료된다.

[영문]

The number of insulin receptor of liver plasma membrane is rapidly increased when the rats are under fasting condition. The increase of insulin secretion by refeeding high carbohydrate diets results in high intracellular insulin concentration brought by receptormediated endocytosis, and in turn, the high intracellular insulin level increases the biosynthesis of lipogenic enzyme such as G-6-PDH and FAS by stimulating the efflux of mRNA from nucleus through insulin receptors of nuclear envelope. In the present study. the major goal was to understand the mechanism of insulin action on the biosynthesis of lipogenic

enzymes, G-6-PDH and FAS, by observing the relation of nuclear RNA efflux and biosynthesis of mRNA using colchicine which inhibits the efflux of RNA from nucleus and actinomycin D which inhibits the synthesis of RNA.

The results are sumarized as follows. The rats fasted for 2 days were treated with insulin, colchicine or actinomrcin D simultaneously by intraperitoneal administration of ((14)**C)-orotate, and polysomal RNA was purified from each group and the radioactivity was counted. Between saline-treated group and

colchicine-treated group the radioactivities of polysomal RNA were not significantly different, but in insulin-treated group it was increased 2.16 fold compared to saline-treated group. The RNA efflux from nucleus was inhibited 35.91% in the group treated with both colchicine and insulin, and the radioactivity was decreased 65.01% in the group treated with both actinomycin D and insulin compared to insulin-treated group. The radioactivities of mRNA purified from polysomal RNA of each group using oligo(dT)-cellulose chromatography were as follows. In saline-treated and colchicine-treated groups the radioactivities of mRNA were 1,400.79±14.60 dpm and 1,420.99±31.58 dpm respectively and there was no significant difference between these groups. In insulintreated group the radioactivity was increased 1.8 fold in comparison to saline-treated group. In the group treated with both colchicine and insulin, mRNA transport from nucleus was inhibited 42.59%, and in the group treated with actinomycin D and insulin, the radioactivity was decreased 63.72% compared to insulin-treated group. Protein was synthesized in vitro from mRNA of each group using rabbit reticulocyte lysate system. After translation, immunoprecipitates were obtained with antiserum against G-6-PDH or FAS, and were electrophoresed using SDS-polyacrylamide gel, the amount of synthesized enzyme was measured by radioactivity of gel slice corresponding to the authentic enzyme. The radioactivity of G-6-PDH was not detected in control

group, colchicine-treated group and the group treated with both actinomycin D and insulin but it was 1,027 dpm in insulin-treated group. This corresponds to the fact that insulin increase the G-6-PDH mRNA amount of the polysomal RNA. The radioactivity of synthesized FAS was not detected in control group and colchicine-treated group, but it was 2,300 dpm in insulin-treated group. It

decreased 54% and 71% in the group treated with both colchicine and insulin and the group treated with both actinomycin D and insulin, respectively.

The present result indicates that insulin increases the amount of the polysomal mRNA for lipogenic enzymes in cytoplasm rather than that of the polysomal RNA, and colchicine inhibits the efflux of polysomal mRNA rather than that of polysomal RNA in some extent, although it was not statistically significant. Insulin increases the amount of polysomal mRNA for FAS more than that for G-6-PDH. It is assumed that insulin increase the biosynthesis of lipogenic enzymes by stimulating synthesis and

efflux of mRNA for lipogenic enzymes.