Ce 2 diet

In this study, 24 female Sprague–Dawley rats were used. The rats, with an average weight of 308.3 ± 4.7 g, were purchased from Japan SLC (Hamamatsu, Japan). After a one‐week acclimatization period, the rats were randomly divided into four groups: control (CON, n = 6), NA supplementation (NA, n = 6), 14‐day HU (HU, n = 6), and NA supplementation during HU (HU + NA, n = 6). Rats in the NA and HU + NA groups received oral administration of NA (146–01235, Wako Pure Chemical Industries, Osaka, Japan) (750 mg/kg/day) dissolved in a 5% arabic gum solution (016–00025, Wako Pure Chemical Industries) via a catheter twice daily. Rats in the other groups received the same volume of 5% gum arabic solution, which served as an emulsifier. After 1 week of administration for acclimatization, the rats in the HU and HU + NA groups underwent HU for 2 weeks. All rats received either the solution or NA during the HU period. The rats were housed in an isolated, environmentally controlled room at a temperature of 22 ± 2°C, with a 12‐h light–dark cycle. They had ad libitum access to food (Diet CE‐2, CLEA Japan, Tokyo, Japan, Table 1) and drinking water.

All animal care procedures and experiments conformed to the Association for Assessment and Accreditation of Laboratory Animal Care guidelines and were approved by the Experimental Animal Care and Use Committee of Takeda Pharmaceutical Company Limited. All rats were housed in individual cages in a room with controlled temperature (23°C), humidity (55%) and lighting (lights on from 7:00 a.m. to 7:00 p.m.) and were fed with a normal chow diet (CE2 diet, CLEA Japan) with free access to water. Rats were sacrificed by exsanguination, and a spine curvature in each rat was confirmed when they were sacrificed. Organs were eviscerated and some of them were measured for weights. The spinal cord was dissected, and the thoracic and lumbar segments were identified using the ribs and vertebrae as a guide.

Animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). All animal care was approved by the animal care committee of the University of Gifu (No. 27-30, approval date: 4 June 2015). Mice were housed at 23 °C on a 12-h light/dark cycle. Chrebp^−/− mice were backcrossed for at least 10 generations onto the C57BL/6J background [19 (link),30 (link)]. Shp^−/− mice were purchased from Lexicon Genetics Inc. (The Woodlands, TX, USA). Shp^+/− mice were backcrossed for at least 12 generations onto the C57BL/6J background. Male mice were used for all studies. Chrebp^−/−Shp^−/− (DKO) mice were intercrossed with Chrebp^−/− and Shp^−/− mice.
Mice had free access to water and were fed an autoclaved CE-2 diet (CLEA Japan, Tokyo, Japan). Wild-type (WT), Chrebp^−/−, Shp^−/−, and DKO mice were housed separately with a total of three mice per cage. Body weight was measured weekly between 7 and 21 weeks of age. Mice were sacrificed at 21 weeks of age by cervical dislocation. All tissue samples were immediately placed into liquid nitrogen and stored at −80 °C until further analysis for hepatic triacylglycerol and cholesterol contents and for quantitative polymerase chain reaction (PCR).

In this study, 2 strains of mice, HO-2-null on the C57BL/6J background and its littermate were utilized.^{(5 (link))} Mice heterozygous for the disruption in the HO-2 gene were the generous gift from Eiichiro Nagata (Department of Neurology, Tokai University School of Medicine). They were bred at Keio University School of Medicine and fed ad libitum a standard CE-2 diet (Clea Japan, Tokyo, Japan). Genotypes were examined by polymerase chain reaction (PCR) of tail genomic DNA (see Supplemental Table 1* for primers used for genotyping). All experiments were approved by the Animal Care and Utilization Committee of Keio University School of Medicine (permission number 08040).

Animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978). All animal care was approved by the Animal Care Committee of the University of Gifu (No. 25-16, approval date: 2 May 2013; No. 22-26, approval date: 8 November 2010). Mice were housed at 23 °C on a 12-h/12-h light/dark cycle. At 8–9 weeks of age, male C57BL/6J mice (n = 6 per group) were intravenously infected with an adenovirus harboring ChREBP lacking the N-terminal 196 amino acids (ChREBP Δ196) or enhanced green fluorescent protein (GFP) as a control [23 (link),24 (link)]. ChREBP Δ196 lacks two nuclear export signals and mimics ChREBP-β [25 (link),26 (link)]. Mice had free access to water and an autoclaved CE-2 diet (25.5% protein, 4.6% fat, 48.9% carbohydrate; CLEA Japan, Tokyo, Japan). Mice were euthanized after 5 days by cervical dislocation. All tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C until further analysis for hepatic TAG and cholesterol contents or quantitative polymerase chain reaction (PCR).