La theta lct 100

Computed tomography scanning was performed using a LaTheta LCT100™ micro‐CT scanner (Hitachi Ltd., Tokyo, Japan) with the parameters set to 50 kV, 1 mA, and a slice thickness of 200 μm, thus allowing high‐resolution 3D images of the skull samples to be obtained. Amira 5.2.2 or 5.4.5 software (FEI Visualization Sciences Group, Burlington, MA, USA) was used to reconstruct 3D images from the scanned cross sections.

Mice were anesthetized with Nembutal (50 mg/kg of body weight, i.p.; Abbott Laboratories, North Chicago, IL, USA). CT analysis was carried out by a micro-CT scanner (LaTheta LCT-100; Hitachi, Tokyo, Japan). The analysis of CT data was carried out by the use of LaTheta software (Hitachi). The vWAT and sWAT, and muscle weights were measured from images at the level of the umbilicus. Subcutaneous WAT was defined as the extraperitoneal fat between skin and muscle. The intraperitoneal part with the same density as the subcutaneous fat layer was defined as vWAT. Proportions of vWAT and sWAT were determined by automatic planimetry as described previously^{29 (link)}.

Intraperitoneal glucose tolerance test (IPGTT) was performed as previously described [20 (link)]. After 16 h of fasting, 1.5 g/kg body weight glucose was intraperitoneally injected into the mice. The blood glucose level of the mice was measured at 0, 15, 30, 60, and 120 min using a Glutest sensor (Sanwa Kagaku Kenkyusyo Co., Ltd. Aichi, Japan). For the high-fat diet study, 8-week-old male mice were fed diets with 60% of calories from fat (high-fat diet, Research Diets D12492) for 10 weeks. Body weight was measured every week. Finally, IPGTT was performed when the mice 18 weeks old. Blood tests for several biological markers were performed by Unitech Co. (Kashiwa, Japan). Muscle muss and body fat mass in mice were measured using magnetic resonance imaging (Latheta LCT-100, Hitachi).