Effect of Adenovirus - mediated NeuroD overexpression by AICAR in MIN

A. Materials

III. RESULTS

8. Effect of Adenovirus - mediated NeuroD overexpression by AICAR in MIN

48h after expose to Ad-AMPKα.α1³¹²/T172D, MIN cells were treated with

400 µM AICAR for 16 h. Expression of c- myc tagged AMPKα.α1³¹²/T172D was verified with anti-c-myc antibody (upper). Activity of AMPK was determined by phosphorylation of Ser79 acetyl-CoA carboxylase (ACC) (middle).

AMPKα.α1³¹²/T172D repressed BETA2/NeuroD (lower).

8. Effect of Adenovirus -mediated NeuroD overexpression by AICAR in MIN cells.

To determine whether the protein level of exogenous NeuroD is decreased by AICAR, western blotting for NeuroD was performed after infection with Ad-flag-NeuroD (Fig. 23). Ad-flag-Ad-flag-NeuroD was used to construct the recombinant adenovirus as described previously.³⁶ The recombinant NeuroD protein contains a flag epitope tag at the C-terminus, allowing detection with an anti flag antibody. MIN cells was infected with Ad-flag-NeuroD for 32 h, and treated with 400 µM AICAR in a time-dependent manner. As shown in Fig. 24, GFP expression was visible 32 h after infection in 50-60 % of the MIN cells. Western analysis showed that the protein

level of BETA2/NeuroD in MIN cells was maintained for at least 6 h, but had decreased to 80 % at 12 h, and disappeared at 18 h after treatment with 400 µM AICAR (Fig 26). As an internal control, expression of GSK3β (Glycogen Synthase Kinase-3β) was not affected by AICAR treatment (Fig. 25). Therefore, AICAR decreased BETA2/NeuroD protein level in time-dependent manner. This result

Fig. 22. Effect of AICAR on the intracellular localization of Pdx-1.

Pdx-1 (Cytosol)

Pdx-1 (Nucleus)

GSK3β 1 2 3 4

48 h after exposure to Ad-LacZ or Ad-AMPKα/T172D and 16 h after

treatment with 400 µM AICAR, nuclear and cytosol extracts were obtained and western blotting was done with an antibody for Pdx-1. GSK3b was used as an internal control. Lane1; control, lane2; Ad- LacZ, lane3; Ad-AMPKα/T172D, lane4;

400 µM AICAR.

Fig. 23. Map of pAdTrack-CMV-FLAG-NeuroD

Fig. 24. Overexpression of NeuroD using MIN cells.

MIN cells were infected with recombinant adenovirus, Ad- flag-NeuroD (1 x 10⁸ PFU/ml). The panel shows representative MIN cells 32 h after infected with Ad-flag-NeuroD. Many cells are infected after exposure to Ad-flag-NeuroD which also contains GFP.

Cytosol

Nucleus

NeuroD

GSK3β

NeuroD

GSK3β AICAR (h) 0 1 3 6 18

Fig. 25. Repression of BETA2/NeuroD level by AICAR in MIN cells.

32 h after exposure to Ad-flag-NeuroD, MIN cells were treated with 400 µM AICAR, nuclear and cytosol extracts were obtained. Expression of flag-NeuroD was verified with anti- flag antibody. An internal control, expression of GSK3β was not affected by AICAR treatment. AICAR reduced the level of BETA2/NeuroD protein.

suggests that AMPK suppressed the expression of BETA2/NeuroD protein in post-transcription level.

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- 국문요약 -

B E T A 2 / N e u r o D 와 A M P - activated protein kinase ( A M P K ) 에 의한 S U R 1 유전자의 발현조절

본 연구에서는 췌장의 베타세포로 분화하는데 중요한 역할을 하는 BETA2/NeuroD 전사인자가 인슐린 분비에 관여하는 베타세포의 특이적인 유전자인 SUR (Sulfonylurea receptor)1을 어떻게 조절하는지 구체적인 메카니즘을 밝히고, 또한 진핵세포의 물질대사를 감지하는

AMP-activated protein kinase (AMPK)가 NeuroD와 SUR1 유전자의 발현에 어떠한 영향을 미치는지 조사 하였다.

