10.1038/s41598-020-64706-wScientific Reports101963

Agrawal, Madhur

Chhay, Vanna

Hoon, Shawn

Huang, Shiqi

Khoo, Chin Meng

Shabbir, Asim

Shabeer, Muhammad

Shrivastava, Manu Kunaal

Shyong Tai, E

Toh, Sue-Anne

Vidal-Puig, Antonio

Yeo, Chia Rou

Yeo, C.R.

Agrawal, M.

Hoon, S.

Shabbir, A.

Shrivastava, M.K.

Huang, S.

Khoo, C.M.

Chhay, V.

Shabeer, M.

Shyong Tai, E.

Vidal-Puig, A.

Toh, S.-A.

ScholarBank@NUS

Reply to: ‘Browning capabilities of human primary adipose-derived stromal cells compared to SGBS cells’

Apollo (Cambridge)

1Scientific RepoRtS |         (2020) 10:9634  | https://doi.org/10.1038/s41598-020-64706-wwww.nature.com/scientificreportsReply to: ‘Browning capabilities of human primary adipose-derived stromal cells compared to SGBS cells’chia Rou Yeo1,7, Madhur Agrawal1,7, Shawn Hoon2, Asim Shabbir3, Manu Kunaal Shrivastava4, Shiqi Huang5, Chin Meng Khoo1, Vanna chhay1, Muhammad Shabeer1, E Shyong tai  1, Antonio Vidal-puig  4,6 & Sue-Anne toh  1 ✉replying to: D. Halbgebauer et al.; Scientific Reports https://doi.org/10.1038/s41598-020-64369-7 (2020).Pharmacological browning/beiging of adipose tissue has the potential to be an important therapeutic strategy to combat obesity and associated co-morbidities. SGBS adipocytes are non-transformed primary human adi-pocytes with an ability to grow and differentiate up to 50 passages. Due to extended growth capacity when com-pared to other available primary human adipocytes, SGBS adipocytes are commonly used as a model for human adipose in in-vitro studies. Tews et al. reported equal browning capacity in SGBS adipocytes when compared with primary human adipocytes derived from the mammary region. Results in the manuscript appear to be discordant from findings in our previously published Scientific Reports paper: “SGBS cells as a model of human adipocyte browning: A comprehensive comparative study with primary human white subcutaneous adipocytes”. We observed a more robust browning signature in SGBS adipocytes as compared to primary human adipocytes derived from human subcutaneous abdominal adipose tissue. Here, we discuss the key differences between study design and methods used in both studies, which may have led to the differences in the browning phenotype observed. Together, observations and inferences from both studies support the presence of functional heteroge-neity of different adipose tissue depots in humans. Collectively, our efforts provide insights into considerations for choosing appropriate cell culture models and experimental conditions to be used in future studies related to human adipose tissue browning.Halbgebauer et al. used different pro-adipogenic stimuli (rosiglitazone vs indomethacin) and compared SGBS adipocytes to primary human ASCs (adipose stem/stromal cells), derived from mammary glands, during and post differentiation. Both stimuli are commonly used as supplements in pre-adipocyte differentiation medium in-vitro1. Rosiglitazone is a PPARgamma agonist and indomethacin is a non-steroidal anti-inflammatory drug. Compared to indomethacin, the presence of rosiglitazone increased differentiation capacity, gene and protein expression of browning markers including UCP1, and oxygen consumption rate (OCR) in both adipocyte cell types. When differentiated with medium containing rosiglitazone, UCP1 expression was higher in SGBS adipo-cytes whereas PGC1α and DIO2 expression were higher in ASCs. Functionally, proton leak, basal respiration and maximal respiration were higher in ASCs compared to SGBS adipocytes, suggesting that ASCs and SGBS bear similar browning capacity.In our experiments, SGBS cells were differentiated in medium containing rosiglitazone2,3 and compared to subcutaneous adipose tissue derived ASCs (referred to as primary human white subcutaneous [PHWSC] in this response and in our manuscript) with medium containing indomethacin (purchased from Lonza)4. Under these conditions, both cell types had a similar morphology and we also observed a similar percentage of differentiated adipocytes. Post differentiation, OCR, proton leak and maximal respiration were significantly higher in SGBS 1Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, 117599, Singapore, Singapore. 2Molecular Engineering Laboratory, Biomedical Sciences Institutes, A*Star, 138668, Singapore, Singapore. 3Department of Surgery, National University Hospital, 119074, Singapore, Singapore. 4Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke’s Hospital, University of Cambridge, Cambridge, CB2 0QC, UK. 5Food Science and Technology Program, Department of Chemistry, National University of Singapore, 117542, Singapore, Singapore. 6Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK. 7These authors contributed equally: Chia Rou Yeo and Madhur Agrawal. ✉e-mail: mdcsates@nus.edu.sgMAttErS AriSingopen2Scientific RepoRtS |         (2020) 10:9634  | https://doi.org/10.1038/s41598-020-64706-wwww.nature.com/scientificreportswww.nature.com/scientificreports/adipocytes as compared to PHWSCs. In both studies, PGC1α and DIO2 expression was higher in ASCs as com-pared to SGBS adipocytes. Here, we discuss different features of the experimental design between the two studies, which may have resulted in differences in results, and the implications on the interpretation of the findings.Differences in the site of adipose explant collection and clinical characteristics of donors. PHWSC donors in our study were obese Asians and adipose tissue samples were collected from the abdominal region from individuals undergoing bariatric surgery; whereas Tews et al. obtained explants from mammary region from females undergoing elective surgery as well as deep neck and subcutaneous neck adipose tissue from patients undergoing neck surgery. For the latter, these adipose tissue samples were excised from patients undergoing neck surgery for malignancies or nodular goitre5. It is important to note that adipose tissue from the neck region are inherently different from most other adipose depots6–8. The neck region is primarily where the browning mechanism was disclosed and beside the UCP1 positive cells, it contains at least two types of adipocytes originating from two distinct mesenchymal stem cell lineages. The classic brown adipocytes with precursors in common with muscle cells and newly recognized beige adipocytes which have the same precursor as white adipose cells7. This unique aspect of neck adipose tissue should be should be taken into consideration when interpreting the results.For in-vitro cell culture experiments, Tews et al. isolated adipocytes by collagenase digestion of mammary region adipose tissue. Adipocytes derived from the mammary fat pad function differently from those in other fat depots9,10. For example, trans-differentiation of subcutaneous white adipocytes derived from mouse mammary gland produce “pink adipocytes”. Pink adipocytes are believed to arise exclusively in female subcutaneous depots during pregnancy and lactation10.Other factors such as BMI, can have a negative impact on the differentiation capacity of ASCs derived from mammary adipose tissue11. In ASCs derived from human subcutaneous adipose tissue depots, a donor with an obese phenotype is associated with reduced capacity of their adipocytes to display characteristics of browning, as measured by UCP1 protein expression12. Compared to adipocytes derived from lean individuals, UCP1 protein expression was close to zero in differentiated adipocytes from obese individuals, irrespective of similar differenti-ation capacity. Even in healthy men, body fat percentage and BMI have been shown to have a negative impact on functional brown adipose tissue13.The SGBS adipocytes used in our study were derived from a 3-month old infant2, whereas Tews et al. reported using SGBS adipocytes derived from a 5-month old infant. Considering the disease heterogeneity of the SGBS syndrome14, cells derived from different donors could also potentially display varying phenotypes in-vitro.Differentiation of SgBS preadipocytes or hASCs in the presence of indomethacin or rosigl-itazone in our experiments. The composition of the differentiation medium can impact on adipocyte differentiation and thus we examined effect of indomethacin and rosiglitazone during the early phase of our experiments. UCP1 gene expression remained similar in SGBS and PHWSCs when differentiated with medium that is optimal for either cell types (expressed as Dct, Fig. 1). To maintain PHWSC viability while differentiating with SGBS differentiation medium, we supplemented the medium with 10% FBS which is commonly used in adipocyte differentiation cocktails for primary human adipocytes12.In results obtained by Tews et al., UCP1 was higher expressed in adipocytes differentiated