고함수탄소 식이와 Insulin이 백서간장 세포내 Acetyl CoA Carboxylase mRNA 함량 조절에 관한 연구

Abstract

[한글]

백서를 장시간 굶기면 간장 세포막의 insulin수용체가 급격히 증가한다. 이런 쥐에 고함수탄수식이를 재투여하여 혈액내 insulin이 증가하면 insulin과 수용체가 결합하여 그 복합체가 세포내로 신속히 이동되어 세포내외 insulin농도가 증가하고, 이 증가된 insuli

n이 핵막의 insulin수용체와 결합하면 핵으로부터 mRNA efflux를 증가시켜 증가된 mRNA가 lipogenic enzyme의 생합성을 증가시키는 요인이라고 보고한 바 있으며(Whang등, 1982; Yoon등, 1983), 그 중에서도 간장 세포질내 glucose-6-phosphate dehydrogenase(G-6-PDH)

를 합성하는 mRNA가 증가되어 G-6-PDH합성증가가 일어나는 기전을 일부 밝힌바 있다(Kim등, 1985). 또한 insulin과 포도당은 간장 세포질내 fatty acid synthetase(FAS)mRNA 함량을 증가시켜 FAS 생합성이 증가된다는 기전일부를 밝힌 바 있다(Suh등, 1986). 또한 백

서간장 세포에서 insulin은 lipogenic enzyme인 G-6-PDH와 FAS의 mRNA생합성 및 핵으로부터 efflux를 자극하여 세포질내에서 lipogenic enzymes생합성을 증가시키는 사실을 실험적으로 구명하였다(Lee 등, 1987).

본 연구는 지방산 생합성의 rate limiting step에 관여하는 효소인 acetyl CoA carboxylase가 장시간 굶긴 백서에 고함수탄소 식이 및 insulin을 처리하면 백서 간장 세포질내 acetyl CoAcarboxylase함량이 어떻게 조절되는지를 밝히기 위하여 이 효소를 정제 분리하

고, 이에 대한 항체를 얻어 이를 가지고 면역화학법에 의하여 이 효소를 합성하는 특이 po1ysome을 정제 분리하여, 이 효소의 합성 증가기전의 일부를 밝히기 위하여 실험에 착수하여 다음과 같은 결과를 얻었다. 백서를 3일간 굶긴다음 고함수탄소 식이를 3일간 재투

여한 후 간장세포에서 acetyl CoAcarboxylase를 polyethylene glycol 및 ammonium sulfate를 처리한 후 Sepharose 2B gelfiltration chromatography를 시행하여 1,552배 정제 분리하였으며 이 효소의 specific activity는 ㎎당 3.88units이었다.

이 효소를 사용하여 토끼에서 얻은 항혈청과 간장 세포 균등액을 20,Oeoxg로 원침하여 얻은 상층액, 3% polyethylene glycol 추출액과 작용시켜 Ouchterlony double immunodiffusion 실험을 한 결과 항혈청이 15㎕는 간장세포 균등액과 반응하여 서로 연결된 한개의

침전대가 형성된 것으로 보아 동일한 항혈청이 생성된 것을 확인하였다. 이 항혈청을 가지고 proteins Sepharose chromatography를 시행하여 얻은 IgG를 간장세포 균등액을 5% p

olyethylene glycol로 추출한 용액에 IgG농도를 증가시켜 첨가하면 acetyl CoA carboxylase활성은 IgG농도에 비례하여 억제된 사실로 미루어 가토 혈청에서 분리한 IgG에는 acetyl CoA carboxylase에 대한 항체가 생성되었음을 확인하였다. 이 항체를 가지고 3일간 굶

긴 백서에 insulin과 고함수탄소 식이를 처리한 후 백서간장 세포질을 분리하여 세포질내 acetyl CoA carboxylase 함량을 면역화학 방법으로 정량한 결과 새로 합성된 이 효소에 ·삽입된 (3**H)-leucine의 활성치는 insulin(1.5 units/100g B.W.) 처리군은 대조군에

비하여 3배, 고함수탄소 식이 처리군은 10배가 증가되고, 세포질내 총 단백질 합성량에 비하여 이 효소 합성 비율은 insulin처리군에서 4배, 고함수탄소 식이 처리군에서 10배 증가하였다.

또한 이들 백서간장 세포질내 acetyl CoA carboxylase를 합성하는 특이 Polysome내 RNA는insulin 처리군은 279%, 고함수탄소 식이 처리군은 365%가 증가하였으며, polysome내 acetyl CoAcarboxylase nascent chain은 insulin 처리군은 153%, 고함수탄소 식이 처리군

은 311%가 증가하였다.

이상과 같은 실험결과로 미루어 보아 insulin과 고함수탄소 식이에 의하여 간장세포내 acetyl CoAcarboxylase 함량 증가는 이 효소를 합성하는 polysome내 mRNA 함량증가로 인한 결과로 사료된다.

Regulation of Acetyl CoA Carboxylase mRNA in Rat Liver by High carbohydrate Diet

and Insulin

Dong Hee Choi

Department of Medical Science The Graduate School Yonsei University

(Directed by Professor Yoon Soo Kim, M.D.,Ph.d.)

It has been reported that insulin receptors in the plasma membrane of liver cell

rapidly increase in fasting rats, and that the intracellular insulin concentration

could be increased by the rapid internalization of insulin-receptor complex formed

from the increase of blood insulin level as rats were refed with high carbohydrate

diet (Whang et al., 1982; Yoon et al.,1983).

