Microwave-assisted extraction of Sudachitin
The green peels of Citrus sudachi Hort. ex Shirai were collected in Tokushima Prefecture, Japan, in September. The dried peels were ground in a grinder. Microwave-assisted extraction was performed using a micro-reactor (2.45 GHz, 700 W; Shikoku Instrumentation Co., Ltd., Kagawa, Japan). A mixture of dried ground sudachi peels (100 g) in MeOH (600 mL) and water (200 mL) was stirred in a separable three-necked round-bottom flask (1 L) with a condenser, thermo-sensor, and mechanical stirrer. The mixture was irradiated under microwaves (MW) for 12 min under reflux with stirring. The reaction mixture was extracted with ethyl acetate and separated into the ethyl acetate layer and water-soluble layer. The ethyl acetate layer was dried with Na2SO4, and ethyl acetate was subsequently removed under reduced pressure. The resulting dark green residue was chromatographed on silica gel column (elution with t-BuOH/hexane/ethyl acetate = 0.5 : 5 : 1.2) to yield sudachitin as yellow needles, which was recrystallized from ethyl acetate to give sudachitin (0.092 g, melting point 238–240°C). A mixture of the water-soluble layer (280 mL), MeOH (280 mL), and concentrated HCl (240 mL) in the round-bottom flask was irradiated by MW for 7 min under reflux with stirring. The reaction mixture was neutralized with NaOH, and MeOH was removed under reduced pressure to give a brown residue, which was extracted with ethyl acetate and dried (Na2SO4). After removal of ethyl acetate, the resulting brown residue was chromatographed on silica gel column (elution with t-BuOH/hexane/ethyl acetate = 0.5 : 5 : 1.2) and further recrystallized from ethyl acetate to give sudachitin (0.376 g, melting point 238–240°C) as colorless needles. The purity of sudachitin was confirmed by HPLC to be above 95%. Sudachitin was dissolved in 0.2% dimethyl sulfoxide (DMSO).
Animals and experiments
All work involving animals was performed in compliance with the Guide for the Care and Use of Laboratory Animals and protocols approved by the Institutional Animal Care and Use Committee at the University of Tokushima Graduate School (Tokushima, Japan).
C57BL/6 mice at 4 weeks of age were purchased from Japan SLC, Inc. (Shizuoka, Japan). Mice were housed in temperature- (23 ± 3°C) and humidity-controlled conditions with a 12-h light/dark cycle. Mice were given free access to water and either a control diet (n = 20; 14% of calories from fat; Oriental Yeast Co., Ltd., Tokyo, Japan) or a high-fat diet (n = 20; 40% of calories from fat; Oriental Yeast Co., Ltd.). Mice were allowed to adapt to these conditions for 1 week before starting the experimental protocol. During the experimental period, the mice were divided into two groups (n = 10) and orally administered either 5 mg/kg sudachitin dissolved in a 0.2% sodium carbonate solution or a corresponding volume of 0.2% DMSO solution mixed with 0.2% sodium carbonate daily for 12 weeks. Body weight and food intake of each mouse were measured weekly.
Ten db/db mice at 4 weeks of age were purchased from Charles River Japan, Inc. (Kanagawa, Japan). Mice were housed under temperature- (23 ± 3°C) and humidity-controlled conditions with a 12-h light/dark cycle. Mice were given free access to water and the control diet, as described above, and allowed to adapt to the conditions for 1 week before the beginning of the experimental protocol. Mice were divided into two groups (n = 5) and orally administered 5 mg/kg sudachitin or 0.2% DMSO, as above, daily for 12 weeks. The body weights and food intake of mice were measured once a week.
Plasma and tissue collection
Before, during, and at the end of the experimental period, blood samples were taken from the tails of the mice fasted overnight for biochemical analyses. At the end of the study, mice were sacrificed by cervical dislocation after blood collection. Plasma samples were prepared by centrifugation at 3,000 rpm for 10 min at 4°C, and were stored at -80°C until analysis. Samples of the liver, gastrocnemius muscle, epididymal (visceral) and inguinal (subcutaneous) WAT, and BAT were collected, rinsed, weighed, frozen in liquid nitrogen, and stored at -80°C until analysis.
