title: Development of a bioassay to screen for chemicals mimicking the anti-aging effects of calorie restriction.
abstract: Suppression of the growth hormone/insulin-like growth factor-I pathway in Ames dwarf (DF) mice, and caloric restriction (CR) in normal mice extends lifespan and delays the onset of age-related disorders. In combination, these interventions have an additive effect on lifespan in Ames DF mice. Therefore, common signaling pathways regulated by DF and CR could have additive effects on longevity. In this study, we tried to identity the signaling mechanism and develop a system to assess pro-longevity status in cells and mice. We previously identified genes up-regulated in the liver of DF and CR mice by DNA microarray analysis. Motif analysis of the upstream sequences of those genes revealed four major consensus sequence motifs, which have been named dwarfism and calorie restriction-responsive elements (DFCR-REs). One of the synthesized sequences bound to hepatocyte nuclear factor-4α (HNF-4α), an important transcription factor involved in liver metabolism. Furthermore, using this sequence information, we developed a highly sensitive bioassay to identify chemicals mimicking the anti-aging effects of CR. When the reporter construct, containing an element upstream of a secreted alkaline phosphatase (SEAP) gene, was co-transfected with HNF-4α and its regulator peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α), SEAP activity was increased compared with untransfected controls. Moreover, transient transgenic mice established using this construct showed increased SEAP activity in CR mice compared with ad libitum-fed mice. These data suggest that because of its rapidity, ease of use, and specificity, our bioassay will be more useful than the systems currently employed to screen for CR mimetics, which mimic the beneficial effects of CR. Our system will be particularly useful for high-throughput screening of natural and synthetic candidate molecules.
authors.affiliation: Department of Investigative Pathology, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. takuya@nagasaki-u.ac.jp
authors.forename: Takuya
authors.initials: T
authors.lastname: Chiba
authors.forename: Tomoshi
authors.initials: T
authors.lastname: Tsuchiya
authors.forename: Toshimitsu
authors.initials: T
authors.lastname: Komatsu
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Mori
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hayashi
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Shimano
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Spindler
authors.forename: Isao
authors.initials: I
authors.lastname: Shimokawa
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D031204:Caloric Restriction; D004353:Drug Evaluation, Preclinical; D004392:Dwarfism / Q000235:genetics; D017930:Genes, Reporter; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000235:genetics; D008136:Longevity / Q000187:drug effects*; D051379:Mice; D008813:Mice, Inbred ICR; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic
chemical_list: D051557:Hepatocyte Nuclear Factor 4; C496593:Hnf4a protein, mouse; D007334:Insulin-Like Growth Factor I; D000469:Alkaline Phosphatase