UCP1 in Sebaceous Glands Corresponds with Increased Antioxidant Potential and Not Brown Adipose Tissue Function

The rodent tail is a thermoregulatory organ. Further the tail is highly sympathetically innervated and when trunk temperatures dip below thermoneutrality there is vasoconstriction in the tail 1. Heat loss occurs most frequently at the base of the tail, close to the rump 2; therefore, blood temperatures circulating in the tip of the tail are the coldest in the body 3, 4. Considering increased sympathetic tone and the relatively colder environment seen in the rodent tail tip, it is reasonable to suggest that uncoupling protein 1 (UCP1), a major mediator of thermoregulation 5, would also be expressed. Regions of UCP1 expression have previously been identified in the skin of humans and include the epidermis, sweat glands and sebaceous glands and suggest the possibility of similar properties within the skin of the rodent tail 6. UCP1 activity in rodent sebaceous glands has yet to be identified. From the background work and conclusions of Mori et al. 6, we hypothesized that UCP1-positive regions within the tail would have properties similar to brown adipose tissue (BAT) and contribute to thermoregulation. For the purpose of this study, 3-month-old male C57BL/6J mice were used to investigate the properties of sebaceous gland UCP1. The distal 6 mm of tail was removed from euthanized mice and immediately frozen for gene expression or immunofluorescence assessment. BAT from the same mice was also excised to compare gene expression patterns of thermogenic genes, uncoupling proteins and antioxidants to the tail by quantitative RT-PCR. Additional assessments compared the gene expression and presence of UCP1 between 3-month-old and aged (>12 months old) mice. Lastly, regional sections of the tail were processed to determine whether the known regional temperature differences within the tail produced variances in the quantity of UCP1 protein and RNA. Detailed information of the experimental procedures can be found within the Supporting Information section. Considering that the tail expressed well-described markers of BAT (see Figures S1 and S2), transcript expression in tail samples was compared to that of scapular BAT. Transcript was normalized to the levels of the general lipid marker fatty acid binding protein 4 (Fabp4). The normalization to Fabp4 permitted us to explore the relative gene expression within a lipid-rich cell type inclusive of adipocytes and sebocytes. As UCP1 was not ubiquitously expressed within the tail section (Figure S2), normalization to a lipid marker permits a more focused measurement of UCP1-rich cells in a mixed-cell-type tissue such as the tail. While the expression of Ucp1 was not significantly different between the tail and BAT, the expression of Ucps 2-5 was up to 1000-fold higher within the tail section relative to BAT (Fig. 1a). UCPs have been hypothesized to play a role in many diseases including both forms of diabetes and Parkinson's disease 7, 8. The protective effects of UCPs in these diseases are attributed to their antioxidant properties in the face of elevated mitochondrial superoxide concentrations. The increased presence of UCP1, in addition to the other UCPs, results in a reduced membrane potential thereby reducing reactive oxygen species formation as part of a negative feedback mechanism to reduce oxidative stress 8. As the UCPs were elevated within the tail section relative to BAT, we explored antioxidant gene expression between the tissues. Similarly, up to 100-fold increases in the antioxidant genes glutathione peroxidase 1 (Gpx1) and superoxide dismutase (Sod1 and Sod2) were found in the tail section relative to BAT (Fig. 1b). Together with the expression patterns of the Ucps, our data indicate that sebaceous glands, which express relatively high amounts of Fabp4, have an antioxidant potential that varies greatly from BAT. Regional temperatures vary widely in the tail, with the temperatures decreasing dramatically from the proximal to the distal end of the tail 4. Redfern et al. found that skin tail temperature proximal to the body started at 35°C, was reduced to 26°C towards the middle region, and dropped to 18°C at the tip. Because UCP1 is a thermogenic protein responsive to temperature fluctuations in BAT, we assayed differences in UCPs among these three distinct temperature zones of the tail. No significant differences in Ucp1 expression or protein staining intensity were measured among the tip, middle and proximal tail regions of 3-month-old mice (Fig. 2a). Intriguingly, the tip did express increased amounts of the antioxidants Ucp2, Gpx1 and Sod1 compared to the other regions. As the amount of sebocytes was controlled for by the use of Fabp4, the increased antioxidant expression in the tip may be due to differences within the hair cycle or for protection from external damage and renders further investigation. Despite this, the lack of a difference in the thermogenic UCP1 protein and RNA within the regions indicates that UCP1 within the sebocyte population is irresponsive to regional temperature variations in the tail. Lastly, because the amount of BAT and UCP1 activity is greatly reduced or even lost during ageing 5 and this same ageing pattern could be recapitulated in tail, Ucp1 expression and protein localization were compared in tails among young (3 months old) and aged (12 months old) mice. Age had no effect on the expression of the well-characterized thermogenic genes Ucp1 or Prdm16 between mice (Figure S3a). In addition, neither the immunofluorescence intensity nor the location of UCP1 protein differed notably in the terminal tail region between young and aged mice (Figure S3b). We extended the results by Mori et al. 6 showing that Ucp1 is expressed in various locations in the tail sebaceous glands. Our data, however, do not indicate that the role of sebaceous gland UCP1 is one of thermoregulation. Further, we provide evidence that UCPs in the sebaceous gland serve an alternative antioxidant role as described in other organs 8. Miller performed the research, designed the study, analysed the data and wrote the manuscript. Yu, Amabti, McKinney and Avra performed the research and analysed associated data. Baile and Meagher participated in the study design and contributed essential reagents and equipment. All of the present authors participated in the drafting and revision of the manuscript and have approved all versions of the manuscript and its associated documents prior to submission. This research was supported by the Atlanta Chapter of the Achievement Awards for College Scientists (CNM) and by the National Institute of Diabetes and Digestive and Kidney Diseases (#DK090300) (RBM). Additional support was provided from the Georgia Research Alliance Eminent Scholar Fund and the UGA Research Foundation (CAB). The authors declare no conflicting interests. Data S1. Materials and Methods. Data S2. Supplementary References. Table S1. Primer sequences used for gene expression assays. 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.