The increased intracellular insulin binds to the insulin receptor in nuclear

membrane and stimulates the efflux of mRNA from nucleus, which may be a cause of

the increased biosynthesis of lipogenic enzymes.

It was an example to demonstrate that the increased biosynthesis of

glucose-6-phosphate dehydrogenase(G-6-PDH) resulted from an increased amount of

mRNA for G-6-PDH (Kim et al., 1985).

It has been also demonstrated that insulin and glucose stimulated biosynthesis of

fatty acid synthetase (FAS) by the increase of cytoplasmic mRNA for FAS (Suh et

al., 1986).

The fact that insulin stimulates the synthesis and nuclear efflux of mRNA for

G-6-PDH and PAS, both lipogenic enzymes, has been proved by Lee et al (1987).

In this study, the change of acetyl CoA carboxylase content in liver cytosol of

rats refed with high carbohydrate diet or treated with insulin after fasting, was

measured by immunoassay method in order to clarify the control mechanism for the

amount of acetyl CoA carboxylase.

The results are as follows. Acetyl CoA carboxylase was purified 1,552 folds with

a specific activity of 3.88 units/㎎ from rat liver refed with a high carbohydrate

diet for 3 days after 3 day fasting, by the precipitation with polyethylene glycol

and ammoium sulfate, and followed by Sepharose 2B gel filtration.

15㎕ of rabbit antiserum obtained by immunization with purified acetyl CeA

carboxylase was precipitated with the 27,000xg supernatant of rat liver homogenate

in Ouchterlony double immunodiffusion and showed a single precipitin, indicating

the antiserum for anti-acetyl CoA carboxylase was produced. Treatment of insulin

and feeding a high carbohydrate diet of rats resulted in the increase of acetyl CoA

carboxylase in liver cytosol by 3 times in the insulin treated group and 10 times

in the high carbohydrate diet group as (3**H) -leucine incorporation into the

enzyme was measured by immunochemical method.

The synthetic ratios of this enzyme to total cytosolic proteins were 4 times

higher in insulin treated group and 10 times higher in high carbohydrate diet

group, compared to the control group.

The polysomal RNA contents for acetyl CeA carboxylase in rat liver cytosol were

279% of the control in insulin treated group and 365% of the control in high

carbohydrate diet group, and acetyl CoA carboxylase nascent chains in polysome were

158% of the control in insulin treated group and 311% of the control in high

carbohydrate diet group.

From these results, it is assumed that the increase of acetyl CoA carboxylase

content in the rat liver cell by insulin and high carbohydrate diet resulted from

the increased polysmal mRNA for the synthesis of this enzyme, which is directly

related to the biosynthesis of this enzyme.

[영문]

It has been reported that insulin receptors in the plasma membrane of liver cell rapidly increase in fasting rats, and that the intracellular insulin concentration could be increased by the rapid internalization of insulin-receptor complex formed from the increase of blood insulin level as rats were refed with high carbohydrate diet (Whang et al., 1982; Yoon et al.,1983).

The increased intracellular insulin binds to the insulin receptor in nuclear membrane and stimulates the efflux of mRNA from nucleus, which may be a cause of the increased biosynthesis of lipogenic enzymes.

It was an example to demonstrate that the increased biosynthesis of glucose-6-phosphate dehydrogenase(G-6-PDH) resulted from an increased amount of mRNA for G-6-PDH (Kim et al., 1985).

It has been also demonstrated that insulin and glucose stimulated biosynthesis of fatty acid synthetase (FAS) by the increase of cytoplasmic mRNA for FAS (Suh et al., 1986).

The fact that insulin stimulates the synthesis and nuclear efflux of mRNA for G-6-PDH and PAS, both lipogenic enzymes, has been proved by Lee et al (1987).

In this study, the change of acetyl CoA carboxylase content in liver cytosol of rats refed with high carbohydrate diet or treated with insulin after fasting, was measured by immunoassay method in order to clarify the control mechanism for the amount of acetyl CoA carboxylase.

The results are as follows. Acetyl CoA carboxylase was purified 1,552 folds with a specific activity of 3.88 units/㎎ from rat liver refed with a high carbohydrate diet for 3 days after 3 day fasting, by the precipitation with polyethylene glycol and ammoium sulfate, and followed by Sepharose 2B gel filtration.

15㎕ of rabbit antiserum obtained by immunization with purified acetyl CeA carboxylase was precipitated with the 27,000xg supernatant of rat liver homogenate in Ouchterlony double immunodiffusion and showed a single precipitin, indicating

the antiserum for anti-acetyl CoA carboxylase was produced. Treatment of insulin and feeding a high carbohydrate diet of rats resulted in the increase of acetyl CoA carboxylase in liver cytosol by 3 times in the insulin treated group and 10 times

in the high carbohydrate diet group as (3**H) -leucine incorporation into the enzyme was measured by immunochemical method.

The synthetic ratios of this enzyme to total cytosolic proteins were 4 times higher in insulin treated group and 10 times higher in high carbohydrate diet group, compared to the control group.

The polysomal RNA contents for acetyl CeA carboxylase in rat liver cytosol were 279% of the control in insulin treated group and 365% of the control in high carbohydrate diet group, and acetyl CoA carboxylase nascent chains in polysome were

158% of the control in insulin treated group and 311% of the control in high carbohydrate diet group.

From these results, it is assumed that the increase of acetyl CoA carboxylase content in the rat liver cell by insulin and high carbohydrate diet resulted from the increased polysmal mRNA for the synthesis of this enzyme, which is directly related to the biosynthesis of this enzyme.