Materials and Methods

 
Compounds
HSG4112 and other glabridin-derivative compounds were prepared at Glaceum Inc. (Suwon, Republic of Korea) following the protocols from Patent US9783551B2 [17]. Glabridin was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
 
Animals and diets
Male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) at 14 weeks of age. The mice were fed high-fat (60% kcal% fat) diets (Research Diets Inc., New Brunswick, NJ, USA) starting at 6 weeks of age or were fed normal diet (10% kcal% fat). Mice were acclimatized for 2 weeks in a controlled environment (12 h light/dark cycle, lights on at 7:00 a.m. , 22 ± 2°C, 54.4 ± 8% humidity, ad libitum access to water and their respective diets) and were randomly divided into groups at 17 weeks of age in a manner in which obese group’s mean body weights were equal. Therefore, the HFD-induced obese group were fed HFD for a total of 11 weeks before the first drug administration. Individual food consumption and body weight were measured weekly unless otherwise noted. Animals in different groups were identified by color-coded cage cards. Animals were dosed via gastric intubation once a day in the afternoon (3:00 to 4:00 p.m.) for 6 weeks unless otherwise noted. At the end of the study, animals were fasted for 14 to 16 hours and anesthetized by isoflurane inhalation at terminal sacrifice. In the phenotypic screening assays, each group consisted of 4 animals (n = 2 for the vehicle group), with the exception of the enantiomerization step, where each group consisted of 5 animals. In the main efficacy study, each group consisted of 10 animals; one animal from the normal group was excluded due to incidental death (the exclusion criteria were pre-established). The sample sizes were chosen to observe weight-reducing trends of multiple compounds at the screening phase, and to detect sufficient statistical significance in the main study. 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. This study was conducted at Biotoxtech Co., Ltd., Republic of Korea (Institutional Animal Care and Use Committees (IACUC) Approval No.: 150289).  
 
Compound stability analysis
Three sets of 10 mg of HSG4112 and glabridin were each added to 10 mL of 1% HCl in MeOH (v/v%) or 1% NaOH in MeOH (w/v%). At 0, 8, 12, 24, 48, and 72-hour time-points, 1 mL aliquots from each condition were placed in HPLC (LC2030C, Shimadzu, Japan) in addition to 9 mL of internal standard (10 mg of (±)-3’’,4’’-dihydro-4’-O-methyl-glabridin dissolved in 100 mL of acetonitrile). HCl, NaOH, MeOH (Sigma-Aldrich Co.), HPLC-grade acetonitrile and formic acid (Thermo Fisher Scientific, Waltham, MA, USA), and deionized water (Millipore, Bedford, MA, USA) were used. The following HPLC condition was used: mobile phase (A:B = 80:20), A: 0.1 formic acid (MeOH:ACN3 = 1:3), B: 0.1 formic acid (DI water), column (Syncronis C18, 150×2.1 mm, 5 µm), flow rate (0.5 mL/min), and UV detection (280 nm). n = 3 per condition.
 
Histological analysis
Paraformaldehyde-fixed periepididymal fat and liver were paraffin-embedded, sectioned, and stained with Mayer’s hematoxylin-eosin (Sigma-Aldrich Co). The NAFLD activity score and fibrosis staging system were applied to liver sections for scoring of steatosis, lobular inflammation, hepatocyte ballooning, and fibrosis as outlined by Kleiner et al. [18]. All histological assessments were performed by a pathologist blind to the treatment.
 
Biochemical analysis
Blood samples were collected at sacrifice from the abdominal vein. The following parameters were measured in the serum using a blood-chemical analyzer (7180, HITACHI, Japan): alanine aminotransferase (ALT; JSCC (UV Kinetic)), aspartate aminotransferase (AST; JSCC (UV Kinetic)), total cholesterol (cholesterol oxidase-HMMPS), triglycerides (GPO-HMMPS glycerol blanking), LDL (low-density lipoprotein) cholesterol (selective protection enzymatic), HDL (high-density lipoprotein) cholesterol (direct), and glucose (GHexokinase-G6PDH). Serum insulin level was measured by using the Mouse Insulin ELISA Kit, TBM TMB type (MIT-696, Shibayagi Co., Ltd., Japan). Serum leptin level was measured using the Mouse Leptin ELISA Kit, TMBBM type (MLP-817, Shibayagi Co., Ltd.).
 
Quantitative real-time RT-PCR analysis
Total RNA was extracted from the liver, muscle, hypothalamus, and interscapular fat tissues and purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). The primers for 68 select genes were designed using Primer3; sequences are provided in Supplementary Table 1. GAPDH was used for normalization. qRT-PCR was performed using the QuantiSpeed SYBR Green Kit (PhileKorea, Seoul, Republic of Korea). n = 4 per group.
 
Western Blot
HFD-induced obese mice were dosed with 100 mg·kg-1 of HSG4112 for 11 days prior to sacrifice. The hypothalamus and interscapular adipose tissues were extracted. Western blotting was performed as previously described [19]. Phospho AMPK alpha 1 (T183) and 2 (T172) and UCP1 (Abcam, Cambridge, United Kingdom) were used as primary antibodies.
 
Metabolic analysis
HFD-induced obese mice were given either vehicle only or vehicle with HSG4112 (0.5% feed mixture) for 4 weeks prior to the metabolic rate analysis. Instead of oral administration, HSG4112 was mixed to the diet in order to avoid effects that may occur from taking the animals out of the indirect calorimetry cage for daily dose administration while maintaining the weight-loss effect equivalent to the oral administration of HSG4112 at 100 mg·kg-1 dose (Supplementary Fig. 1). The mice were placed inside an Oxymax/CLAMS (Columbus Instruments, Columbus, OH, USA) for 24 hours prior to the experiment for environmental adaptation, and for an additional 49 hours for the experiment. All parameters – VO2, VCO2, respiratory exchange ratio, energy expenditure, and locomotor activity – were calculated using a built-in software and previously reported method [20]. n = 4 per group. This study was conducted at Asan Medical Center, Republic of Korea (approval from the Institutional Animal Care and Use Committee of Asan Institute for Life Sciences).
 
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8.3.0 (GraphPad Software Inc., San Diego, CA, USA) by one-way or two-way ANOVA followed by Student’s, Dunnett’s, Tukey’s, or Sidak’s post-hoc test as appropriate. A P value of less than 0.5 was considered significant. All density values were quantified using ImageJ.