MBKO mice were generated by crossing BDNF Flox/Flox mice (catalog no. 004339; the Jackson Laboratory, Bar Harbor, ME) with transgenic mice carrying the human α-skeletal actin promoter–driven Cre (catalog no. 006149; the Jackson Laboratory, USA). Because of the sex-specific response in obesity development, all in vivo experiments were performed using female mice. Genotyping was performed via PCR using genomic DNA extracted from the tail and using primers suggested by the Jackson Laboratory. Mice were housed in environmentally controlled conditions under a 12-hour light/dark cycle with ad libitum access to standard rodent pellet food and water. All in vivo assays were performed using 8-week-old female mice because of the sex-dimorphic effect and were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center and Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong.

Blood glucose levels were measured using the ACCU-CHEK Advantage Blood Glucose Meter (Roche, Basel, Switzerland). Serum insulin, FGF21, and BDNF were measured by enzyme-linked immunosorbent assay (ELISA) (Crystal Chem, Elk Grove Village, IL; Abcam; and Abnova, Taipei, Taiwan, respectively). Serum TG and free FA levels were measured using the Triglyceride Quantification Colorimetric Kit and the Free Fatty Acid Quantification Colorimetric Kit, respectively (BioVision, Milpitas, CA). Alanine and β-hydroxybutyrate serum concentrations and CK activities were detected using commercially available kits (Cayman Chemical, Ann Arbor, MI). Hepatic glycogen content was determined by the Glycogen Assay Kit (Cayman Chemical). The glucose tolerance test (GTT) was performed after intraperitoneally injecting d-glucose (2 g/kg of body weight) into female mice that had fasted overnight (16 hours).

Tissue CREB activities were determined by CREB TFact DNA-binding ELISA (Assay Biotechnology Company Inc., Fremont, CA). Muscle glycogen and ATP levels were measured enzymatically using commercially available kits (Cayman Chemical). AMP concentration determination and profiling of various lipid and glucose metabolites were performed at the West Coast Metabolomics Center at the University of California Davis Genome Center (USA) using liquid chromatography–mass spectrometry (LC-MS). LC/quadrupole time-of-flight (Q-TOF) MS analyses were performed using an Agilent 1290 Infinity LC system (Santa Clara, CA) coupled to either an Agilent 6530 (positive ion mode) or an Agilent 6550 mass spectrometer equipped with an ion funnel (iFunnel; negative ion mode). Lipids were separated on an Acquity UPLC CSH C18 column (Waters, Milford, MA) maintained at 65°C at a flow rate of 0.6 ml/min. The mobile phases consisted of 60:40 acetonitrile:water with 10 mM ammonium formate and 0.1% formic acid (A) and 90:10 propan-2-ol:acetonitrile with 10 mM ammonium formate and 0.1% formic acid. The gradient was as follows: 0 min, 85% (A); 0 to 2 min, 70% (A); 2 to 2.5 min, 52% (A); 2.5 to 11 min, 18% (A); 11 to 11.5 min, 1% (A); 11.5 to 12 min, 1% (A); 12 to 12.1 min, 85% (A); and 12.1 to 15 min, 85% (A). A sample volume of 3 μl was used for the injection and sample temperature was maintained at 4°C in the autosampler. The Q-TOF MS were operated with electrospray ionization (ESI) performing full scans in the mass range m/z from 65 to 1700 in positive (Agilent 6530, equipped with a JetStreamSource) and negative (Agilent 6550, equipped with a dual JetStream Source) modes producing both unique and complementary spectra. Instrument parameters were as follows (positive mode): gas temperature, 325°C; gas flow, 8 liter/min; nebulizer, 34264.5 Pa; sheath gas, 350°C; sheath gas flow, 11; capillary voltage, 3500 V; nozzle voltage, 1000 V; fragmentor, 120 V; and skimmer, 65 V. Data (both profile and centroid) were collected at a rate of 2 scans/s. In negative ion mode, the parameters were identical to positive ion mode except for the following: gas temperature, 200°C; gas flow, 14 liter/min; and fragmentor, 175 V. For 6530 Q-TOF, a reference solution generating ions of 121.050 and 922.007 m/z in positive mode and 119.036 and 966.0007 m/z in negative mode was used for continuous mass correction. For 6550 Q-TOF, the reference solution was introduced by a dual-spray ESI, with the same ions and continuous mass correction.

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