[논문]3T3-L1 지방세포에서 PI3K/AKT 및 AMPK 경로의 활성화를 통한 루페올의 포도당 흡수촉진 효과

이현아; 한지숙

doi:10.5352/jls.2022.32.2.86

3T3-L1 지방세포에서 PI3K/AKT 및 AMPK 경로의 활성화를 통한 루페올의 포도당 흡수촉진 효과
Facilitation of Glucose Uptake by Lupeol through the Activation of the PI3K/AKT and AMPK Dependent Pathways in 3T3-L1 Adipocytes 원문보기

생명과학회지 = Journal of life science, v.32 no.2, 2022년, pp.86 - 93

초록
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Lupeol은 pentacyclic triterpene의 일종으로 다양한 질병에 약리 효과가 있는 것으로 보고되어 있으나, lupeol이 포도당 흡수에 미치는 영향은 아직 보고된 바 없다. 본 연구에서 3T3-L1 지방세포에서 포도당 흡수에 대한 lupeol의 효과를 조사하였다. 그 결과, Lupeol은 3T3-L1 지방세포에서 GLUT4를 원형질막으로 이동시켜 포도당 흡수를 촉진하였으며, 이는 PI3K/AKT 및 AMPK 경로의 활성화와 관련되어 있었다. PI3K/AKT 경로에서 lupeol은 PI3K를 활성화시키는 insulin receptor substrate 1의 인산화와 AKT의 인산화를 촉진하지만 비정형 단백질 키나아제 C isoforms ζ 및 λ의 인산화는 촉진하지 않았다. 반면, lupeol은 5 'AMP-activated protein kinase의 인산화를 촉진하였고, Lupeol의 의한 AMPK의 활성화는 원형질막-GLUT4의 발현과 세포내 포도당 흡수를 증가시키는 것으로 확인되었다. 3T3-L1 지방세포에서 lupeol에 의한 포도당 흡수 효과는 PI3K 억제제인 wortmannin 및 AMPK 억제제인 Compound C에 의해 억제됨을 통해 확인하였다. 본 연구 결과는 lupeol이 3T3-L1 지방세포에서 PI3K/AKT 및 AMPK 경로를 통해 원형질막 GLUT4의 발현을 자극함으로써 인슐린 감수성을 증가시켜 포도당 흡수를 촉진할 수 있음을 제시하고 있다.

Abstract ▼ AI-Helper

Lupeol is a type of pentacyclic triterpene and has been reported to have pharmacological activities against various diseases; however, the effect of lupeol on glucose absorption has not been elucidated yet. This study aimed to investigate the effect of lupeol on glucose uptake in 3T3-L1 adipocytes. Lupeol significantly facilitated glucose uptake by translocating glucose transporter type 4 (GLUT4) to the plasma membrane of the 3T3-L1 adipocytes, which was related to activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and 5 'adenosine monophosphate-activated protein kinase (AMPK) pathways. In the PI3K/AKT pathway, lupeol stimulates the phosphorylation of insulin receptor substrate 1 (IRS-1), which activates PI3K. Its activation by lupeol promotes the phosphorylation of AKT, but not the atypical protein kinase C isoforms ζ and λ. Lupeol also promoted the phosphorylation of AMPK. The activation of AMPK increased the expressions of the plasma membrane GLUT4 and the intracellular glucose uptake. The increase in the glucose uptake by lupeol was suppressed by wortmannin (PI3K inhibitor) and compound C (AMPK inhibitor) in the 3T3-L1 adipocytes. The results indicate that lupeol can facilitate glucose uptake by increasing insulin sensitivity through the stimulation of the expression of plasma membrane glucose transporter type 4 via the PI3K/AKT and AMPK pathways in the 3T3-L1 adipocytes.

주제어

표/그림 (6)

