Orlistat

TBS consists of C. rotundus L., C. unshiu Markovich, and Poria cocos. The three herbs were purchased from Nanum Pharmaceutical Company (Seoul, Republic of Korea). The herbal samples were performed for sensory test according to ‘The Korean Herbal Pharmacopoeia’ by Prof. Yun-Yeop Cha, and only those that passed the Korean Pharmacopoeia standard were selected and used for this experiment. TBS was made using a 1: 1: 1 ratio of these herbs (400 g each). The herbs were then extracted in water at 99 °C for 3 h. The extract was freeze-dried, and the yield rate was calculated at 33.20% (33.20 g per 100 g of liquid extract). The powder was dissolved in distilled water for this experiment, and the residual powder was stored at − 20 °C. The 30% HFD was obtained from Research Diets (New Brunswick, NJ, USA). The p-AMPK and AMPK antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). PPARγ, C/EBPα, SREBP1, AMPK, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) oligonucleotide primers were purchased from Bioneer Corporation (Daejeon, Republic of Korea), and SYBR Premix Ex Taq was purchased from Takara Bio Inc. (Otsu, Japan). Orlistat was purchased Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan) and other reagents were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA).

A chemical library of 658-natural compounds was kindly provided by Dr. Sang Jeon Chung of Sungkyunkwan University (Suwon, Korea). Kaempferide (69545), dimethylsulfoxide (D2650), bafilomycin A1 (B1793), rapamycin (553210), tiliroside (79257), chloroquine (C6628), orlistat (O4139), palmitic acid (P5585), oleic acid (O1383), acridine orange (A6014), oil-red-O (O0625), dexamethasone (D8893), insulin (I0516), and 3-isobutyl-1-methylxanthine (I5879) were purchased from Sigma-Aldrich. BODIPY 493/503 (D3922), Hoechst33342 (H3570), lipofectamine LTX (94756), lipofectamine 2000 (52887), Plus reagent (10964), protease and phosphatase inhibitor solution (78441), M-PER kit (89842Y), DMEM, fetal bovine serum (FBS), bovine serum, and antibiotics were purchased from Invitrogen ThermoFisher Scientific. For in vivo experiments, Kaempferide (K0057) was purchased from TCI Chemicals. siRNA targeting TUFM was purchased from Dharmacon. mRFP-GFP-LC3B plasmids were kindly provided by Dr. Jaewhan Song of Yonsei University (Seoul, Korea).

CCA quantification of Drosophila homogenates was essentially done as described in [22] (link). If not described differently eight flies per replicate were homogenized in a 2 ml screwcap tube containing 1 ml 0.05% Tween-20 and a ceramic cylinder using a peqlab Precellys 24 instrument (10 sec at 5000 rpm). Homogenates were heat-inactivated (5 min at 70°C) and debris pelleted in a Beckmann GS6KR centrifuge (3 min at 3500 rpm). Of the supernatants 50 µl samples were transferred to a 96 well microtiter plate and homogenate (blank) absorbance was measured at 540 nm in a Biorad Benchmark Microplate Reader. Prewarmed Triglyceride solution (200 µl; Thermo Fisher Scientific #981786) was added to each homogenate sample and incubated at 37°C with mild shaking for 30–35 min. Total absorbance at 540 nm was measured and corrected by subtraction of blank and substrate absorbance prior to triglyceride equivalent content calculation using 0–40 µg of triolein (Sigma T7140) as TAG standard, which was treated like the samples.
For experiments with inactive CCA reagent shown in Fig. 1C the Triglyceride solution was heat-inactivated (5 min at 96°C) or incubated with 200 µM of the lipase inhibitor Orlistat (Sigma O4139) prior to use.
For homogenate absorbance determination prior to CCA assay (Fig. 2A), the 540 nm absorbance of 250 µl 0.05% Tween-20 was subtracted as blank value. Homogenate absorbance values were calculated per mg fly wet weight.
For experiments shown in Fig. 2B, 16 flies per replicate were homogenized in 1 ml 0,05% Tween-20. Homogenate supernatants (150 µl) were added to equal volumes of 0.05% Tween-20 containing increasing amounts of triolein and treated once more in the peqlab Precellys 24 instrument as described. Aliquots (50 µl) of the resulting homogenate samples were subjected to CCA measurement as described.
Shown are representative experiments with average values of triplicate measurements and corresponding standard deviations. Experiments were repeated at least twice.
For fly free glycerol content determination eight male flies were homogenized in 0.5 ml 0.05% Tween-20 as described above. Free glycerol content of 50 µl homogenate supernatants was determined with the Free Glycerol Reagent (Sigma F6428) using 0–50 µg triolein equivalents (Glycerol Standard Solution, Sigma G7793) as standard. Total free glycerol and glyceride content was determined by diluting 25 µl of the aforementioned homogenate with 25 µl 0.05% Tween-20 before using the Free Glycerol Reagent combined with the Triglyceride Reagent (Sigma T2449+F6428) using 0–40 µg triolein as standard. Free glycerol content and total free glycerol+glyceride content both expressed as µg triolein equivalent/mg fly wet weight were calculated as described above.
Shown are average values of triplicate measurements of three independent experiments.