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Effects of Cold Exposure Revealed by Global Transcriptomic Analysis In www.nature.com/scientificreports OPEN Efects of cold exposure revealed by global transcriptomic analysis in ferret peripheral blood mononuclear cells Bàrbara Reynés1,2,3, Evert M. van Schothorst 4, Jaap Keijer 4, Andreu Palou1,2,3* & Paula Oliver 1,2,3 Animal studies, mostly performed in rodents, show the benefcial anti-obesity efects of cold studies. This is due to thermogenic activation of brown adipose tissue (BAT), a tissue also recently discovered in adult humans. Studies in humans, however, are hampered by the accessibility of most tissues. In contrast, peripheral blood mononuclear cells (PBMC) are accessible and share the expression profle of diferent sets of genes with other tissues, including those that refect metabolic responses. Ferrets are an animal model physiologically closer to humans than rodents. Here, we investigated the efects on ferrets of one-week acclimation to 4 °C by analysing the PBMC transcriptome. Cold exposure deeply afected PBMC gene expression, producing a widespread down-regulation of genes involved in diferent biological pathways (cell cycle, gene expression regulation/protein synthesis, immune response, signal transduction, and genes related to extracellular matrix/cytoskeleton), while thermogenic and glycogenolysis-related processes were increased. Results obtained in PBMC refected those of adipose tissue, but hardly those of the liver. Our study, using ferret as a model, reinforce PBMC usefulness as sentinel biological material for cold-exposure studies in order to deepen our understanding of the general and specifc pathways afected by cold acclimation. This is relevant for future development of therapies to be used clinically. Te efects of cold exposure on mammals have mainly been studied in terms of its relevance for body temperature and body weight maintenance. It is well established that sympathetic nervous system activation in brown adipose tissue (BAT) by cold exposure increases energy expenditure and induces adaptive thermogenesis in order to preserve body temperature at the expense of mobilisation of fat stores, which helps to maintain body weight1,2. More recently, BAT-thermogenic activation capacity by cold exposure has also been described in humans, which opens new possibilities for protection against obesity3. In addition, cold acclimation has been widely studied in relation to modulation of the infammatory response, and although some research points to an immunosuppres- sive efect, there are contradictory results4,5. Furthermore, the modulatory efect of environmental temperature is more complex, and has an important regulatory role in overall cellular processes6,7. Mechanistic research on the efects of cold exposure mostly examine gene expression responses of selected specifc tissues in animal models, mainly rodents, or cell lines6,8, which makes it difcult to gain a broad idea of the whole metabolic efect of cold on our metabolism. In general, these studies show that low temperature stress induces a slowing in a range of cellular processes, but potentiates pathways involved in heat production9. Peripheral blood mononuclear cells (PBMC), a fraction of blood cells composed of lymphocytes and mono- cytes, constitute an important source of biomarkers of biological efects in internal tissues10. Studies by our group performed in rodents reveal that PBMC are able to refect BAT thermogenic activation as well as the acquisition of brown-like properties in white adipose tissue (browning process) in response to cold exposure, pointing towards the possibility of performing thermogenic studies with minimum invasiveness11. In general, these circulating blood cells express most of the genes encoded by the genome (including tissue-specifc transcripts), and can 1Laboratory of Molecular Biology, Nutrition and Biotechnology (Nutrigenomics and Obesity group), University of the Balearic Islands, Palma, Spain. 2Health Research Institute of the Balearic Islands (IdISBa), Palma, Spain. 3CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Madrid, Spain. 