Izumi-Mishima_Cell metabolism_2023

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This data sets are manuscript of the cell metabolim.

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Data were collected by the following methods. Acute cold tolerance trest: Core body temperature was measured with a Homeothermic Monitor by gentle insertion of a thermal probe into the rectum of the mouse. Oxygen consumption and RER measurement: Analysis of respiratory gases was performed with a mass spectrometer (ARCO-2000, ARCO System). Locomotor activity recording: The activity level of mice was assessed with an infrared activity monitor (ACTIMO-100N, Shin Factory). A cage containing one mouse was placed inside the activity monitor with infrared beams at 20-mm intervals. Mouse movements were counted every 0.5 s for 24 h. RNA isolation and RT and real-time PCR analysis: Total RNA was extracted from mouse tissues and cells with the use of RNAiso (#9109, Takara Bio) and subjected to RT with the use of a TaKaRa PrimeScript II 1st Stand cDNA Synthesis Kit (Takara Bio). The resulting cDNA was subjected to real-time PCR analysis with Fast SYBR Green Master Mix (3485612, Applied Biosystems) in a Step One Plus Real-Time PCR System (Applied Biosystems). The abundance of target mRNAs was normalized by that of Gapdh mRNA. Protein extraction and immunoblot analysis: Band intensity was quantified with ImageJ software. Metabolomics analysis: MassHunter software for TOF/MS (Agilent Technologies) was used for CE-TOF/MS system control and data acquisition. All target metabolites were identified by comparison of their m/z values and migration times with the normalized m/z values and migration times of the corresponding authentic standards. Measurement of [3H]leucine uptake: Leucine uptake in each organ was quantified by measurement of radioactivity with a scintillation counter (LSC-7400, Hitachi). Hepatic glycogen assay: The liver homogenate was centrifuged at 14,000 × g for 5 min at 4°C, and the resulting supernatant was assayed for glycogen with an EnzyChrom Glycogen Assay Kit (BioAssay Systems). ELISAs: Serum IL-6 and TNF- concentrations were determined with ELISA kits (Legend Max Mouse IL-6 ELISA Kit, BioLegend, and Mouse TNF Alpha Uncoated ELISA, Invitrogen, respectively). Serum noradrenaline was extracted with the use of a cis-diol–specific affinity gel, acylated, enzymatically converted, and then measured with an ELISA kit (Noradrenaline Research ELISA, ImmuSmol). Serum corticosterone was determined with a Corticosterone ELISA Kit (Enzo Life Sciences). Data are presented as means ± SEM unless indicated otherwise and were analyzed with statistical software (JMP or Statcel–The Useful Add-in Forms on Excel 4th ed.). Comparisons between two groups were performed with the paired or unpaired t test, as appropriate. Those among more than two groups were performed with Dunnett's test, by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test, or by two-way ANOVA followed by Tukey's post hoc test or the post hoc paired/unpaired t test with Bonferroni’s correction.

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