Bioenergetic profiling in the skin Maria Fernanda Forni, Bruno Chausse, Julia Peloggia and Alicia J. Kowaltowski Departamento de Bioqu  ımica, Instituto de Qu  ımica, Universidade de S~ ao Paulo, S~ ao Paulo, SP, Brazil Correspondence: Alicia J. Kowaltowski, Departamento de Bioqu  ımica, Instituto de Qu  ımica, Universidade de S~ ao Paulo, Avenida Prof. Lineu Prestes, 748 sala 1065, bloco 10 superior, 05508-900 S~ ao Paulo, SP, Brazil, Tel.: +55 11 30912922, Fax: +55 11 38155579, e-mail: alicia@iq.usp.br Key words: bioenergetic profiling – dermis – epidermis – mitochondrial isolation – respiratory rates Accepted for publication 2 September 2015 Background The skin is a large organ which presents important thermoregula- tory and metabolic functions (1,2) and should thus be a focus of studies involving energy metabolism. Nevertheless, bioenergetic studies in epithelia-containing organs such as the skin are rare (3,4), probably due to difficulties in organelle isolation (5) or in situ stud- ies in these tissues, added to the lack of published protocols. Fur- thermore, the skin is subdivided into two tissues: the dermis and the epidermis, as is typical in the structure of composite organs with an epithelial tissue on top of a mesenchymal layer. Results obtained with whole-organ homogenates in composite tissues such as the skin tend to be the average of the individual responses of the differ- ent types of cells resident in the tissue (6). It is thus also important to establish techniques that allow for measurements of bioenergetic characteristics in different tissues of the skin. Questions addressed We established techniques to study skin mitochondrial bioenerget- ics in isolated organelles and in situ in different cell types, produc- ing a bioenergetic profile of this tissue. These methods can also be easily adapted to human skin with evident clinical relevance (S1). Experimental design Techniques used are described in detail in the Supporting infor- mation. Mitochondrial isolation and oxygen consumption Mitochondria were isolated from mouse skin samples using an adaptation of standard differential centrifugation methods. Oxygen concentrations and consumption rates (OCR) were measured using Oroboros high-resolution respirometry (3). Cell isolation, culture and oxygen consumption The dermis and epidermis of mouse back skins were separated by scraping, and the two tissues were cultured separately. The epider- mal fraction consisted mainly of keratinocytes (89 Æ 2.2%) and the dermal fraction of fibroblasts (88 Æ 1.6%, Table S1). OCR and extracellular acidification rates (ECAR) were determined using an XF24 extracellular flux analyser (Seahorse Bioscience, North Billerica, USA). Results By adding trypsin digestion and fur-filtering steps to standard dif- ferential centrifugation protocols, we were able to isolate highly functional mitochondria from murine whole-skin samples. Figure 1 shows a typical oxygen tension trace (Panel a) and quantified ADP- maximized OCR (Panel b) supported by different respiratory sub- strates (NADH-linked pyruvate and malate, Complex II electron donor succinate and Complex IV electron donor TMPD). The relative contribution of each respiratory complex was uncovered using specific inhibitors: rotenone for Complex I and antimycin A for Complex III. Skin mitochondria respire well with either pyruvate plus malate or succinate as substrates. To assess the quality of the preparations, we measured respira- tion in the presence and absence of ATP synthesis (7). Figure 1c and d indicates that skin mitochondria are highly coupled, pre- senting a large increase in OCR when ADP is added, in a manner inhibited by oligomycin, an ATP synthase inhibitor. The respira- tory control ratio was on average 4.39, a value similar to that obtained in preparations from mesenchymal tissues (7) and indi- cating that integrity was maintained. The addition of the protono- phore CCCP stimulated the oligomycin-inhibited OCR, further indicating that these mitochondria were fully coupled. Overall, we find that the method described provides adequate quantities of high-quality mitochondria. 0 3 6 9 12 150 200 250 300 Pyruvate + Malate Rotenone Succinate Antimycin A TMPD + Ascorbate (a) Time (min) O 2 (nmol/ml) OCR (nmol/min/mg protein) OCR (nmol/min/mg protein) Pyr/Mal + Rot + Suc + AA +TMPD/Asc 0 30 60 90 120 (b) 0 2 4 6 8 120 140 160 180 ADP Oligomycin CCCP Pyruvate + Malate (c) Time (min) O 2 (nmol/ml) Baseline + ADP + Oligomycin + CCCP 0 30 60 90 (d) Figure 1. Isolated skin mitochondrial respiration. Mitochondria (0.25 mg/ml) were incubated as described in the Appendix S1, and oxygen tensions were followed over time. (a) Oxygen consumption maximized by ADP. Pyruvate plus malate, rotenone, succinate, antimycin A and TMPD plus ascorbate were added where indicated. (b) OCR from experiments such as those depicted in Panel (a). (c) Oxygen consumption measured in different respiratory states. Pyruvate and malate, ADP, oligomycin and CCCP were added where indicated. (d) Quantification of experiments such as those in Panel (c). ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2016, 25, 147–148 147 DOI: 10.1111/exd.12856 www.wileyonlinelibrary.com/journal/EXD Methods Letter to the Editor