Generation of Mgat2-deficient mice
Mgat2-targeted 129/SvJae embryonic stem cells and, subsequently, mice carrying this mutation with a targeting vector designed to replace exon 1 of Mgat2 with a neomycin-resistance cassette (Figure 1A) were generated. Mgat2 deficiency was confirmed by Southern blotting of genomic DNA, which was digested with MfeI and SoeI using a probe hybridizing to sequences upstream of those included in the targeting vector, and by quantitative PCR. Mgat2−/− and wild-type littermates were generated by breeding heterozygotes that were first backcrossed with C57BL/6 J mice. Mice were housed in a pathogen-free facility. Diets included a normal diet (ND, CRF-1, Oriental Yeast Co., 3.59 kcal g−1) and a high-fat diet (HFD, Oriental Yeast Co., 59% cal fat (lard), 5.578 kcal/g). Animals were maintained in a 12/12-h light–dark cycle. All experiments were performed on male animals. Experimental protocols concerning the use of laboratory animals were reviewed and endorsed by the Institutional Animal Care and Use Committee of Mitsubishi Tanabe Pharma Corporation.
MGAT enzyme assays
MGAT activity was determined by measuring the incorporation of the [14 C]palmitoyl moiety into triacylglycerol with [14 C]palmitoyl-CoA (ARC, St. Louis, MO) and 2-monooleoylglycerol. Crude membranes (10 μg) of the small intestine were used as the enzyme source. Assays were performed in 200 μl buffer (100 mM Tris–HCl, pH 7.4, 200 mM sucrose, 5 mM MgCl2, 1.25 mg/ml BSA) containing 10 μM 2-monooleoylglycerol and 20 μM [14 C]palmitoyl-CoA. Reactions were performed for 5 min at 30°C and the products were extracted with 1 ml of chloroform:methanol (2:1, v/v). The extracts were dried and separated by thin layer chromatography with hexane:diethyl ether:acetic acid (80:20:1, v/v/v). Areas containing [14 C]triacylglycerol were visualized and quantitated using the FLA3000 fluorescence detection system (Fujifilm, Tokyo, Japan).
Oral fat tolerance test (OFTT)
Mice were fasted overnight and then 10 ml/kg of Intralipos containing 20 % soybean oil (v/v) (Otsuka, Tokyo, Japan) was orally administered. Plasma samples were obtained from the tail vein before and 75, 150, 225, and 300 min after the fat challenge for determination of plasma triglyceride levels.
Long-term feeding studies
Animals were weaned at three weeks of age and maintained on an ND or switched to an HFD at seven weeks of age. They were weighed and their food intake was monitored. After 10 weeks of HFD feeding, blood samples in the fed state and feces were collected for 3 days. After overnight fasting, the mice were killed by whole blood collection from the abdominal aorta under ether anesthesia. The small intestine and liver were quickly removed from each mouse, immediately frozen in liquid nitrogen, and stored at −80°C for quantitative PCR and lipid measurements.
Determination of body composition
After 10–12 weeks of HFD feeding, the body composition was analyzed by dual-energy X-ray absorptiometry with PIXImus2 (GE Healthcare, Tokyo, Japan) under ether anesthesia.
Determination of plasma parameters
Plasma triglyceride, total cholesterol, and non-esterified fatty acid (NEFA) levels were determined using an enzymatic assay kit (Wako Chemicals, Osaka, Japan). Blood glucose was determined using commercially available kits based on the glucose oxidase method (Wako Chemicals, Osaka, Japan). Plasma insulin was assayed using an enzyme-linked immunosorbent assay (ELISA) kit (Morinaga Co. Ltd., Yokohama, Japan).
Oral glucose tolerance test (OGTT)
An oral glucose tolerance test (OGTT) was performed after 10 weeks of HFD feeding. Mice were fasted for 3 h and then 2 g/kg glucose solution was orally administered at a volume of 10 ml/kg. Blood samples were obtained from the tail vein before and 15, 30, 60, and 120 min after the glucose challenge for determination of blood glucose levels. Plasma samples were obtained before and 15, 30, and 60 min after the glucose challenge for determination of plasma insulin levels. HOMA-IR was calculated .
Determination of lipid levels in feces and the small intestine
Fecal lipids were extracted with a 30-fold volume of chloroform:methanol (2:1, v/v) with vigorous shaking. Then, a 1/3.75-fold volume of water was added and the mixture was shaken vigorously. The organic phase was collected and again a 30-fold volume of chloroform:methanol (2:1, v/v) was added to the water phase. After vigorous shaking, the organic phase was collected, and the combined organic phase was evaporated. The dried residue was dissolved in isopropanol:Triton X-100 (9:1, v/v) and measured for triglyceride and NEFA levels using an enzymatic assay kit. The small intestine was homogenized in 20-fold volume of saline. Then, lipids were extracted with a 3.75-fold volume of chloroform:methanol (2:1, v/v) with vigorous shaking. The organic phase was collected and evaporated. The dried residue was dissolved in isopropanol:Triton X-100 (9:1, v/v) and measured for triglyceride levels using an enzymatic assay kit.
Determination of oxygen consumption
After 10 weeks of HFD feeding, oxygen consumed by each mouse was measured using an ARCO-2000 analyzer (Arco System, Chiba, Japan) every 3 min for a 24-h period.
Portions of duodenum were fixed in 10 % (v/v) neutral buffered formalin and embedded in paraffin. Paraffin sections (3–5 μm thick) were stained with hematoxylin and eosin and examined under a light microscope.
Total RNA of the duodenum and the liver was extracted without using DNase. Total RNA was reverse transcribed by TaqMan Reverse Transcriptase Reagents (Applied Biosystems, Foster City, CA). Real-time PCR was performed with SYBR Green PCR mix (Applied Biosystems, Foster City, CA) and analyzed with an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). The relative expression levels were compared after normalization to B-actin. The primers used for B-actin and Mgat2 were from QIAGEN (Tokyo, Japan). The following primer pairs were used (5′ to 3′): Cpt-1a, forward: AAGCTGTTCAAGATAGCTTG, reverse: TGCTGATGACGGCTATGGTGT; Acot1, forward: GGCTGGGAATGGAGTTTCAT, reverse: GCTATCCAAGAAAAGTGCCAGG; Acot2, forward: AGTGCCTATGAAGGACTGAGGA, reverse: GGTAAAGGTGCTTTCTGCC; Acox1, forward: GGTAAAGGTGCTTTCTGCC, reverse: AGATAAACTCCCCAAGATTCAAGAC; Hmgcs2, forward: TGTCCCCTGAGGAATTCACAGAA, reverse: AACGAGTGGATGAGATGCATCG; Lcad, forward: CTGGTTAAGTGATCTCGTGATCGTCG, reverse: CTGGCAATCGGACATCTTCAAAGAATAGT; Mcad, forward GCTCTGATGTGGCGGCCATTA, reverse: AAGGCTTTACTAGCGGGTACTTTAGG
All data were expressed as mean ± SEM. The comparison of mean values between two groups was performed by the Student’s t-test, and the Dunnett’s test was used for comparing more than two groups. P values of <0.05 were considered statistically significant.