Many active components derived from natural resources such as plant extracts have recently attracted the attention of scientific world for their potential use as drugs in treating metabolic diseases such as diabetes. In this study, I investigated the effect of two phytochemicals, genistein and nepodin, on type 2 diabetes and analyzed their modes of actions. Genistein, an isoflavone of soybean components, was studied with collaborators at Tokyo Noko University. Nepodin, acetyl-1, 8-dihydroxy-3-methyl naphthalene, is a component found from the root of Rumex japonicus, a common perennial herb that grows in East Asia. It has traditionally been used by the local people in Okinawa, Japan for treatment of acute and chronic cutaneous diseases, and by the Chinese as an effective drug for the treatment of constipation, jaundice, uterine hemorrhage, and hematemesis. I identified nepodin, a small molecular activator of glucose uptake in cultured L6 myotubes through high throughput screening of small compound library consisting of 2400 compounds.

As for the effect of nepodin on type 2 diabetes in vivo, I used male C57BL/KsJ-db/db mice (5 weeks of age), a genetically obese diabetic mouse strain, and C57BL/KsJ-m/m mice as nondiabetic lean group, to investigate the regulatory mechanism of nepodin on glucose homeostasis. During 5 weeks of oral nepodin administration (2 mg/kg/day and 10 mg/kg/day), I found that nepodin attenuates the increase of fasting blood glucose level dose-dependently without changes of food intake and weight in db/db mice. I conducted an intraperitoneal glucose tolerance test (IPGTT) to examine the effect of nepodin on the glucose tolerance. I found that nepodin improves the impaired glucose tolerance of C57BL/KsJ-db/db mice. Moreover, I found that nepodin decreases lipid levels such as triglyceride and cholesterol in serum and liver and inhibits plasma concentrations of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation and oxidative stress in db/db mice. In addition, I found that in quantitative RT-PCR assay, nepodin inhibits the expression of genes related to fatty acid synthesis, including fatty acid synthase (FAS) and stearoyl-Coenzyme A desaturase 1 (SCD1), while increasing the expression of glycogen synthase (GS) in the liver of db/db mice. To confirm direct effect of nepodin on gene expression associated with glucose homeostasis and lipid metabolism, I performed quantitative RT-PCR assay using C2C12, a mouse skeletal muscle cell line. I found that nepodin increases the expression of GLUT4 and genes related to fatty acids oxidation such as carnitine palmitoyltransferase I (CPT1α), Pyruvate dehydrogenase kinase (PDK1) and medium-chain acyl-CoA dehydrogenase (MCAD).

In vitro, I investigated the effect of genistein and nepodin on glucose uptake and the mechanism of glucose uptake enhancement by cultured L6 myotubes under both normal glucose (5.5 mM) and high glucose (25 mM) conditions, mimicking the normoglycemic condition and the hyperglycemic condition in diabetes, respectively. I found that both genistein and nepodin significantly stimulate glucose uptake in differentiated L6 myotubes under both glucose conditions. Through western blotting of GLUT4 in plasma membrane fraction by subcellular fractionation in L6 myotubes, and via the immunocytochemistry of GLUT4 in L6 myoblast transfected with pFC21A-Glut4-Halo, I found that both genistein and nepodin stimulated the translocation of GLUT4, a main glucose transporter responding to insulin signaling, to plasma membrane in differentiated L6 myotubes under both glucose conditions. As for nepodin, I found robust immunofluorescent signals for Glut4-Halo. Through further study using inhibitors of kinases related to glucose uptake in skeletal muscle cells, I found that the stimulatory effect of genistein on glucose uptake was be dependent on the PI3-kinase, mTOR, PKC and AMPK pathway, and that the stimulatory effect of nepodin on glucose uptake was dependent on the AMPK and Sirt1pathways under both glucose conditions. Moreover, I found that specifically both genistein and nepodin stimulate the phosphorylation of AMPK time-dependently under both glucose conditions. In skeletal muscle of db/db mice, nepodin rescued the impaired phosphorylation of AMPK.

In summary, these results suggest that both genistein and nepodin have antihyperglycemic potentials, including inhibition of the increase of fasting blood glucose and improvement of impaired glucose tolerance in db/db mice, and that this effect is mediated through the regulation of gene expression related to glucose homeostasis by a mechanism including both the activation of AMPK and GLUT4 translocation.

植物エキス由来の天然物は最近、糖尿病などの代謝性疾患薬の候補として注目を集めている。本研究では、植物由来成分の低分子化合物であるゲニステインと、低分子化合物ライブラリーを用いて糖取り込みを指標としたスクリーニングにより見出したネポジンを用いて、培養筋管細胞の糖取り込み対するに効果、2型糖尿病モデルマウスを用いた抗糖尿病効果、およびその調節メカニズムを検討した。

下記の結果を得ている。

1.通常のグルコース濃度培地と高グルコース濃度培地の2種類の培地を用いて分化させたL6筋管細胞を用い, ゲニステインとネポジンによる糖取り込みを検討した結果, ゲニステインとネポジンは濃度依存的に糖取り込みを促進した。

2.2型糖尿病マウスモデルであるKKAy/Ta JclとC57BL/KsJ-db/dbマウスを用いた実験で、ゲニステインとネポジンは、抗糖尿病作用を示した。特にネポジンの場合、ブドウ糖負荷試験においてC57BL/KsJ-db/dbマウスの耐糖能異常を改善させることが明らかとなった。

3.ゲニステインとネポジンの糖尿病モデルマウスにおける血中トリグリセリドおよびコレステロール値を検討した結果、ゲニステインとネポジンはこれらの値を抑制した。ネポジンは肝組織におけるグリコーゲン合成関連遺伝子の発現を増加させ、脂肪酸合成関連遺伝子の発現を抑制した。マウス筋肉細胞由来のC2C12細胞において、ネポジン曝露により、糖の細胞内吸収に関連するglucose transporter 4(GLUT4)の発現と糖と脂質メタボリズム制御に関連するSirt1、Sirt4の発現が増加し、さらに、CPT1α、PDK1、MCADなどの脂肪酸酸化に関連する遺伝子発現も増加した。

4. ゲニステインとネポジンは、筋管細胞において、GLUT4の細胞膜移動を促進した。糖取り込みに関与する複数のキナーゼの阻害剤を用いた解析の結果、ゲニステインによる糖取り込みは、PI-3K、PKC、AMPK、mTORシグナル伝達が関与し、ネポジンによる糖取り込みは、AMPK、Sirt1のシグナル伝達が関与することが明らかとなった。さらに、ゲニステインとネポジンはAMPKのリン酸化を時間依存的に増加させた。

以上、本論文は植物由来成分の低分子化合物であるゲニステインとネポジンの抗糖尿病活性を、2型糖尿病のマウスモデルを用したin vivo実験およびin vitro実験により明らかにした。本研究は、新たな抗糖尿病治療の候補物質として、糖尿病治療に重要な貢献を果たす可能性を秘めており、学位の授与に値するものと考えられる。