Chronic administration of Eucommia leaf extract (ELE) and asperuloside(ASP), the major component of ELE, prevents adipocyte hypertrophy in white adipose tissues

1Laboratory of Molecular Prophylaxis and Pharmacology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3 Suzuka, Minamitamagakicho, Suzuka, Mie 513-8670, Japan 2R&D Center, Kobayashi Pharmaceutical Co., Ltd. 1-30-3, Toyokawa, Ibaraki, Osaka 567-0057, Japan 3Faculty of Acupuncture & Moxibustion, Suzuka University of Medical Science, Mie, Japan 4Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, 1757 Kanazawa, Tobetsu-cho, Ishikari, Hokkaido 061-0293, Japan 5Department of Biochemistry and Proteomics, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan


Introduction
Obesity is known to be a risk factor for life style-related diseases such as cardiovascular disease, diabetes, musculoskeletal disorders (especially osteoarthritis), hypertension, kidney disease, gout, and so on. Preventing obesity has a beneficial influence on not only health problems, but also economic problems. Obesity is characterized as hypertrophy and proliferation of adipocytes in white adipose tissue (WAT) [1,2]. Eucommia leaf extract (ELE) has been reported to have various bioactive properties [3][4][5][6][7][8][9][10][11], such as anti-obesity effects [6][7][8][9][10][11]. Asperuloside (ASP) is indicated as the major ingredient of ELE; it contributes to its anti-obesity effect [8,11]. In the previous study, the administration of ELE or ASP significantly reduced the ratio of WAT weight to the body weight in perirenal WAT and tended to reduce this ratio in epididymal WAT [9,11]. The aim of this study is to ascertain whether ELE and its major component, ASP, affect the size of adipocytes in WAT.

Abstract
Obesity is known to be a risk factor for life style-related diseases like cardiovascular diseases, hypertension, kidney diseases and so on. Preventing obesity is beneficial for health problems and economic problems. ELE and asperuloside (ASP) have been reported to have the anti-obesity effects. In the previous study, ELE and ASP significantly reduced the ratio of WAT weight to the body weight in perirenal WAT and tended to reduce this ratio in epididymal WAT. In this study, we observed the size of the stained adipocyte in visceral WAT (perirenal and epididymal) with a microscope. ELE and ASP markedly decreased the size of adipocytes in perirenal WAT. ELE slightly decreased the size of adipocytes in epididymal WAT. ASP (0.1, 0.3%) markedly and concentration-dependent decreased in the size of adipocytes in epididymal WAT. ELE and ASP could be beneficial for curbing metabolic disorders, as they prevent the harmful effects, which are caused by the hypertrophy of adipocytes in the visceral WAT.
(7.7%), protein (23.6%), lipid (35.0%), ash (6.1%), dietary fiber (2.9%), and nitrogen-free extract (24.7%). Test foods were prepared by adding 5% ELE and 0.1 and 0.3% ASP, or by adding 5% casein as a control. The test foods were provided ad libitum for 3 months. The doses given to HFD fed animals were determined based on previous studies [9,11]. After chronic administration of ELE and ASP, the body weight was measured, and the rats were sacrificed without stress; the WATs (perirenal WAT and epididymal WAT) were immediately removed. The Institutional Animal Care and Use Committees at Mie University Faculty of Medicine and the Suzuka University of Medical Science approved the animal facilities and study protocols. All procedures were performed in accordance with the National Institutes of Health guidelines for animal care.

Morphological observations
The WAT was fixed with formalin and stained with hematoxylin and eosin, as reported previously [8]. We then observed the size of the stained adipocyte in visceral WAT (perirenal and epididymal) with a microscope.

Results
Compared with the Control-HFD group, the size of adipocytes in perirenal WAT decreased markedly in 3 months in the ELE5%-HFD group. This decrease was also observed in the ASP (0.1, 0.3%)-HFD group, compared to the Control-HFD group (Figure 1). However, a dose-dependent decrease was not observed in the ASP (0.1%-0.3%)-HFD group. On the other hand, the size of adipocytes in epididymal WAT decreased slightly in 3 months in the ELE5%-HFD group, compared to the Control-HFD group ( Figure 2). The ASP (0.1, 0.3%)-HFD group showed a marked decrease in the size of adipocytes in epididymal WAT, and a dose-dependent decrease was observed in the ASP (0.1%-0.3%)-HFD group ( Figure 2).

Discussion
In the previous study, ELE and its major component, ASP, were reported to result in the reduction of epididymal and perirenal WAT [9,11]. In this study, we found that ELE and ASP inhibited the HFDinduced hypertrophy of adipocytes in both perirenal and epididymal WAT. When WAT expansion occurs with obesity, the size, and/or the number of adipocytes is known to increase [2]. In such conditions, both ELE and ASP seemed to prevent obesity through the prevention of the hypertrophy of adipocytes in the visceral WAT. The hypertrophy of adipocytes in the visceral WAT is correlated with a high incidence of metabolic disorders. The hypertrophy of adipocytes in the visceral WAT increased basal fatty acid release, pro-inflammatory cytokine release, immune cell recruitment, hypoxia, fibrosis, decreased adiponectin, and impaired insulin sensitivity [1]. ELE and ASP could be beneficial for curbing metabolic disorders, as they prevent these harmful effects, which are caused by the hypertrophy of adipocytes in the visceral WAT.