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in vitroおよびin silico分析によるアディポネクチン受容体アゴニストジ・トリペプチドに関する研究

イ, ユナ LEE, YUNA 九州大学

2022.09.22

概要

The incidence of insulin-independent Type Ⅱ diabetes (T2DM), characterized by insulin resistance and hyperglycemia, and its related complications continues to increase worldwide. Although most blood glucose is uptaken in skeletal muscle in the postprandial period in an insulin-dependent manner, considerable evidence indicates that alterative glucose uptake pathway such as adiponectin-dependent manner is also involved in the blood glucose regulation; the adiponectin-related glucose uptake via the activation of adenosine monophosphate-activated protein kinase (AMPK) is in a manner distinct from that of insulin signaling. Adiponectin is an adipocyte-derived hormone that acts via two adiponectin receptor R1/2 (AdipoR1/R2). AdipoR1 is ubiquitously expressed and is mainly abundant in skeletal muscle, whereas AdipoR2 expression is mainly restricted to the liver. The adiponectin intracellular signaling for glucose uptake by skeletal muscle primarily includes the AdipoR1-mediated AMPK/glucose transporter 4 (GLUT4) translocation pathway. Thus, targeted stimulation of the adiponectin signaling pathway or the development of skeletal muscle AdipoR1 agonists may be an effective strategy for preventing and managing insulin-independent T2DM. However, adiponectin-like drugs are not currently available as direct therapeutics, except for AdipoRon (2-(4-benzoylphenoxy)-N-[1-(phenylmethyl)-4-piperidinyl]acetamide), because of the difficulties in converting full-size adiponectin (244 amino acids) into a viable agent. Therefore, the goal of the present study was to investigate AdipoR1-agonsitic di- and tripeptides rather than longer oligopeptides using in vitro L6 cells and in silico MD simulation analyses because of their favorable absorption.

This study primarily aimed to clarify possible AdipoR1 agonistic dipeptides using skeletal muscle cells. Fifteen dipeptides with the structural similarity to AdipoRon were targeted for glucose uptake experiments in rat skeletal muscle L6 cells. Among the dipeptides, YP showed a significant promotion of glucose uptake, while other dipeptides, including PY, failed to exert the effect. YP significantly induced GLUT4 translocation to the plasma membrane, along with AMPK activation. By knocking down AdipoR1 expression in L6 cells, the increased glucose uptake by YP was abolished, indicating that YP may play a promoting role in the glucose uptake through the AdipoR1-mediated AMPK/GLUT4 translocation pathway in L6 cells. The preferable molecular dynamics simulation of YP toward the AdipoR1 was also confirmed by the CHARMM-GUI-aided in silico analysis, in which YP was stably positioned in the two potential binding sites 1/2 of AdipoR1 embedded in a virtual phospholipid membrane. Consequently, these findings indicate the anti-diabetic function of the dipeptide YP as a possible AdipoR1 agonist.

It was demonstrated that dipeptide YP can serve as an AdipoR1 agonist. According to the dipeptide YP study, further experiments were performed to investigate the AdipoR1-agonistic potential of YP-related natural tripeptides by in vitro L6 cells and in silico MD simulation analyses. Seventeen YP-related tripeptides in the sequence of soybean proteins were synthesized and subjected to glucose uptake experiment in rat skeletal muscle L6 cells. Among the tripeptides, those elongated at the C-terminal of YP (YPG, YPE, YPP, and YPQ) significantly promoted glucose uptake in L6 cells, comparable to the effect of AdipoRon. Knockdown of AdipoR1 expression in L6 cells abrogated the effect for YPG and YPP, indicating that the two tripeptides had an AdipoR1 agonistic action. Moreover, the CHARMM-GUI-aided MD simulation in a virtual phospholipid membrane revealed that YPG and YPP were stably positioned in the two potential binding sites 1/2 of AdipoR1 (The Gibbs free energy of binding, ΔGbind < -10 kcal/mol). By the aid of mass spectrometric analysis, YPP was confirmed to be present in soybean hydrolysate at a concentration of 29.8 µg/g.

Taken together all, the present study demonstrates for the first time that di-/tripeptides may be alternative-medicinal food compounds against insulin-independent T2DM, with their mechanism of action involving adiponectin-like glucose uptake in skeletal muscle.

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参考文献

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