그림 Fig. 1. Effect of lupeol on glucose uptake in 3T3-L1 adipocytes. Lupeol of 5-20 μM were treated in 3T3-L1 adipocytes for 24 hr prior to the 2-deoxyglucose uptake assay. Each value is shown as the mean ± standard deviation (n=3). ^a-eValues composed of different alphabets indicate significant differences (p<0.05) by Duncan’s multiple range test. 2-DG, 2-deoxyglucose.
그림 Fig. 2. Effects of lupeol on the expression of IRS-1 and PI3K in 3T3-L1 adipocytes. Lupeol of 10, 20 μM or insulin of 100 nM were treated in 3T3-L1 adipocytes for 24 hr, after that, lysed cells were performed to Western blotting. (A) Phosphorylation value of insulin receptor substrate-1, (B) Expression value of phosphoinositide 3-kinase. Each value is shown as the mean ± standard deviation (n=3). ^a-dValues composed of different alphabets indicate significant differences (p<0.05) by Duncan's multiple range test.
그림 Fig. 3. Effects of lupeol on the expression of AKT and PKC ζ/λ in 3T3-L1 adipocytes. Lupeol of 10, 20 μM or insulin of 100 nM were treated in 3T3-L1 adipocytes for 24 hr, after that, lysed cells were performed to Western blot analysis. (A) Phosphorylation value of AKT, (B) Phosphorylation value of protein kinase C ζ/ λ. Each value is shown as the mean ± standard deviation (n=3). ^a-cValues composed of different alphabets indicate significant differences (p<0.05) by Duncan's multiple range test.
그림 Fig. 4. Effects of lupeol on the expression of AMPK and ACC in 3T3-L1 adipocytes. Lupeol of 10, 20 μM or AICAR of 0.5 mM were treated in 3T3-L1 adipocytes for 24 hr, after that, lysed cells were performed to Western blot analysis. (A) Phosphorylation value of 5 'AMP-activated protein kinase, (B) Phosphorylation value of acetyl-CoA carboxylase. Each value is shown as the mean ± standard deviation (n=3). ^a-dValues composed of different alphabets indicate significant differences (p<0.05) by Duncan's multiple range test.
그림 Fig. 5. Effect of lupeol on PM-GLUT4 expression in 3T3-L1 adipocytes. 20 μM lupeol and its combination with 20 μM WM or 10 μM CC and 100 nM insulin were treated in 3T3-L1 adipocytes for 24 hr, after that, lysed cells were performed to Western blot analysis. Each value is shown as the mean ± standard deviation (n=3). ^a-cValues composed of different alphabets indicate significant differences (p<0.05) by Duncan's multiple range test. WM, wortmannin; CC, compound C.
그림 Fig. 6. Effect of lupeol combined with wortmannin of compound C on glucose uptake in 3T3-L1 adipocytes. 20 μM lupeol, its combination with 20 μM WM or 10 μM CC were treated in 3T3-L1 adipocytes, after that, 2-deoxyglucose uptake assay was conducted. Each value is shown as the mean ± standard deviation (n=3). ^a-dValues composed of different alphabets indicate significant differences (p<0.05) by Duncan's multiple range test. WM, wortmannin; CC, compound C.

AI 본문요약
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제안 방법

However, the effects of lupeol on glucose uptake and mechanisms have not been elucidated yet. Therefore, this study was conducted to investigate the effect of lupeol on glucose uptake and clarify the cellular mechanisms about this effect in 3T3-L1 adipocytes. The pathogenesis of type 2 diabetes reduced glucose uptake into cells due to impaired insulin action [6].

대상 데이터

Louis, MO, USA). 3T3-L1 preadipocytes were purchased from Korean Cell Line Bank (KCLB, Seoul, Korea). Dulbecco modified Eagle medium (DMEM), fetal bovine serum (FBS), wortmannin, and compound C were purchased from Sigma (St.

데이터처리

The differences were evaluated by the one-way ANOVA test, followed by the post hoc Duncan’s multiple range tests.

성능/효과

In conclusion, lupeol improves glucose uptake by stimulating the PI3K/AKT and AMPK pathways in 3T3-L1 adi- pocytes. Activation of PI3K and AMPK by lupeol was confirmed by PI3K inhibitor or AMPK inhibitor, and these inhibitors inhibited the expression of PM-GLUT4 by lupeol. It indicated that lupeol enhanced glucose uptake by up-regu- lating insulin sensitivity in 3T3-L1 adipocytes.
In conclusion, lupeol improves glucose uptake by stimulating the PI3K/AKT and AMPK pathways in 3T3-L1 adi- pocytes. Activation of PI3K and AMPK by lupeol was confirmed by PI3K inhibitor or AMPK inhibitor, and these inhibitors inhibited the expression of PM-GLUT4 by lupeol.
Moreover, lupeol increased phosphorylation of AKT through PI3K activation in 3T3-L1 adipocytes, but it was not effective in phosphorylation of PKC ζ /λ
Phosphorylation of AKT translocated GLUT4 to plasma membrane and promoted intracellular glucose uptake. The data suggest that lupeol might improve glucose uptake through the PI3K/AKT pathway in adipocytes.
We found that treatment of wortmannin and compound C, which are PI3K and AMPK inhibitor, respectively, inhibited the expression of PM-GLUT4 and decreased glucose uptake by lupeol in 3T3-L1 adipocytes. These data showed lupeol may increase the activation of the PI3K/AKT and AMPK dependent pathways and stimulate GLUT4 translocation to plasma membrane, thereby enhancing glucose uptake.
Akt or PKC ζ /λ activation causes translocation of GLUT4 to the plasma membrane and finally intracellular glucose uptake is enhanced [19, 22]. This study confirmed that lupeol significantly increased phosphorylation of IRS-1 and activation of PI3K in 3T3-L1 adipocytes. Moreover, lupeol increased phosphorylation of AKT through PI3K activation in 3T3-L1 adipocytes, but it was not effective in phosphorylation of PKC ζ /λ.

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