Biosci. Biotechnol. Biochem., 77 (3), 566–571, 2013 Potent Lipolytic Activity of Lactoferrin in Mature Adipocytes y Tomoji ONO,1;2; Chikako FUJISAKI,1 Yasuharu ISHIHARA,1 Keiko IKOMA,1;2 Satoru MORISHITA,1;3 Michiaki MURAKOSHI,1;4 Keikichi SUGIYAMA,1;5 Hisanori KATO,3 Kazuo MIYASHITA,6 Toshihide YOSHIDA,4;7 and Hoyoku NISHINO4;5 1Research and Development Headquarters, Lion Corporation, 100 Tajima, Odawara, Kanagawa 256-0811, Japan 2Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan 3Food for Life, Organization for Interdisciplinary Research Projects, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 4Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyou-ku, Kyoto 602-8566, Japan 5Research Organization of Science and Engineering, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan 6Department of Marine Bioresources Chemistry, Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minatocho, Hakodate, Hokkaido 041-8611, Japan 7Kyoto City Hospital, 1-2 Higashi-takada-cho, Mibu, Nakagyou-ku, Kyoto 604-8845, Japan Received October 22, 2012; Accepted November 26, 2012; Online Publication, March 7, 2013 [doi:10.1271/bbb.120817] Lactoferrin (LF) is a multifunctional glycoprotein resistance, high blood pressure, and dyslipidemia. To found in mammalian milk. We have shown in a previous prevent progression of metabolic syndrome, lifestyle clinical study that enteric-coated bovine LF tablets habits must be improved to achieve a balance between decreased visceral fat accumulation. To address the energy intake and consumption. In addition, the use of underlying mechanism, we conducted in vitro studies specific food factors as helpful supplements is attracting and revealed the anti-adipogenic action of LF in pre- increasing attention. adipocytes. The aim of this study was to assess whether Lactoferrin (LF) is an iron-binding glycoprotein LF could increase the lipolytic activity in mature found in a high concentration in mammalian breast adipocytes. Pre-adipocytes were prepared from rat milk. This multifunctional protein has anti-bacterial, mesenteric fat and differentiated into mature adipocytes anti-viral, immunostimulatory, anti-oxidative, and can- for assays of lipolysis. The addition of LF significantly cer-preventive activities.1–5) Commercially available LF increased the glycerol concentration in the medium in a is produced from bovine milk, and it has been approved dose-dependent manner, whereas pepsin-degraded LF as a food additive in Japan and is included in the did not. A DNA microarray analysis demonstrated that generally recognized as safe (GRAS) category in USA. LF decreased the expression of perilipin and affected bLF is currently added to infant formula, yoghurt, skim the cAMP pathway. These findings are supported by the milk and nutraceuticals for the many purposes. We have results of quantitative RT-PCR of perilipin and assays initially focused on the anti-bacterial activities of LF and of cAMP. These data collectively indicate that visceral found that it potently ameliorated periodontal diseases.6) fat reduction by LF may result from the promotion A reduction in visceral fat was subsequently noted in the of lipolysis and the additional anti-adipogenic activity animals of that study, leading to the identification of of LF. the novel function of LF as a treatment for metabolic syndrome. Key words: lactoferrin; adipocyte; lipolysis; cAMP; We conducted clinical trials to confirm that LF perilipin supplements could decrease visceral fat and ameliorate metabolic syndrome.7) Using enteric-coated LF (eLF) Metabolic syndrome is a combination of medical tablets that are not degraded in the stomach, we have disorders that increase the risk of developing cardiovas- found that a treatment for 8 weeks resulted in decreased cular disease and other chronic ailments. Dietary excess, abdominal fat accumulation, particularly of visceral fat, lack of exercise, and increasing psychological stress in Japanese men and women with abdominal obesity. have rapidly increased the number of affected individ- Some previous animal studies have provided further uals worldwide. Visceral obesity is central to the evidence of this effect. In particular, Shia et al. have development of metabolic syndrome. Excessive visceral reported the benefits of LF on body weight and fat fat accumulation disrupts the production of adiponectin, content under energy restriction.8) To elucidate the plasminogen activator inhibitor type 1, tumor necrosis mechanism of LF, its distribution in rats has been factor (TNF), and non-esterified fatty acids (NEFA), and investigated after an oral administration.9) We have thus leads to high blood glucose, induction of insulin subsequently detected immunoreactive LF most prom- y To whom correspondence should be addressed. Tel: +81-465-49-4472; Fax: +81-465-48-4079; E-mail: [email protected] Abbreviations: LF, lactoferrin; GO, gene ontology Lipolytic Action of Lactoferrin in Mature Adipocytes 567 inently in mesenteric fat tissue, suggesting that LF may used