Animals
Ten-weeks-old female trehalase KO mice (RBRC00857, background strain C57BL/6J) and WT mice were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan) and fed a standard diet (CE-2; CLEA Japan, Inc.) and water ad libitum for two weeks. The mice were kept in a temperature-controlled room with a 12-hour light cycle. This study was approved by the Laboratory Animal Care Committee of the Hayashibara Co., Ltd. (Okayama, Japan) and all experiments involving animals were conducted in accordance with the Guidelines for Care and Use of Laboratory Animals of the Hayashibara Co., Ltd.
Test substance
Trehalose (Reagent grade; Hayashibara Co., Ltd.) containing >98.0% trehalose dihydrate was used as the source of trehalose.
Study design
The experimental protocol is shown in Figure 1. A total of 40 mice were acclimated for 2 weeks and then the 12-weeks-old mice were randomly divided into 5 groups and matched for average body weight. Two groups of trehalase KO mice and two groups of WT mice were fed a high fat diet (HFD32, CREA Co., Ltd., Japan) and then given drinking water ad libitum that either lacked (control) or contained 0.3% (w/v) trehalose (KO/HFD/Water, KO/HFD/Tre, WT/HFD/Water, WT/HFD/Tre, n=8 for each group). As the experimental control between trehalase KO group, another group were given a normal diet and water ad libitum (KO/CE-2/Water, n=8). Four mice were grouped per cage with each cage containing animals from a single experimental group. Food and water were replaced every other day and food intake was monitored; body weights were recorded weekly throughout the experimental period. After 8 weeks of treatment, the animals were euthanized under pentobarbital anesthesia. The adipose tissue was weighed, and blood samples were collected from the abdominal vena cava for measurement of serum lipids and chylomicrons (CM). Serum TG and non-esterified fatty acids (NEFA) were measured using Triglyceride E-test kits and NEFA C-test kits (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), respectively. CM was measured by high sensitivity gel filtration HPLC (Skylight-biotech, Akita, Japan).
Calculation of energy intake
The energy densities of the HFD, a normal diet, and the drinking water containing 0.3% (w/v) trehalose were 20.9 kJ, 14.2 kJ, and 0.05 kJ per gram, respectively. Based on these data, the mean energy intake per mouse in each group was calculated using the following formulas:
Energy intake (kJ/mouse/day):
Mice consuming HFD and 0.3% (w/v) trehalose:
Food intake (g) ×20.9 (kJ) + water intake (g) × 0.05 (kJ)
Mice consuming HFD and water:
Food intake (g) ×20.9 (kJ) + water intake (g) × 0 (kJ)
Mice consuming normal diet and water:
Food intake (g) × 14.2 (kJ) + water intake (g) × 0 (kJ)
Histological analysis of adipocyte size
Mesenteric adipose tissue samples were fixed in 10% (v/v) buffered formalin and embedded in paraffin. The sections were deparaffinized with xylene, stained with hematoxylin and eosin, and then examined by light microscopy. Photographs of 5 random areas per section in the respective adipose tissue were taken at 200 × magnification. More than 200 adipocyte sizes were measured by image analysis software (cellSens, Olympus Corporation, Tokyo, Japan).
Rates of CLDs in jejunal epithelium
Sections of intestine were stained with hematoxylin and eosin, and then examined using light microscopy. Jejunal cytoplasmic vacuoles were demonstrated to contain neutral lipids by Oil Red O staining of frozen sections. Photographs of 5 random areas in the respective jejunal section were taken at 400x magnification. The proportion of area containing lipid (%) was measured in more than 20 intestinal villi/mouse by image analysis software (cellSens).
Cell culture
We prepared Caco-2 cells, a human colon carcinoma cell line, according to the method described by Vidal et al. [15] and Morel et al. [16]. The cells were seeded at 1.5×104cells/well in a 24-well insert cup (0.4 μm pore size, polyethylene terephthalate) treated with atelocollagen, and cultured for one week in Dulbecco’s modified Eagle’s medium (DMEM) containing 20% fetal bovine serum (FCS) at 37 °C. Confluency was confirmed by measuring electric resistance before serum-free DMEM and DMEM containing 20% FCS DMEM were added to the apical and basal side, respectively. The cells were cultured for an additional week to promote differentiation into intestinal epithelial cells, which were then divided into groups according to the electrical resistance values before use in experiments.
Micelle treatment of Caco-2 cells
As reported by Hernell et al. [17], micelles in the duodenum consist of 0.3 mM oleic acid, 0.025 mM cholesterol, 0.1 mM 2-monooleylglycerol, 1.0 mM taurocholic acid and 0.1 mM α-lysophosphatidylcholine. Lipids at these concentrations were dissolved in ethanol in a glass test tube, dried with N2 gas, and stored at -80 °C until use. After adding serum-free DMEM medium to the lipids and sonicating for 20 minutes, they were mixed with the same amount of serum-free DMEM medium with or without trehalose (50 mM final concentration) and sonicated for 5 minutes. The medium on the apical side of the differentiated Caco-2 cells was removed, and 300 µL/well of the micelle solution was added. After incubating for 24 hours, basal side culture medium was collected in a tube, mixed with polyoxyethylene (10) octylphenyl ether and EDTA at a final concentration of 1% and 5 mM, respectively, and protein inhibitors before storage at -80 °C until use. When assessing the amount of intracellular lipid accumulation, the content of micelle components was reduced by half to prevent cytotoxicity and mixed with the fluorescently labeled fatty acid BODIPYTM FL C16 (Thermo Fisher Scientific Inc., Waltham, MA, USA) at a final concentration of 10 μM.
Measurement of lipid droplet accumulation in Caco-2 cells
The cultured cells were fixed by treatment with 4% formaldehyde for 5 minutes. The membrane at the bottom of the insert cup was cut with a scalpel, transferred to a slide glass, and encapsulated with mounting medium (50% glycerol, 0.05% NaN3 in phosphate buffered saline). Then, the specimen was observed with a fluorescence microscope (Olympus BX53-FK, exposure time: 10 milliseconds), and the ratio of BODIPYTM FL C16 that accumulated in areas containing lipid droplets was analyzed with Olympus cellSens.
Evaluation of secreted ApoB-48 and ApoB-100 by western blotting
Basal side culture medium was collected and mixed with ×1.25 sample buffer (2.08% sodium lauryl sulfate, 6.25% glycerol, 1.94% dithiothreitol in 0.073 M Tris-HCl buffer, pH 6.8) and denatured by boiling for 5 minutes. The protein was subjected to 5% SDS-PAGE and transferred to a PVDF membrane. The membrane was treated with blocking agent, and labeled with a mouse anti-human ApoB monoclonal antibody (7B8) (ab39560, Abcam plc, Cambridge, UK) as the primary antibody, and horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody (Nr.P.0447, Dako, Agilent Technologies, Santa Clara, CA) as the secondary antibody, which was detected by ECL prime detection agent (GE Healthcare, Little Chalfont, UK). The resulting images were analyzed with Image J (version 1.52a).
Statistics
Data are expressed as means ± standard deviations. A power analysis (G*Power 3.1.9.4, http://www.gpower.hhu.de/) showed that a sample size of 8 mice per group was suitable to detect a difference between 5 experimental groups (1-β=0.95, effect size=0.8, α=0.01). In addition, the calculated p values were described. Statistically significant effects of trehalose on dispersion uniformity and normality were examined using Tukey-Kramer (Statcel, ver. 3). Non-parametric data were analyzed by the Steel-Dwass test (Statcel, ver. 3). A p-value less than 0.05 was considered significant.