당뇨병이 유발된 백서 간장 세포에서 인슐린에 의한 ATP-citrate lyase mRNA 함량 조절

Abstract

[영문]

[한글]

본 연구는 인슐린이 백서 간장 세포에서 ATP-citrate lyase를 합성하는 mRNA 함량 조절

기전을 밝히기 위하여 백서를 streptozotocin (6mg/100g BW)을 정맥내 주입하여 당뇨병이

유발된 백서에 인슐린 투여 후 시간별로 백서 간장을 적출하여 세포질내 총 RNA 및 poly

(A)-RNA, 핵 및 세포질을 분리하여 총 RNA 및 poly (A)RNA를 Northern blot를 시행한 다

음 ATP-citrate lyase에 대한 (32)**P-cDNA를 가지고 hybridization을 시행하였다.

ATP-citrate lyase mRNA 함량은 인슐린 투여 후 6시간에 약간 증가하여 16시간에 최고

치에 달하고 72시간에는 급격히 감소하였다.

각군의 간장 세포 핵으로부터 전사 활성 측정은 pGACL1 (cDNA, 1.6 kbp) 및 pGACL2(cDN

A, 2.3 kbp)를 probe로 이용하여 정량하였다.

인슐린 투여 후 전사 활성은 3시간 까지는 변화가 없었으며 pGACL1은 6시간에 2배, 16

시간에 5배 증가하고, pGACL2는 6시간에 5배, 16시간에 22배 증가하였으며 이런 활성 증

가 차이는 probe의 크기와 위치가 중요하다는 사실을 시사한다.

또한 각군의 백서 간장으로부터 세포질을 분리하여 Western blot를 시행한 다음 ATP-ci

trate lyase에 대한 항체를 사용하여 ATP-citrate lyase 효소 함량을 정량하였든 바 인슐

런 투여 후 6시간 까지는 변화가 없었으며 16시간에 급격히 증가하여 72시간에 최고치에

달하였다.

이상과 같은 실험 결과로 미루어 인슐린을 투여한 후 ATP-citrate lyase 효소 함량 증

가는 유전자 선상에서 일차적으로 전사 활성의 증가에 의한 세포질내 ATP-citrate lyase

를 합성하는mRNA 함량 증가가 주 조절 기전으로 사료된다.

Reflulation of liver ATP-citrate lyase mRNA expression by insulin in the diabetic

rat liver

Jin Soo Kim

Department of Medical Science The Graduate School, Yonsei University

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

A study on the regulation of liver ATP-citrate lyase mRNA expression by insulin

in the diabetic rat liver was attempted. Experimental diabetes in rats was induced

by the injection of streptozotocin (6mg/100g BW) I.V. and livers were removed from

rats at 0, 1, 3, 6, 16 and 72hours after subcutaneous administration of insulin

(2.0 unit/100g B.W). Total RNA, poly (A)-RNA, nuclei and cytosol were isolated, and

Northern blot, hybridizations of total RNA and poly(A)-RNA were carried out using

(32)**P-cDNA for ATP-citrate lyase as a probe. The level of ATP-citrate lyase mRNA

increased slightly at 6 hours and reached the highest level at 16 hours; then the

level decreased rapidly at 72 hours after insulin administration. The pGACL1 (cDNA,

1.6 kbp)and the pGACL2 (cDNA, 2.3 kbp) were used as probes for the detection of

transcriptional activities in rat liver nuclei of each group. No change in the

transcriptional activity was observed until 3 hours after the administration of

insulin, however, the activities increased two fold at 6 hours, five fold at 16

hours, five fold at 6 hours and 22 fold at 16 hours after the administration of

insulin when pGACL1 and pGACL2 were used as a probe; this indicates the

transcriptional activity level is dependent on the size and part of cDNA.

The amounts of ATP-citrate lyase in the liver cytosol of rats in each group were

determined by Western blot, immunostaining using an anti-ATP-citrate lyase

antibody. There was no detectable change in the amount of ATP-citrate lyase until 6

hours after the administration; then, a rapid increase occurred at 16 hours after

the administration, followed by the highest value at 72 the amounts of ATP-citrate

lyase by insulin administration might be primarily due to the increased

transcriptional activity of the gene, followed by the increased amount of mRNA for

the synthesis of ATP-citrate lyase.