SUR은 당뇨병의 치료제로 사용되는 sulfonylurea의 약리작용을

이해하는 과정에서 밝혀진 세포막 단백질로서 Kir 6.2와 함께 KATP

채널을 구성한다. SUR은 혈당량의 증가를 감지하여 인슐린이 분비되도록 유도하는데 핵심적으로 작용하며 베타세포에 특이적으로 발현된다고 알려졌다. SUR1의 특이적인 발현이 BETA2/NeuroD라는 basic helix-loop-helix (bHLH) transcription factor에 의해 조절되어짐을 명백히 밝힌바 있다. 인슐린 유전자는 베타세포의 특이적인 유전자로 알려져 있을 뿐만 아니라 베타세포의 여러 전사인자들의 post-translational modification 을 통해 extracelluar signaling molecule에 의해 조절되는 것으로 알려져 있다. 또한 인슐린의 합성과 분비는 세포 내의 포도당의 농도에 의해 조절되어지는데 포도당에 반응하는 신호전달물질로써 AMPK가 있다. AMPK는 세포 내의 ATP 고갈에 의한 AMP 농도에 중가에 의해 활성화되면서 ATP 생성 경로를 활성화시키고 ATP 소모 경로를 차단시킴으로 세포 내의 ATP 항상성을 유지시켜준다. 따라서 AMPK는 뇌, 간, 췌장 등 다양한 조직에서 metabolic sensor로써 역할을 한다. AMPK는 간세포에서 지질과 포도당 대사를 조절한다고 알려져

있다. 특히 고농도 포도당에서 lipogenesis에 관여하는 유전자들의 발현을 저해한다. 간에서의 지질과 포도당 대사가 유사한 췌장의 베타세포에서도 AMPK의 기능, 즉 특이적인 유전자의 발현을 조절할 수 있으리라 기대되어 진다.

따라서 본 연구에서는 BETA2/NeuroD가 SUR1 유전자의 발현을 조절하는 구체적인 메카니즘을 밝히고 AMPK가 베타 세포의 특이적인 유전자로 알려져 있는 NeuroD와 SUR1 유전자의 발현에 어떠한 영향을 밝히는데 그 목적이 있다.

본 실험에서 BETA2/NeuroD는 SUR1 프로모터에 존재하는 putative E-box 중에 세 번째 E-box에만 특이적으로 반응하였다. 또한 BETA2/NeuroD가 특이적으로 반응하는 E-box에 BETA2/NeuroD와 같은 bHLH transcription factor이면서 베타 세포의 발생과정에서 발현되어지는 ngn3 (neurogenin3)는 HeLa 세포에서 SUR 프로모터의 발현을 유도하지 않는 반면에 MIN 세포에서는 SUR1의 발현을 증가시켰다. 따라서 ngn3는 SUR1 프로모터에 직접적으로 결합하는 것이 아니라 BETA2/NeuroD에 결합하는 간접적인 방법으로 SUR1의 발현을 증가시킨다. 또한 단 사간동안 고농도의 포도당을 처리하였을 때 SUR1과 BETA2/NeuroD 유전자들의 mRNA와 단백질 발현이 증가했고 이렇게

증가된 발현이 AMPK의 활성제인 AICAR의 농도 의존적으로 저해되었다.

그러나 Pdx-1의 mRNA는 AICAR에 의해 조절되지 않았다. 이러한 결과로 베타세포에 특이적인 모든 유전자가 AMPK에 의해 조절되어지는 것이 아님을 확인할 수 있었다.

종합적으로 정리해보면 위와 같은 실험을 통해 인슐린 분비에 중요한 역할을 하는 SUR1 유전자는 베타세포의 특이적인 전사인자인 BETA2/NeuroD에 의해서만 발현한다는 사실을 보여준다. 또한 세포 내의 포도당 농도가 KATP 채널의 발현과 BETA2/NeuroD와 이를 매개로 발현되는 SUR1의 전사를 조절하는 것으로 추측되어지기 때문에 AMPK는 베타 세포의 특이적인 유전자들의 발현을 조절하는데 중요한

문서에서 Regulation if SUR1 gene by BETA2/NeuroD and AMP-activated protein kinase (페이지 68-95)