Mk a110d

Rats weighing ~130 g were assigned randomly to either a starch ad libitum, starch pair-fed, sucrose ad libitum, or ISO ad libitum group. Just before the dark cycle (18:00), the food was changed to experimental diets containing the indicated sugars, and the rats had access to the food container until the end of the dark cycle (06:00). During the dark cycle, locomotor activity was continuously measured (Supermex; Muromachi Kikai, Tokyo, Japan). After 3-h fasting (09:00), some rats underwent exercise consisting of eight 20-s bouts of swimming while loaded with a weight equal to 18% of body weight, with 40 s of rest between bouts.^{(12 (link)–14 (link))} These exercised and non-exercised rats in a comparison were accustomed to swimming for 10 min/day, 2 days before the experiment. Rats were anesthetized with by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) or volatile isoflurane (MK-A110D; Muromachi Kikai) according to the manufacturer’s instructions, followed by dissection of the epitrochlearis muscles. Some of these muscles were clamp-frozen in liquid nitrogen for measurement of muscle metabolites [glycogen and triglyceride (TG)] and Western blot analysis, and the remaining samples were used for subsequent incubation, as described below. After muscle dissection was completed, retroperitoneal and epididymal fat-pads were removed and weighted.

Lipopolysaccharides (LPS) from Escherichia coli O26:B6, fluorescein isothiocyanate (FITC)-labeled wheat germ agglutinin (WGA) lectin from Triticum vulgaris, and tetramethylrhodamine (TMR)-labeled dextran (average molecular weight, 75 kDa [TMR-dex75]) were purchased from Sigma-Aldrich Co. (St Louis, MO). The mouse-soluble syndecan-1 (Sdc-1) enzyme-linked immunosorbent assay (ELISA) kit from Diaclone SAS (Besancon Cedex, France) was purchased. Ketamine hydrochloride, xylazine hydrochloride, and rhodamine 6G were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sevoflurane was purchased from Pfizer Japan Inc. (Tokyo, Japan), and used for anesthesia using a small animal anesthetizer (MK-A110D, Muromachi Kikai Co., Ltd., Tokyo, Japan).

Seven-week-old specific pathogen-free (SPF) male C57BL/6 mice were purchased from Charles River Japan (Yokohama, Japan). The animals were kept in the filtered-air ventilated cage rack and were fed with a wheat-containing standard rodent diet (CE-2; CLEA Japan Inc., Tokyo, Japan) until the experiment. All mice were allowed free access to diet and water, with no special indications. When performing invasive procedures including cardiac puncture and euthanasia, anesthesia was always conducted using isoflurane anesthetizer (MK-A110D, Muromachi Kikai, Tokyo, Japan). Anesthesia was induced at 4% isoflurane with 20% oxygen using chamber and maintained at 1.5–2.75% isoflurane with 20% oxygen using anesthetic mask. Euthanasia was performed by cervical dislocation with deep euthanasia. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments were carried out with confirmation of The Regulations on Animal Experiments and with approval of the Institutional Animal Care and Use Committee of Osaka City University Graduate School of Medicine. All invasive procedures were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.