Sucrose octaacetate

DMEM (Dulbecco’s Modified Eagle Medium), FBS (fetal bovine serum), Collagenase Type I were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Restriction enzyme BglII and HindIII were bought from New England Biolabs (Ipswich, MA, USA). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA, USA). Antibodies for C/EBPα, PPARγ and FABP4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for HSL, p-HSL, ERK, p-ERK, S6 and p-S6 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Methylisobutylxanthine, dexamethasone, Insulin, Oil Red O, Quinine hydrochloride dehydrate, Caffeine, Salicin, 6-propyl-2-thiouracil (PROP), Sucrose octaacetate, Hesperidin and Sodium Benzoate were bought from Sigma-Aldrich Co. (St. Louis, MO, USA). All tested bitter compounds were selected based on common knowledge and previous publications mentioned above. The bitter agonists were either dissolved in DPBS or DMSO, and final DMSO concentration did not exceed 0.1% (v/v) to avoid toxic effect on cells.

Subjects from both experimental groups received a 500 ml high‐dose protein–carbohydrate drink (Table 2) 30 min after each exercise session. In addition, immediately following each session and 30 min before sleep, KE subjects received 25 g of ketone ester [96% (R)‐3‐hydroxybutyl (R)‐3‐hydroxybutyrate] to elevate post‐exercise circulating plasma ketone concentrations, as previously shown by our lab (Vandoorne et al. 2017). The ketone ester supplements were purchased from TdeltaS Ltd (Thame, UK). Subjects in CON received an isocaloric drink (CON) containing 16.4 g pure medium‐chain triglycerides (Now Foods, Bloomingdale, IL, USA). To equalize the taste and appearance of CON and KE drinks, 1 mm bitter sucrose octaacetate (Sigma‐Aldrich, Bornem, Belgium) was added to the CON, whilst a red colorant (AVEVE Bloem, Merksem, Belgium) was added to both the drinks to obtain similar appearance. Subjects received 50 ml of diet coke for mouth rinsing immediately following the KE or CON drink to improve palatability (Leckey et al. 2017). Capillary blood samples from the earlobe were obtained before and after exercise and 30 min following ingestion of the supplements to assess circulating blood β‐hydroxybutyrate concentrations (Glucomen Lx plus‐meter with Lx β‐ketone strips, Menarini Diagnostics, Firenze, Italy) during IMT sessions on days 6, 13 and 20 of the training period.

Caffeine, (+)-catechin, (-)-epicatechin, naringin, sucrose octaacetate, L-tryptophan, L-phenylalanine, magnesium sulfate heptahydrate, sodium bicarbonate, quinine monohydrochloride dihydrate and methanol were purchased in food grade or primary reference standard quality from Sigma-Aldrich (St. Louis, MO). Urea was purchased from Spectrum Chemical (Gardena, CA). sodium bicarbonate was purchased from Fisher Chemical (Fair Lawn, NJ). Cyclo(-leu-pro), cyclo(-pro-val), cyclo(-phe-pro) were synthetized by Bachem Americas (Torrance, CA) in high purity (>99%) and further underwent Solid Phase Extraction (SPE) to ensure high purity and removal of any residual solvent. Briefly, 0.6 g of material were dissolved in 200 mL of 90/10 water/methanol and loaded onto a 6g/35cc HLB Prime cartridge (Waters, Milford, MA). The cartridge was further washed with 60 mL of 95/5 water/methanol and elution was carried out with 60 mL of 95/5 water/methanol. Sample was freed from solvent and freeze-dried twice to ensure safety for consumption.