title: Immunomodulatory activity of enzymatically synthesized glycogen and its digested metabolite in a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages.
abstract: Previously, we developed enzymatically synthesized glycogen (ESG) from starch, and showed its immunomodulatory and dietary fiber-like activities. In this study, we investigated the metabolism of ESG and its immunomodulatory activity using differentiated Caco-2 cells as a model of the intestinal barrier. In a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages, mRNA expression of IL-6, IL-8, IL-1β and BAFF cytokines was up-regulated in Caco-2 cells and IL-8 production in basolateral medium was induced after 24 h apical treatment with 5 mg ml(-1) of ESG. The mRNA level of iNOS was also up-regulated in RAW264.7 macrophages. After characterization of the binding of anti-glycogen monoclonal antibodies (IV58B6 and ESG1A9) to ESG and its digested metabolite resistant glycogen (RG), an enzyme-linked immunosorbent assay (ELISA) system was developed to quantify ESG and RG. Using this system, we investigated the metabolism of ESG in differentiated Caco-2 cells. When ESG (7000 kDa, 5 mg ml(-1)) was added to the apical side of Caco-2 monolayers, ESG disappeared and RG (about 3000 kDa, 3.5 mg ml(-1)) appeared in the apical solution during a 24 h incubation. Neither ESG nor RG was detected in the basolateral solution. In addition, both ESG and RG were bound to TLR2 in Caco-2 cells. In conclusion, we suggest that ESG is metabolized to a RG-like structure in the intestine, and this metabolite activates the immune system via stimulation of the intestinal epithelium, although neither ESG nor its metabolite could permeate the intestinal cells under our experimental conditions. These results provide evidence for the beneficial function of ESG as a food ingredient.
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Nada-ku, Kobe 657-8501, Japan. yasuda@port.kobe-u.ac.jp
authors.forename: Michiko
authors.initials: M
authors.lastname: Yasuda
authors.forename: Takashi
authors.initials: T
authors.lastname: Furuyashiki
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Nakamura
authors.forename: Ryo
authors.initials: R
authors.lastname: Kakutani
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takata
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Ashida
mesh_terms: D000818:Animals; D018938:Caco-2 Cells; D002454:Cell Differentiation / Q000187:drug effects*; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D004043:Dietary Fiber / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D006003:Glycogen / Q000138:chemical synthesis* / Q000493:pharmacokinetics*; D006801:Humans; D056747:Immunomodulation / Q000187:drug effects*; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D004043:Dietary Fiber; D053583:Interleukin-1beta; D015850:Interleukin-6; D016209:Interleukin-8; D012333:RNA, Messenger; C495342:TLR2 protein, human; D051195:Toll-Like Receptor 2; D006003:Glycogen; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse