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A protocol for the isolation and cultivation of brown bear (Ursus arctos) adipocytes

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Abstract

Brown bears (Ursus arctos) exhibit hyperphagia each fall and can become obese in preparation for hibernation. They do this without displaying the physiological problems typically seen in obese humans, such as Type 2 diabetes and heart disease. The study of brown bear hibernation biology could therefore aid in the development of novel methods for combating metabolic diseases. To this end, we isolated mesenchymal stem cells from subcutaneous fat biopsies, and culture methods were developed to differentiate these into the adipogenic lineage. Biopsies were taken from 8 captive male (N = 6) and female (N = 2) brown bears, ages 2–12 years. Plastic adherent, fibroblast-like cells were proliferated and subsequently cryopreserved or differentiated. Differentiation conditions were optimized with respect to fetal bovine serum content and time spent in differentiation medium. Cultures were characterized through immunostaining, RT-qPCR, and Oil red O staining to quantify lipid accumulation. Adiponectin, leptin, and glycerol medium concentrations were also determined over the course of differentiation. The culturing protocol succeeded in generating hormone-sensitive lipase-expressing, lipid-producing white-type adipocytes (UCP1 negative). Serum concentration and time of exposure to differentiation medium were both positively related to lipid production. Cells cultured to low passage numbers retained similar lipid production and expression of lipid markers PLIN2 and FABP4. Ultimately, the protocols described here may be useful to biologists in the field investigating the health of wild bear populations and could potentially increase our understanding of metabolic disorders in humans.

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Acknowledgments

Funding was provided by Amgen Inc., the Interagency Grizzly Bear Committee, the Raili Korkka Brown Bear Endowment, the Bear Research and Conservation Endowment, and a National Science Foundation Graduate Research Fellowship (KSR, 1347943). We thank the scientists at Washington State University’s Department of Integrative Physiology and Neuroscience including Jamie Gaber and Marina Savenkova for their mentorship and technical expertise. We also thank Danielle Rivet, Joy Erlenbach, and the other dedicated researchers at WSU’s Bear Research, Education, and Conservation Center for their assistance in data collection and captive bear care.

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    1. School of the Environment and School of Biological Sciences, Washington State University, Pullman, WA, 99164, USA

      J. L. Gehring, K. S. Rigano & C. T. Robbins

    2. Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, WA, 99164, USA

      B. D. Evans Hutzenbiler & H. T. Jansen

    3. Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA, 99164, USA

      O. L. Nelson

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    1. J. L. Gehring
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    Correspondence to J. L. Gehring.

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    J. L. Gehring and K. S. Rigano have contributed equally to this work.

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    Gehring, J.L., Rigano, K.S., Evans Hutzenbiler, B.D. et al. A protocol for the isolation and cultivation of brown bear (Ursus arctos) adipocytes. Cytotechnology 68, 2177–2191 (2016). https://doi.org/10.1007/s10616-015-9937-y

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