4Human and Animal Physiology, Wageningen University, Wageningen, The Netherlands. *email: [email protected] SCIENTIFIC REPORTS | (2019) 9:19985 | https://doi.org/10.1038/s41598-019-56354-6 1 www.nature.com/scientificreports/ www.nature.com/scientificreports 5 MGA LCOR USP13 DAPK1 ) 4 FAM214B e TULP4 u l a 3 HBM MLLT11 v CTSG - TREML1 p AHSP ( 2 CTS9L g o l - 1 0 -4 -2 0 2 4 Fold Change (2log) Figure 1. Volcano plot of unique genes expressed in PBMC plotted as 2 log fold change of cold over control exposed ferrets versus the P-value. Non-signifcant genes are shown in black, signifcant genes (p < 0.01; Top 50 shown in Table 2) in red. Gene symbols are shown for a subset of signifcant genes on the perimeter. respond to internal and external signals refecting gene expression patterns of several tissues (reviewed by10,12). So, in addition to revealing the thermogenic adaptations to cold exposure, PBMC refect gene expression changes in response to nutritional and pharmacological interventions, and are increasingly used in nutrigenomic and clin- ical research as sentinel biological material to study metabolic responses of other internal tissues that are difcult to obtain10. In this sense, we have demonstrated, in rats and ferrets, that PBMC can even be useful to study hypo- thalamic responses to cold exposure13. Ferrets (Mustela putorius furo) are an alternative animal model to rodents that are closer to humans in various aspects, including adipose tissue organisation and thermogenic and immune response to cold exposure14–17. Recently, we showed in ferrets that an immunosuppressive gene expression pattern was specifcally induced by cold acclimation in perivascular adipose tissue but not in inguinal adipose tissue18. A limited set of infammation markers was also analysed in PBMC and refected the perivascular adipose tissue response18. Given the deleterious impact of infammation on vascular biology, this could be indicative of a cardi- ovascular protective efect18. PBMC, given their particular characteristics, constitute an ideal surrogate tissue to study the global biological and molecular responses to cold exposure in mammals. Taking into consideration that PBMC are immune cells, and since infammation is the basis of several metabolic diseases – such as obesity or cardiovascular disease19 – detailed analysis of PBMC gene expression analysis could serve to better understand immune modulation by cold. Terefore, here we performed a global transcriptome gene expression study (microarray analysis) of PBMC in control and cold-exposed ferrets to characterise the efect of cold exposure on metabolism. Te most outstand- ing gene expression patterns were refected in a homeostatic tissue with a key role in cold response – adipose tissue – but hardly in the liver. Results Body weight, adiposity, and serum parameters. Data of body weight, adiposity, and circulating NEFA levels of the same set of animals have previously been published18. Briefy, cold exposure induced a reduction in fat mass, with a signifcant decreased size of both subcutaneous (inguinal and interscapular) and visceral (retro- peritoneal) adipose tissue depots in cold-exposed animals. Despite the lower fat mass, fnal body weight was not afected, although animals in the cold group had a lower body weight increase (−16%) in comparison to controls during the week in which they were exposed to cold. As expected, and in agreement with the mobilisation of fat depots, circulating NEFA levels were increased by 35% in the cold group. Circulating glucose levels were not afected by cold acclimation (187 ± 21 mg/dL for cold vs 127 ± 17 mg/dL for control groups; p = 0.06 by Students’ t-test). PBMC transcriptomic analysis. In order to gain a global, comprehensive overview of the impact of cold exposure on global gene expression, we developed a ferret microarray consisting of 45,328 sequences, encoding a total of 19,299 unique genes. We then performed a global gene expression analysis of PBMC of ferrets acclima- tised to 4 °C (cold) or 22 °C (control) for one week. Our results show that PBMC gene expression was afected by cold exposure with a total of 611 genes changing their expression in cold vs control animals (considering a p-value < 0.01), 61% of which were down-regulated. A volcano plot of these genes is shown in Fig. 1, and the heatmap in Supplementary Fig. 1. For interpretation, a bioinformatics network analysis was performed (Fig. 2A), which identifed immune response as being highly diferentially expressed. Within the top10 networks, fve were immune related, with three additional ones being related to blood vessel morphogenesis/angiogenesis/signalling. For a more specifc view, the 611 top regulated genes were manually classifed in biological processes, using scientifc literature and databases. Each individual gene was included in one process only. Tis classifcation resulted in 11 processes: gene expression, cell cycle and immune response, extracellular matrix or cytoskeleton, signal transduction, cell transport, energy metabolism, enzymes/metabolism, DNA repair, organ and tissue development and protein SCIENTIFIC REPORTS | (2019) 9:19985 | https://doi.org/10.1038/s41598-019-56354-6 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Number of genes Classifcation Sub-classifcation Up-regulated Down-regulated Gene expression Transcription factor 13 53 RNA maturation 2 10 DNA structure 2 9 Epigenetic regulation 0 6 Coactivator 0 5 Post-transcriptional regulation 0 5 Corepressor
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