Bioenergetic profiling in the skin

The skin is a large organ which presents important thermoregulatory 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 studies in these tissues, added to the lack of published protocols. Furthermore, 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 different 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. We established techniques to study skin mitochondrial bioenergetics in isolated organelles and in situ in different cell types, producing a bioenergetic profile of this tissue. These methods can also be easily adapted to human skin with evident clinical relevance (S1). Techniques used are described in detail in the Supporting information. 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. The dermis and epidermis of mouse back skins were separated by scraping, and the two tissues were cultured separately. The epidermal fraction consisted mainly of keratinocytes (89 ± 2.2%) and the dermal fraction of fibroblasts (88 ± 1.6%, Table S1). OCR and extracellular acidification rates (ECAR) were determined using an XF24 extracellular flux analyser (Seahorse Bioscience, North Billerica, USA). By adding trypsin digestion and fur-filtering steps to standard differential 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 substrates (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 respiration in the presence and absence of ATP synthesis 7. Figure 1c and d indicates that skin mitochondria are highly coupled, presenting a large increase in OCR when ADP is added, in a manner inhibited by oligomycin, an ATP synthase inhibitor. The respiratory control ratio was on average 4.39, a value similar to that obtained in preparations from mesenchymal tissues 7 and indicating that integrity was maintained. The addition of the protonophore 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. Although it is useful to study many mitochondrial characteristics, isolation of this organelle from the whole skin does not adequately assess differences that may exist between the tissues that the skin is composed of. Thus, we also measured mitochondrial bioenergetics in intact cells isolated from the epidermis and dermis using extracellular flux measurements 8. These primary cultures were composed mainly of fibroblasts and keratinocytes, with a small contingent of other cell types (Table S1). Figure 2a shows typical in situ bioenergetic measurements. The basal OCR, which represents O2 consumption under physiological conditions, was substantially higher in the dermis (Fig. 2b), indicating that these cells present a more respiratory phenotype than epidermal cultures. After oligomycin was added, a decrease in basal respiration proportional to ATP-generating mitochondrial activity is observed (Fig. 2a). Dermal cells displayed higher ATP production-dependent OCR (Fig. 2c), suggesting that epidermal cells rely more on glycolysis. Extracellular acidification (ECAR) was significantly higher in the epidermis than in the dermis (Fig. 2h), corroborating the suggestion that the epidermis has higher glycolytic activity than the dermis. CCCP-stimulated maximal respiration is higher in the dermis (Fig. 2d), but no significant changes were evident in the reserve capacity, or the difference between basal and maximal respiration (Fig. 2e). This leads to the speculation that, when challenged, both tissues are equally capable of responding with increased respiration to compensate for a higher ATP demand 8. The proton leak across the inner membrane, estimated in the presence of oligomycin, was significantly increased in the epidermis (Fig. 2f). To determine the extent of non-mitochondrial oxygen consumption, the respiratory chain was inhibited with antimycin A and rotenone. This non-mitochondrial OCR was subtracted from all rates calculated previously, is linked to the activity of non-mitochondrial oxidases present in the cell and was increased in the dermis (Fig. 2g). Overall, our data show that it is possible to reliably measure mitochondrial characteristics as isolated organelles and in situ, in both the dermis and epidermis, which present distinctive bioenergetic profiles. The same experimental approaches can be adapted for human skin explants and help elucidate metabolic alterations associated with clinical conditions of the skin (S1). The authors are in debt to Silvania M.P. Neves, Renata S. Fontes, Flavia M.P. Ong and Maria de Fátima Rodrigues for animal care; and Camille C. Caldeira da Silva and Edison Alves Gomes for technical support. This work was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 10/51906-1, 13/04871-6, 14/17270-3), Centro de Pesquisa, Inovação e Difusão de Processos Redox em Biomedicina (13/07937-8), Núcleo de Pesquisa em Processos Redox em Biomedicina (NAP-Rexodoma), Instituto Nacional de Ciência e Technologia de Processos Redox em Biomedicina (INCT Redoxoma) and Conselho Nacional de Pesquisa e Desenvolvimento (CNPq, 153560/2011-8, 302898/2013-1). MFF and AJK designed the research. MFF, BC and JP helped in data acquisition, analysis and interpretation. MFF, BC, JP and AJK participated in critical revision and approval of the final manuscript version. The authors have declared no conflicting interests. Appendix S1. Methods. Table S1. Immunophenotypic characterization of primary epidermal and dermal cultures. Data S1. Supplementary References. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.