as a negative control. The culture supernatant was collected for a act directly on adipocytes. We then examined the lipolysis assay 24 h and 48 h after the addition treatment. The lipolytic 10) activities of LF against pre-adipocytes derived from rat activity was analyzed by measuring the glycerol concentration in the medium with a commercially available 148270 F-kit glycerol assay kit mesenteric fat tissue and found that LF inhibited (Roche Diagnostics, Tokyo, Japan) according to the manufacturer’s adipogenic differentiation in a dose-dependent manner. protocol. However, a treatment with pepsin abrogated this activity, indicating the need for enteric-coating of LF Isolation of RNA for the gene expression analysis of adipocytes. The to maximize its delivery to adipocytes. gene expression analysis using DNA microarrays was conducted by 2 The balance between lipid synthesis and degradation culturing adipocytes as already described in 25-cm flasks (Sumitomo determines the lipid content of adipocytes. Although we Bakelite Co., Tokyo, Japan). The cells were harvested 24 h after adding 1000 mg/mL of LF. A sample without any additive was used as a found that LF inhibited lipid synthesis, the lipolytic control. Total RNA was extracted by using an RNeasy Mini kit action of LF has not currently been reported. We (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. therefore analysed in the present study the lipolytic The quality of purified total RNA was analyzed with a 2100 action of LF and conducted a DNA microarray analysis Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and only to investigate the mechanism by which LF affected samples with an 18s:28s ribosomal RNA ratio of 2 or more were used. mature adipocytes. DNA microarray analysis. The DNA microarray analysis was conducted by amplifying and labeling 0.5 mg of total RNA with an Materials and Methods Amino Allyl Message AmpTM II aRNA amplification kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s Materials. Bovine LF was purchased from Friesland Campina instructions. Each sample of aRNA labeled with Cy3, and reference Domo (The Netherlands). The typical protein purity was 98% aRNA labeled with Cy5, was co-hybridized on to a 3D-GeneÔ Rat according to the manufacturer’s data. 12K Oligo chip (Toray Industries, Tokyo, Japan) at 37 C for 16 h. After hybridization, the DNA chips were washed and dried. The Animals and diets. Male Sprague-Dawley rats (eight-week-old) were hybridization signals derived from Cy3 and Cy5 were scanned by using purchased from Japan SLC (Shizuoka, Japan), and were maintained in a Scan Array Express (Perkin Elmer, Waltham, MA, USA), the scanned barrier room at 23 C with a 12-h light/12-h dark cycle (light on 7:00 image being analyzed by using GenePix Pro (Molecular Devices, a.m.–7:00 p.m.). The animals were fed a CE-2 solid laboratory diet Sunnyvale, CA, USA). All the analyzed data were scaled by global (Clea Japan, Tokyo, Japan) and tap water ad libitum. All animal normalization. Affymetrix NetAffix Gene Ontology and Ingenuity experiments were performed in accordance with the Guide for Care Pathway analysis systems (Ingenuity Systems, Red Wood City, CA, and Use of Laboratory Animals of Lion Corporation, Japan. USA) were respectively used for the gene ontology (GO) analysis and pathway analysis. Preparation of the LF degradation products by pepsin. The LF products degraded by pepsin were prepared as previously described.9) Confirmation of gene expression by quantitative RT-PCR. Total In brief, 5 g of LF was dissolved in 100 mL of water, and the pH value RNA was reverse transcribed with a Reva-Tra Ace kit (Toyobo, Osaka, was adjusted to 2.5 by using HCl. Pepsin (45,000 units, 1:10000 from Japan). Quantitative real-time RT-PCR analyses of perilipin and porcine stomach mucosa; 162-20752, Wako Pure Chemical Industries, peroxisome proliferator-activated receptor gamma (PPAR ) mRNA Tokyo, Japan) was subsequently added, and the solution was incubated were performed with a MyiQ single-color real-time PCR detection at 37 C for 24 h. The degradation products were visualized and system (Bio-Rad Laboratories, Tokyo, Japan), using the SYBR Green quantified by using SDS–PAGE. Real-time PCR Master Mix (Toyobo) according to the manufacturer’s instructions. Hypoxanthine phosphoribosyltransferase 1 (HPRT) was used as an endogenous control to normalize the results, since the gene Preparation of mature adipocytes. Mature adipocytes were obtained expression level of HPRT was not affected by the addition of LF from primary cell cultures of mesenteric fat-derived pre-adipocytes during the culture period (data not shown). The PCR primers for HPRT as previously described.9) Sprague-Dawley rats were euthanized by (QT00365722) were purchased from QuantiTect Primer Assay exsanguination of the abdominal aorta under anesthesia. The mesen- (Qiagen, Hilden, Germany). The primer sequences for perilipin, teric fat pads were removed and washed with ice-cold PBS containing PPAR , and PPAR 2 are listed in Table 1.