Izumi-Mishima_Cell metabolism_2023

Published: 25 July 2023| Version 1 | DOI: 10.17632/vsxkjcvxfn.1
Contributors:
Hiroshi Sakaue, Izumi Yuna

Description

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.

Institutions

Tokushima Daigaku

Categories

Cell Metabolism

Funding

Japan Society for the Promotion of Science

21J14844

Japan Society for the Promotion of Science

22H03535

Japan Society for the Promotion of Science

21K11724

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