Thermoneutral housing is a critical factor for immune function and diet-induced obesity in C57BL/6 nude mice

1,4*Kerstin Stemmer, 1Petra Kotzbeck, 1Fabio Zani, 1Michaela Bauer, 1Christina Neff,2Timo D. Müller,3Paul T. Pfluger, 4Randy J. Seeley, 5Senad Divanovic

1 Division of Metabolism and Cancer, Institute for Diabetes and Obesity, Helmholtz Centre Munich, Neuherberg, Germany

2 Division of Molecular Pharmacology, Institute for Diabetes and Obesity, Helmholtz Centre Munich, Neuherberg, Germany

3Division of Nutrition Biology, Institute for Diabetes and Obesity, Helmholtz Centre Munich, Neuherberg, Germany

4Metabolic Diseases Institute, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA

5Division of Immunobiology, Cincinnati Children's Hospital Medical Center, and the University of Cincinnati College of Medicine, Cincinnati, Ohio, USA

* Corresponding Author

Address all correspondence and requests for reprints to:

Kerstin Stemmer, Institute for Diabetes and Obesity, Helmholtz Centre Munich, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.

Email:

Phone: +49-89-3187-2109, Fax: +49-89-3187-2182

Running Head: Dietinduced obesity in nude mice

Conflict of interest: The authors declare no conflict of interest.

Supplemental Methods

Mice

Male homozygous nude mice on a C57BL/6 background (B6.Cg-Foxn1nu/J) and corresponding C57BL/6 wild type (WT) mice were assigned to the following groups, as depicted in Suppl. Table 1:

Mice were acclimatized to thermoneutrality for at least two weeks and ad libitum fed with regular chow diet. Mice were moved to the combined indirect calorimetry system (PhenoMaster, TSE Systems GmbH) and kept at 33°C on low fat diet (LFD (D12450B, Research Diets Inc., New Brunswick, NJ), for a recording time of 4 days before being switched to high fat diet (HFD (D12492, Research Diets Inc., New Brunswick, NJ)). After 3 additional recording days at 33°C and HFD exposure, the temperature was switched to 23°C for the last three days of the experiment. Scales integrated into the sealed cage environment continuously measured cumulative food intake.

Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under animal study proposals approved by the CCHMC or by the University of Cincinnati Institutional Animal Care and Use Committee.

Statistical Analysis

Energy expenditure was analyzed by analysis of covariance (ANCOVA) using body weight as covariate.Statistical differences in body morphometry and age between nude andWT mice were assessed by One-Way ANOVA and Dunnet’s post-hoc test with nude mice as controls, and indicated by asterics, with *p<0.05, #p<0.01, and §p<0.001.

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