Plasma biochemical analyses
Plasma non-esterified fatty acid (NEFA), triglyceride, and total cholesterol levels were measured using enzymatic commercial assay kits (NEFA C-Test, Triglyceride E-Test, and Cholesterol E-Test, respectively; Wako Pure Chemical Industries, Osaka, Japan). Plasma insulin, adiponectin, and leptin levels were measured by enzyme-linked immunosorbent assays (ELISA; Mouse Insulin ELISA Kit, Mouse/Rat High Molecular Weight Adiponectin ELISA Kit, and Mouse Leptin ELISA Kit; Shibayagi, Gunma, Japan).
X-ray CT scan
Total body fat, subcutaneous fat, and visceral fat were measured in mice under isoflurane anesthesia using LaTheta X-ray CT scanner LCT-200 (Hitachi-Aloka Medical, Ltd., Tokyo, Japan). Data was analyzed using Visualization LaTheta software (Hitachi-Aloka Medical Ltd., Tokyo, Japan).
Blood glucose, oral glucose tolerance tests (OGTT), and insulin tolerance tests (ITT)
Blood glucose levels were measured in tail vein blood using a glucometer (Arklay, Kyoto, Japan). For OGTT, blood glucose levels were measured 0, 30, 60, 90, and 120 min after an oral glucose load (1 g/kg) following an overnight fast. For ITT, blood glucose levels were measured 0, 30, 60, and 90 min after an intraperitoneal injection of regular human insulin (Novo Nordisk Pharma Inc., Tokyo, Japan). Insulin was administered to C57BL/6 J mice at 0.75 mU/g body weight and 3 mU/g to db/db mice, following a 6-h fast.
Total RNA isolation
Total RNA was extracted from WAT and the gastrocnemius muscle using RNeasy Lipid Tissue and RNeasy Plus Universal Mini Kits (QIAGEN, Valencia, CA, USA) according to manufacturer’s instructions. Total RNA was extracted from skeletal muscle myocytes using RNAiso and amplified using CellAmp Whole Transcriptome Amplification Kit Version 2 (Takara Bio Inc., Shiga, Japan).
Gene expression analyses
Total RNA (1 μg) was reverse-transcribed to cDNA in a final volume of 20 μL using the Primescript RT Reagent kit (Takara). Real-time polymerase chain reaction (PCR) was performed in a final volume of 10 μL containing 50 ng of the cDNA template and primers, using a StepOnePlus Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). The primers used for PCR are listed in Additional file 1: Table S1. We evaluated the RNA levels of glucose transporters (Glut 1–4), adiponectin, PPARγ, adipocyte protein 2 (AP2), cluster of differentiation 36 (CD36, fatty acid translocase), uncoupling protein 1, 2, and 3 (UCP1, UCP2, and UCP3), and Sirt1 in tissue samples. In cultured skeletal muscle myocytes, we measured the transcription of genes involved in the synthesis of fatty acids (fatty acid synthase (FAS), acetyl-coA carboxylase (ACC1)), triglycerides (diglyceride acetyltransferase (DGAT1 and DGAT2)), or both (SREBP-1), as well as genes associated with hepatic cholesterol synthesis (farnesyl diphosphate synthase (FDS), squalene synthase (SS), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-R), and synthase (HMG-S)), cholesterol sensitivity (microsomal triglyceride transfer protein (MTTP), low-density lipoprotein receptor (LDLR)), lipid oxidation (UCP2, acyl-coenzyme A oxidase (ACOX), PPARγ coactivator 1α (PGC1α)), lipolysis (hormone-sensitive lipase (HSL), carnitine palmitoyl transferase-α (CPT1α), adipose triglyceride lipase (ATGL)), and glycogenolysis (glucose 6-phosphatase (G6Pase)), phosphoenolypyruvate carboxykinase (PEPCK)). Evaluation of mitochondrial biogenesis in primary myocytes involved measurement of nuclear respiratory factor 1 and 2 (NRF1 and NRF2) and mitochondrial transcription factor A (mtTFA).
