脂肪滴形成へのオートファジーの関与について

• Project/Area Number: 21659050
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: General anatomy (including Histology/Embryology)
• Research Institution: Juntendo University
• Principal Investigator: 内山 安男 順天堂大学, 医学部, 教授 (10049091)
• Co-Investigator(Kenkyū-buntansha): 小池 正人 順天堂大学, 医学部, 准教授 (80347210) ; 佐々木 光穂 順天堂大学, 医学部, 助教 (20432536)
• Project Period (FY): 2009 – 2010
• Project Status: Completed (Fiscal Year 2010)
• Budget Amount: ¥3,100,000 (Direct Cost: ¥3,100,000); Fiscal Year 2010: ¥1,200,000 (Direct Cost: ¥1,200,000); Fiscal Year 2009: ¥1,900,000 (Direct Cost: ¥1,900,000)
• Keywords: 脂肪滴 / LC3B / LC3A / オートファジー / 欠損マウス / LC3 / 小胞体膜 / エネルギー代謝 / 培養細胞 / 3T3-L1細胞

Research Abstract

前年度に、オートファジー関連タンパク質のLC3が脂肪滴表面に局在することを絶食負荷時に出来る肝細胞や心筋細胞に形成される脂肪滴で証明した。特に、Atg7を欠損するとLC3が絶食負荷時に脂肪滴形成が抑制されることを示した。本年度は、培養がん細胞(PC12, HeLa, HepG2, 3T3-L1細胞)を用いて、これら細胞を70%コンフルエントの条件で培養し、12時間後にBODIPY陽性の脂肪滴と中性脂肪の量を調べると有意に増加することを利用し、この脂肪滴にLC3が局在することを示した。次いで、LC3をRNAi法でノックダウンしてタンパク質レベルで10～20%まで発現を抑制した細胞株を樹立した。これら細胞を用いて、同様に培養して脂肪滴が形成されるかを検討した結果、LC3mRNAがノックダウンされた細胞では、脂肪滴のみならず中性脂肪の量もMock細胞と比べて、50%以下に抑制されることが分かった。さらに、脂肪細胞の前駆細胞である3T3-L1細胞をホルモン刺激して脂肪細胞に分化させた。その結果、正常細胞では、LC3mRNAは、ペルオキシソーム増殖因子活性化受容体Y等の分化因子と共に、処理後、時間と共に上昇することが分かった。また、LC3mRNAノックダウンL1細胞株では、脂肪細胞に分化した処理後6日目で検討した所、BODIPY陽性の脂肪滴の形成は全く起きないことが明らかとなった。現在、LC3欠損マウスを用いて、本結果を検証中である。

Research Products

• [Journal Article] Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse. (2010)
  • Author(s): Sun L, Wu Z, Baba M, Peters C, Uchiyama Y, Nakanishi H
  • Journal Title: Biochem Biophys Res Comm Volume: 399 Pages: 391-395
  • Peer Reviewed
• [Journal Article] Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. (2010)
  • Author(s): Lee J-H, Yu WH, Kumar A, Lee S, Mohan PS, Peterhoff CM, Wolfe DM, Martinez-Vicente M, Massey AC, Sovak G, Uchiyama Y, Westaway D, Cuervo AM, Nixon RA
  • Journal Title: Cell Volume: 141 Pages: 1146-1158
  • Peer Reviewed
• [Journal Article] Induction of autophagy by cystatin C: a mechanism that protects murine primary cortical neurons and neuronal cell lines. (2010)
  • Author(s): Tizon B, Sahoo S, Yu H, Gauthier S, Kumar AR, Mohan P, Figliola M, Pawlik M, Grubb A, Uchiyama Y, Bandyopadhyay U, Cuervo AM, Nixon RA, Levy E.
  • Journal Title: ProS One
  • Peer Reviewed
• [Journal Article] Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1^<H46R>-Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking. (2010)
  • Author(s): Hadano S, Otomo A, Kunita R, Suzuki-Utsunomiya A, Akatsuka A, Koike M, Aoki M, Uchiyama Y, Itoyama Y, Ikeda JE
  • Journal Title: PLoS One Volume: 5
  • Peer Reviewed
• [Journal Article] Cathepsin L in bone marrow-derived cells is required for retinal and choroidal neovascularization. (2010)
  • Author(s): Shimada N, Ohno-Matsui K, Iseki S, Koike M, Uchiyama Y, Wang J, Yoshida T, Sato T, Peters C, Mochizuki M, Morita I
  • Journal Title: Am J Pathol Volume: 176 Pages: 2571-2580
  • Peer Reviewed
• [Journal Article] Involvement of cathepsin B in the processing and secretion of interleukin-1 in chromogranin A-stimulated microglial. (2010)
  • Author(s): Terada K, Yamada J, Hayashi Y, Wu Z, Uchiyama Y, Peters C, Nakanishi H
  • Journal Title: Glia Volume: 10.1002 Pages: 20906-20906
  • Peer Reviewed
• [Journal Article] LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation. (2010)
  • Author(s): Shibata M, Yoshimura K, Tamura H, Ueno T, Nishimura T, Inoue T, Sasaki M, Koike M, Arai H, Kominami E, Uchiyama Y
  • Journal Title: Biophys Biochem Res Comm Volume: 393 Pages: 274-279
  • Peer Reviewed
• [Journal Article] Chondrogenesis during the ES cell differentiation in vitro. (2010)
  • Author(s): Yagita Y, Horie K, Yamanaka I, Koinuma S, Shigeyoshi Y, Kawakami K, Takeda J, Uchiyama Y
  • Journal Title: PNAS Volume: 107 Pages: 3846-3851
  • Peer Reviewed
• [Journal Article] Atg9A protein, an autophagy-related membrane protein, is localized in the neurons of mouse brains (2010)
  • Author(s): Tamura H, Shibata M, Koike M, Sasaki M, Uchiyama Y
  • Journal Title: J Histochem Cytochem Volume: 58 Pages: 443-453
  • Peer Reviewed
• [Journal Article] JE Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1^<H46R>-Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking (2010)
  • Author(s): Hadano S, Otomo A, Kunita R, Suzuki-Utsunomiya A, Akatsuka A, Koike M, Aoki M, Uchiyama Y, Itoyama Y, Ikeda
  • Journal Title: PLoSONE (in press)
  • Peer Reviewed
• [Journal Article] Cathepsin L in bone marrow-derived cells is required for retinal and choroidal neovascularization (2010)
  • Author(s): Shimada N, Ohno-Matsui K, Iseki S, Koike M, Uchiyama Y, Wang J, Yoshida T, Sato T, Peters C, Mochizuki M, Morita I
  • Journal Title: Am J Pathol (in press)
  • Peer Reviewed
• [Journal Article] LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation (2010)
  • Author(s): Shibata M, Yoshimura K, Tamura H, Ueno T, Nishimura T, Inoue T, Sasaki M, Koike M, Arai H, Kominami E, Uchiyama Y
  • Journal Title: Biophys Biochem Res Comm 393 Pages: 274-279
  • Peer Reviewed
• [Journal Article] Chondrogenesis during the ES cell differentiation in vitro (2010)
  • Author(s): Yagita Y, Horie K, Yamanaka I, Koinuma S, Shigeyoshi Y, Kawakami K, Takeda J, Uchiyama Y
  • Journal Title: PNAS 107 Pages: 3846-3851
  • Peer Reviewed
• [Journal Article] Nesfatin-1-regulated paraventricular oxytocinergic signaling causes anorexia through a leptin-independent melanocortin pathway (2009)
  • Author(s): Maejima Y, Sedbazar U, Suyama S, Kohno D, Onaka T, Takano E, Yoshida N, Koike M, Uchiyama Y, Fujiwara K, Yashiro T, Horvath TL, Dietrich MO, Tanaka S, Dezaki K, Oh-I S, Hashimoto K, Shimizu H, Nakata M, Mori M, Yada T
  • Journal Title: Cell Metabolism 10 Pages: 355-365
  • Peer Reviewed
• [Journal Article] Ablation of C/EBP beta alleviates ER stress and pancreatic beta cell failure through induction of GRP78 (2009)
  • Author(s): Matsuda, T., Kido, Y., Nakano-Hashimoto, N., Kaisho, T., Tanaka, T., Asahara, S., Shigeyama, Y., Takeda, A., Inoue, T., Shibutani, Y., Koyanagi, M., Hosooka, T., Matsumoto, M., Inoue, H., Uchida, T., Koike, M., Uchiyama, Y., Akira, S., Kasuga, M.
  • Journal Title: J Clin Invest 120 Pages: 115-126
  • Peer Reviewed
• [Journal Article] Mini Screening of Kinase Inhibitors Affecting Period-length of Mammalian Cellular Circadian Clock (2009)
  • Author(s): Yagita K, Yamanaka I, Koinuma S, Shigeyoshi Y, Uchiyama Y.
  • Journal Title: Acta Histochem Cytochem 42 Pages: 89-93
  • NAID: 130000118206
  • Peer Reviewed
• [Journal Article] Acquisition of T regulatory function in cathepsin L-inhibited T cells by eye-derived CTLA-2alpha during inflammatory conditions (2009)
  • Author(s): Sugita S, Horie S, Nakamura O, Maruyama K, Takase H, Usui Y, Takeuchi M, Ishidoh K, Koike M, Uchiyama Y, Peters C, Yamamoto Y, Mochizuki M.
  • Journal Title: J Immunol 183 Pages: 5013-5022
  • Peer Reviewed
• [Journal Article] Caspase-9 activation revealed by semaphorin 7A cleavage is independent of apoptosis in the aged olfactory bulb (2009)
  • Author(s): Ohsawa S, Hamada S, Asou H, Kuida K, Uchiyama Y, Yoshida H, Miura M.
  • Journal Title: J Neurosci 29 Pages: 11385-11292
  • Peer Reviewed
• [Journal Article] The MAP1-LC3 conjugation system is involved in lipid droplet formation (2009)
  • Author(s): Shibata M*, Yoshimura K*, Furuya M, Koike M, Ueno T, Komatsu M, Arai H, Tanaka K, Kominami E, Uchiyama Y(*equally contributed)
  • Journal Title: Biophys Biochem Res Comm 382 Pages: 419-423
  • Peer Reviewed
• [Journal Article] The novel lipid raft adaptor p18 controls endosome dynamics by anchoring the MEK-ERK pathway to late endosomes (2009)
  • Author(s): Nada S*, Hondo A*, Kasai A*, Koike M*, Saito K, Uchiyama Y, Okada M(*equally contributed)
  • Journal Title: EMBO J 28 Pages: 477-489
  • Peer Reviewed
• [Journal Article] Participation of autophagy in the initiation of graft dysfunction after rat liver transplantation (2009)
  • Author(s): Gotoh K, Lu Z, Morita M, Shibata M, Koike M, Waguri S, DonoK, DokiY, Kominami E, Sugioka A, Monden M, Uchiyama Y
  • Journal Title: Autophagy 5 Pages: 351-360
  • Peer Reviewed
• [Journal Article] Phosphorylation of highly conserved NF-M KSP repeats is not required for myelin-dependent radial axonal growth (2009)
  • Author(s): Garcia M, Rao M, Fujimoto J, Garcia V, Shah S, Crum J, Gotow T, Uchiyama Y, Ellisman MH, Calcutt N, Cleveland D
  • Journal Title: J Nurosci 29 Pages: 1277-1284
  • Peer Reviewed
• [Journal Article] Very-high-dose alpha-tocopherol supplementation increases blood pressure and causes possible adverse central nervous system effects in stroke-prone spontaneously hypertensive rats (2009)
  • Author(s): Miyamoto K, Shiozaki M, Shibata M, Koike M, Uchiyama Y, Gotow T
  • Journal Title: J Neurosci Res 87 Pages: 556-566
  • Peer Reviewed
• [Journal Article] (2009)
  • Author(s): Uchiyama Y, Koike M, Shibata M, Sasaki M
  • Journal Title: Chapter 3 Autophagic neuron death. In : Autophagy in Disease and Clinical Applications, Part C Vol.453(Edited by : Daniel J.Klionsky)(Elsevier Inc.) Pages: 33-51
• [Journal Article] (2009)
  • Author(s): Uchiyama Y, Koike M, Shibata M
  • Journal Title: Autophagy and cell death. In : Molecular imaging for integrated medical therapy and drug development(Ed.Yuji Kuge)(Springer Japan) Pages: 176-188
• [Presentation] Cell death and autophagy. (2010)
  • Author(s): Uchiyama Y
  • Organizer: 22nd International Symposium on Morphological Sciences
  • Place of Presentation: イタリア タオルミナ
  • Year and Date: 2010-09-19
• [Presentation] オートファジー/リソソームを追って (2010)
  • Author(s): 内山安男
  • Organizer: 平成22年度日本組織細胞化学会高松賞受賞講演(第51回日本組織化学会)
  • Place of Presentation: 秋葉原コンベンションホール
  • Year and Date: 2010-09-04
• [Presentation] Morphological features of programmed cell death across the eukaryotic kingdom (2009)
  • Author(s): 内山安男
  • Organizer: 6^<th> ISEM09 Committee
  • Place of Presentation: 神戸大学
  • Year and Date: 2009-09-17
• [Presentation] Cell Death and Autophagy (2009)
  • Author(s): 内山安男
  • Organizer: International Symposium on Autophagy and Cell Death
  • Place of Presentation: 順天堂大学医学研究科老人性疾患病態・治療研究センター
  • Year and Date: 2009-07-04
• [Presentation] Autophagy and other pathomechanisms (2009)
  • Author(s): 内山安男
  • Organizer: 12^<th> International Congress on Neuronal Ceroid Lipofuscinoses(NCL)
  • Place of Presentation: ドイツ ハンブルグ
  • Year and Date: 2009-06-05
• [Presentation] Morphological aspects of autophagy : Identification and possible mechanism of autophagosome formation in neuronal cells (2009)
  • Author(s): 内山安男
  • Organizer: Microscopy Conference 2009
  • Place of Presentation: オーストリア グラーツ
• [Presentation] Autophagy and cell death (2009)
  • Author(s): 内山安男
  • Organizer: 5^<th> International Symposium on Autophagy
  • Place of Presentation: 大津プリンスホテル コンベンションホール