Animals
All animal experiment procedures were in compliance with the Animal
Protection Act of Korea and the Guide for the Care and Use of Laboratory
Animals. All studies were conducted at Biotoxtech Co., Ltd., Republic of
Korea (Institutional Animal Care and Use Committees (IACUC) Approval
No.: 150289) except for the metabolic assay and western blot assay,
which were conducted at Asan Medical Center, Korea (approval from the
Institutional Animal Care and Use Committee of Asan Institute for Life
Sciences). Male C57BL/6J mice of (Jackson Laboratory, Maine, USA) 14
weeks of age (supplier: ORIENTBIO Inc., Republic of Korea) – fed HFD
starting at 6 weeks of age – were purchased and housed in a controlled
environment (12h light/dark cycle, light on at 7AM, 22 ± 2°C, humidity
54.4% ± 8%). After acclimation period of two weeks and randomized
grouping, each animal was identified by individual color-coded cage card
displaying group and dose level. Mice had ad libitum access to
tap water and either pelleted rodent chow containing 10% fat diet
(normal control) or 60% fat diet (negative control, positive control,
and HSG4112 groups) (Research Diets Inc., USA). Individual food and
drinking water consumptions were measured weekly for all groups unless
noted otherwise. Animals were randomly separated into groups. Body
weight was measured weekly, at minimum. Animals were dosed via gastric
intubation with disposable syringes (1mL, Doowon meditec Co., Republic
of Korea) fitted with an intubation tube once a day in the afternoon
(3–4 PM), unless otherwise noted. All animals were fasted for 14-16
hours and anesthetized by inhalation of isoflurane before terminal
sacrifice.