Measurement of adipocyte size
Subcutaneous and visceral adipose tissue samples were fixed in 2% osmium tetroxide and shaken at 37°C for 7 days. The fixed tissues were filtered through a 250-μm and 25-μm mesh to obtain a single cell suspension. The diameter and distribution of adipocytes were measured using a Coulter Multisizer 3 particle counter (Beckman Coulter, Inc., Brea, CA, USA).
Analysis of visceral adiposity
The volume and distribution of visceral fat at 4 weeks after starting sudachitin administration were evaluated by X-ray computed tomography (CT; LaTheta, LCT-200, Aloka, Tokyo, Japan) of the region between the first lumbar vertebra and the pubic bone, under isoflurane anesthesia. Data were analyzed from continuous 2-mm slice images (for quantitative assessment) using LaTheta software.
Total protein was extracted as described previously . Lysates were separated by SDS-PAGE on 10% polyacrylamide precast gels (Invitrogen, Carlsbad, CA, USA) and transferred to polyvinylidene difluoride membranes by electroelution. Membranes were blocked in 20 mmol/L of TBS solution with 1% Tween containing 5% skim milk and incubated with primary antibodies overnight at 4°C. Polyclonal antibody sensitive to AMPKα1 and phospho-AMPKα1 (Thr172) was obtained from Cell Signaling Technology (Danvers, MA, USA) and antibodies against GLUT4 and β-tubulin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein density were visualized on membranes using horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized using the enhanced chemiluminescence reagent (GE Healthcare; Waukesha, WI, USA), according to manufacturer’s directions.
Four-week-old male C57BL/6 J mice were fed high-fat diet and treated with sudachitin (5 mg/kg) or vehicle for 4 weeks. Oxygen consumption was continuously measured during the 12-h light–dark cycles using a comprehensive laboratory animal open-circuit indirect calorimetry monitoring system (Columbus Instruments, Columbus, OH, USA). Data were corrected for 2 days after 2 days of adaptation to the metabolic cages.
Skeletal muscle enzyme activities
Skeletal muscle samples were homogenized with extraction buffer (0.1 mol/L KH2PO4, 0.1 mol/L NaPHO4, and 2 mmol/L EDTA, pH 7.2). Citrate synthase activity was determined using a Citrate Synthase Assay Kit (Sigma-Aldrich, St. Louis, MO, USA).
ATP content measurement
ATP content was measured using the AMERIC-ATP kit (Applied Medical Enzyme Research Institute Corporation, Tokushima. Japan) according to manufacturer’s instructions. Briefly, a sample of skeletal muscle (100 mg) tissue was homogenized in 10× volume of extraction buffer (0.25 mol/L sucrose, 10 mmol/L HEPES-NaOH, pH 7.4) and centrifuged at 1000 g for 10 min. The supernatant was diluted 8-fold in phenol-based extraction buffer and ATP extraction reagent was added. Following addition of luciferase solution, ATP content was quantified by measuring the luminescence using a luminometer (Lumat LB9507, Berthold Technologies, Tokyo, Japan).
Primary myoblasts were isolated from the calf region, thigh, and pelvic girdle of 8-week-old C57BL/6 J mice. Satellite cells were isolated by the digestion of muscle tissue by the type 2 collagenase (Worthington Biochemicals, Lakewood, NJ, USA), followed by pre-plating to purify cells and remove fibroblasts. Experiments were conducted after myoblasts had reached 99% purity. Cells were cultured at 37°C in 5% CO2 in Dulbecco modified Eagle medium (DMEM), supplanted with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were differentiated in DMEM/20% horse serum supplanted with basic fibroblast growth factor for 3 days. The primary cells were treated with vehicle (0.2% DMSO) or 30 mmol/L sudachitin for 48 h.
Skeletal muscle myocytes were grown and differentiated on a glass coverslip coated with Cell-Tak (BD Bioscience, Franklin Lakes, NJ, USA). The myocytes were stained with Mito-tracker dye (Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 30 min and mitochondrial density was determined using optical microscopy (Eclipse TE 2000-U; Nikon, Tokyo, Japan). The density of mitochondria was quantified using Image J software (NIH, Bethesda, MD, USA).
Data were expressed as means ± standard deviation (SD). Unpaired Student’s t test was used to statistically compare vehicle- and sudachitin-treated groups. Statistical significance was set a priori at P < 0.05. All analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA).