title: Increase in tumor suppressor Arf compensates gene dysregulation in in vitro aged adipocytes.
abstract: Deterioration of adipocyte function due to increased oxidative stress predisposes patients to metabolic disorders in advanced age. However, the roles of tumor suppressors in such conditions remain largely unknown. Therefore, we aimed to address their dynamics in aged adipocytes using a long-term culture model. We compared 3T3-L1 adipocytes at 17-19 days (long-term) with those at 8-10 days (short-term) after initiation of adipogenic induction for mimicking 'aged' and 'young' adipocytes, respectively. H2O2release and dihydroethidium (DHE) staining was increased, while superoxide dismutase (SOD) activity was reduced in long-term cultured adipocytes, which is suggestive of enhanced oxidative stress in this group. Moreover, qRT-PCR revealed increased mRNAs of Nox4 (a subunit of NADPH oxidase complex), Ccl2 (a proinflammatory chemokine) and Il6 [a marker of senescence-associated secretory phenotype (SASP)] along with decreased levels of Pparγ, Adipoq and Slc2a4 (genes related to glucose metabolism). These alterations were associated with increased expression of the tumor suppressors alternate-reading-frame protein p19Arf(Arf) and p16Ink4a. However, silencing of Arf reduced mRNAs of Adipoq and Slc2a4 and enhanced release of Il6. The effect was opposite in Arf overexpressing adipocytes, which showed reduced superoxide production as assessed with DHE staining and SOD activity. Western blots showed that Arf negatively regulates the phosphorylation of Akt. Luciferase assay in Hela cells additionally suggested that Arf negatively regulates Il6 transcriptional activity through a PI3 K/Akt mediated pathway. These findings strongly suggest that the enhanced Arf expression in oxidative stress plays compensatory protective roles against aging-related dysregulation of gene expression in adipocytes.
authors.affiliation: Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan. ahasan@med.kagawa-u.ac.jp.|Department of Pharmacology, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan. ahasan@med.kagawa-u.ac.jp.
authors.forename: Arif U
authors.initials: AU
authors.lastname: Hasan
authors.affiliation: Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.|Department of Cardiovascular Medicine, JCHO Ritsurin Hospital, 3-5-9, Ritsurin-cho, Takamatsu-shi, Kagawa, 760-0073, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Ohmori
authors.affiliation: Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hashimoto
authors.affiliation: Department of Cell Physiology, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Kazuyo
authors.initials: K
authors.lastname: Kamitori
authors.affiliation: Department of Cell Physiology, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Fuminori
authors.initials: F
authors.lastname: Yamaguchi
authors.affiliation: Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Kumi
authors.initials: K
authors.lastname: Konishi
authors.affiliation: Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Takahisa
authors.initials: T
authors.lastname: Noma
authors.affiliation: Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Junsuke
authors.initials: J
authors.lastname: Igarashi
authors.affiliation: Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Yamashita
authors.affiliation: Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Hirano
authors.affiliation: Department of Cell Physiology, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Tokuda
authors.affiliation: Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Minamino
authors.affiliation: Department of Pharmacology, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Nishiyama
authors.affiliation: Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.|Department of Cardiovascular Medicine, Hattori Memorial Hospital, 4244 Kanmaki, Kanmaki-cho, Kitatsuragi-gun, Nara, 639-0214, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Kohno
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D000375:Aging / Q000378:metabolism*; D000818:Animals; D016922:Cellular Senescence / Q000502:physiology*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology
chemical_list: C417743:Cdkn2a protein, mouse; D019941:Cyclin-Dependent Kinase Inhibitor p16; D017382:Reactive Oxygen Species
keywords: 3T3-L1 adipocytes; Cyclin-dependent kinase inhibitor; NADPH oxidase complex; Tumor suppressor; Type 2 diabetes mellitus