SAMS-GFP 펩티드에 의한 AMP-activated protein kinase 기능 차단이 골격근 내 당 수송에 미치는 영향
Inhibition of AMP-activated protein kinase by SAMS-GFP peptide on glucose transport in skeletal muscle.
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초록
운동시 골격근 내 당 수송은 주로 AMP-activated protein kinase (AMPK)와 Ca2+/ calmodulin-dependent protein kinase (CaMK) 신호전달체계를 통해 이루어진다. AMPK의 세 가지 subunit 중 α 는 α1과 α2 isoform이 존재하는데, α2가 골격근 내 주요 isoform으로 알려져 있다. 그렇지만 AMPKα2를 knockout (KO) 하더라도 AMPKα1의 효과까지는 완벽하게 차단되지 않을 뿐 아니라 AMPKα1의 활성이 오히려 반등하게 된다. 따라서 최근 AMPKα2 KO 생쥐를 이용하여 AMPK가 운동시 골격근 내 당 수송에 중요한 역할을 하지 않는다는 연구결과는 골격근 내 당 수송에 대한 AMPK 역할을 확실히 규명하는데 한계가 있다. 이에 본 연구는 가성 펩티드 (pseudo peptide)인 SAMS-GFP를 이용하여 AMPKα1과 α2의 기능을 완전히 차단한 후 AMPK가 골격근 내 당 수송에 어떠한 역할을 하는지 알아보았다. 이를 위해 electric pulse-mediated gene transfer 기술을 이용하여 SAMS-GFP peptide를 epitrochlearis 근육에 과 발현 시켰다. 그 결과 SAMS-GFP 저해 펩티드 (inhibitory peptide)는 AICAR에 의해 증가된 골격근 내 당 수송을 완벽히 차단하였다. 그리고 저산소증과 근 수축에 의해 각각 6.5배 그리고 4.3배 증가된 당 수송이 SAMS-GFP 유전자에 의해 각각 50% 가까이 감소되었다. 또한 SAMS-GFP 저해 펩티드는 GLUT4 생합성에 아무런 영향을 미치지 않았다. 이러한 결과를 종합해 볼 때 AMPK가 근 수축에 따른 골격근 내 당 수송에 CaMK 신호전달체계와 함께 여전히 핵심적인 역할을 하며, SAMS-GFP 저해 펩티드가 AMPKα2 KO 생쥐와는 달리 AMPKα1과 AMPKα2 기능을 모두 완벽하게 차단하였다는 것을 알 수 있다.
Abstract
Glucose transport in skeletal muscle during exercise is mainly accomplished by AMP-activated protein kinase (AMPK) and Ca2+/calmodulin-dependent protein kinase (CaMK) signaling pathways. AMPK exists as heterotrimeric αβγ complexes, while the catalytic subunit has two isoforms (α1 and α2) with different tissue distributions. AMPK complexes containing the α2, rather than the α1 isoform, have a greater dependency on AMP both in direct allosteric activation and in covalent activation by the upstream kinase, LKB1. Therefore, AMPKα2 is thought to be the main isoform in skeletal muscle. Although in AMPKα2 KO mice AMPKα2 activity is abolished, AMPKα1 activity is compensatorily increased. To overcome these obstacles, we designed a SAMS-GFP pseudo peptide vector to block the function of both AMPKα1 and AMPKα2 in skeletal muscle. SAMS-GFP peptide overexpression in rat epitrochlearis muscle was accomplished by using an electric pulse-mediated gene transfer technique. Induction of AMPK pseudo peptide, SAMS-GFP completely blocked AICAR stimulated glucose transport activity. Hypoxia and muscle contraction induce an increase of glucose transport by 6.5 and 4.4 times, respectively. But, this hypoxia- and contraction- induced increase of glucose transport is decreased about 50% by SAMS-GFP. SAMS-GFP peptide overexpression did not change muscle GLUT4 content. These findings suggestthat AMPK with CaMK signaling pathways play a key role on glucose transport by contraction in skeletal muscle. And SAMS-GFP inhibitory peptide completely blocked the function of AMPKα1 and AMPKα2, contrary to AMPKα2 KO mice, in which only α2 is blocked.
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