Glucose solution

The mice underwent a 6-hour fasting period (from 7 am to 1 pm) for both the GTT and ITT. Blood glucose levels were assessed using the AlphaTRAK glucometer and test strips. Following the initial blood glucose measurement, the GTT was initiated by an i.p. injection of a 10% glucose solution (1 g/kg; MilliporeSigma), and blood glucose levels were monitored at set time points over the next 2 hours. The ITT was initiated by an i.p. injection of human insulin (0.75 U/kg; MilliporeSigma), and blood glucose levels were measured at designated time points over the subsequent 2 hours. Magnetic resonance spectroscopy (MRS) was conducted on live mice using EchoMRI Body Composition Analyzers with default settings. A cold tolerance test was performed as previously described (43 (link)). In brief, internal body temperatures of the mice were tracked by implanting a temperature transponder (IPTT-300, Bio Medic Data Systems) under the skin and recording temperatures with a portable reader (DAS-8007-IUS, Bio Medic Data Systems). To subject the mice to acute cold exposure, they were individually housed in 6°C cold chambers without access to food, and body temperature was assessed at specified time points. Metabolic cage measurements were performed using the TSE PhenoMaster system. Mice were acclimatized for 5 days in metabolic cages before the actual measurement. For the pair-feeding experiment, WT mouse (pair-fed group) was offered the amount of HFD eaten by the Gpr75^–/– mouse (comparison group) on the previous day, beginning at 6 weeks of age.

Glucose tolerance tests (GTTs) were carried out on each animal during BL and at the end of the study (~11 weeks). GTT was carried out in a fasted state (9 h). Blood glucose was measured from tail blood using the OneTouch® Ultra® 2 glucose monitor (Johnson and Johnson, New Jersey, USA). Each mouse received an intraperitoneal injection of 2% glucose solution (Sigma, UK) equivalent to 2 mg/g body weight. Blood was sampled at exactly 15, 30, 60, and 120 min after injection, and blood glucose levels (mmol/l) were recorded.

OCR was measured using an XFe96 Extracellular Flux Analyzer (Seahorse Biosciences, Inc., Billerica, MA, USA). The cells were seeded in 6-well plates at a density of 1.4 × 10⁵ cells/well. On day 3, the cells were washed and treated with ICM diluted in serum-free media with DCA or GM10395 for 24 h. Next, cells were transferred into the Xfe96 cell culture plate (Agilent Technologies, Santa Clara, CA, USA, 103794-100) with assay medium at a density of 1.0 × 10⁴ cells/well, and the plate was incubated in 5% CO₂ at 37 °C for 5 h. The OCR assay medium consisted of XF DMEM medium pH 7.4 (Agilent Technologies, 102353) supplemented with 25 mM Glucose Solution (Sigma-Aldrich, G7528), 1 mM Pyruvate Solution (Sigma-Aldrich, S8636), and 2 mM Glutamine Solution (Thermo Fisher Scientific, 35050061). The ECAR assay medium consisted of XF DMEM medium pH 7.4 supplemented with 2 mM Glutamine Solution. Uncouplers and inhibitors were used at the indicated concentrations: oligomycin A (1 μM, Sigma-Aldrich, 75351), FCCP (carbonyl cyanide 4-(trifluoro-methoxy)phenylhydrazone, 1 μM, Sigma-Aldrich, C2920), rotenone (1 μM, Sigma-Aldrich, R8875), 2-DG (50 mM, Sigma-Aldrich, D6134) and Glucose (10 mM, Sigma-Aldrich, G7528). The nuclei were stained with DAPI solution (1 μg/mL, Thermo Fisher Scientific, 62248) and counted automatically using an ImageXpress Micro Confocal Microscope (Molecular Devices, San Jose, CA, USA) to normalize by cell number.

At 8 and 24 weeks into the dietary intervention, mice were fasted for 12 h before receiving an intraperitoneal injection of 20% glucose solution dosed at 2 g/kg body weight (Sigma, St. Louis, MO, USA). Blood samples were taken from tail veins immediately before (0 min) and at 5, 15, 30, 60, 90, and 120 min after glucose injection. Blood glucose levels were measured with an AimStrip Plus glucometer (Cat.#. 37,321, Germaine Laboratories, San Antonio, TX, USA).

Collagen type I obtained from rat tail tendon—RTC (2 mg/mL)—was pre-glycated by incubation with 500 mM of glucose solution (Merck, Rahway, NJ, USA) in PBS at pH 7.4, containing 0.02% NaN₃ for 1 and 5 days at 37 °C as described elsewhere [56 (link)]. The samples were dialyzed versus 0.05 M acetic acid before being designed as RTC GL1 and RTC GL5, respectively, and stored at 4 °C until use.