with rosiglitazone compared to indomethacin, where UCP1 expression was hardly detectable. SGBS cells showed stronger UCP1 expression compared to hASCs when differentiated with rosiglitazone. Interestingly, in our experiments UCP1 expression remained detectable and the amount of UCP1 were similar between control and the ones without rosiglitazone when we omitted rosiglitazone (Fig. 2). Further studies will be needed to explain these observed differences.technical considerations. In our results obtained from SGBS adipocytes, isoproterenol (ISO) treatment led to a sharp rise in OCR and as expected, introduction of (fatty acid free-bovine serum albumin) FAF-BSA Figure 1. Differentiation of PHWSC and SGBS preadipocytes in both SGBS and Lonza medium, respectively. To rule out the possibility of UCP1 expression being induced by differing media compositions, SGBS (n = 2 in duplicates) and SC (n = 3 in duplicates) were differentiated in vitro using both sets of media, on two separate occasions. Statistical differences were analysed by Kruskal–Wallis one-way ANOVA followed by Dunn’s test for multiple comparisons. Values were expressed as mean ± SD and P < 0.05 was considered statistically different. # P < 0.05 compared to SC with SGBS medium and *:P < 0.05 compared to SC with Lonza medium.3Scientific RepoRtS |         (2020) 10:9634  | https://doi.org/10.1038/s41598-020-64706-wwww.nature.com/scientificreportswww.nature.com/scientificreports/diminished the effect of ISO in a dose dependent manner. ISO-induced leak respiration, which is considered a better representation of UCP1 activity, was nearly two folds higher than basal leak respiration in absence of FAF-BSA. As a confirmative marker of browning in cells using the Agilent Seahorse flux analyzer, it is now com-monly recommended to measure OCR after stimulating cells with lipolytic stimuli combined with the presence or absence of BSA in the medium, since it measures the increase in OCR due to free fatty acid metabolism derived from cellular lipolysis. However, we acknowledge that the oligomycin-protonophore based assay used here is not specific to UCP1- dependent proton leak. This is a technical limitation, and future studies that assess direct measures of UCP1 function should take this into consideration.Another consideration to note is that the absorbance obtained from Janus Kinase B method is dependent on the number of mitochondria in the cell. The number of cells obtained from Janus Kinase B method is accurate for a broad range of cell numbers, but the methodology needs to be optimised for each cell type before absorbance can be considered directly proportional to the cell number15. In any given experimental setup, using the absolute absorbance to normalize cell numbers in SeaHorse experiments may not be ideal as differences in the number of mitochondria per cell may mask variation in OCR observed in adipocytes. Alternative methods include nuclear staining such as DAPI or Hoechst or using total protein concentration as a basis for normalizing cell numbers.Overall, key differences in the (i) clinical characteristics of the donor from which the cells were derived from, (ii) adipose tissue depot from which samples were collected from, (iii) composition of cell culture media, and (iv) approaches to the analyses of functional readouts can explain the phenotypic differences observed in primary human adipocytes, when comparing findings from both studies. In our opinion, both manuscripts provide valua-ble insights into how these differences can affect the resultant characteristics observed from different human cells lines, and underscores the importance of understanding the strength and limitations of different human derived in vitro models and the conditions used. Applying these considerations can then guide the selection of appropri-ate models and the conditions used, to optimally address specific research questions of interest.Data availabilityAll data generated or analysed during this study are included in this published article.Received: 4 December 2019; Accepted: 16 April 2020;Published: xx xx xxxxReferences 1. Merlin, J. et al. Rosiglitazone and a beta3-Adrenoceptor Agonist Are Both Required for Functional Browning of White Adipocytes in. Culture. Frontiers in endocrinology 9, 249, https://doi.org/10.3389/fendo.2018.00249 (2018). 2. Wabitsch, M. et al. Characterization of a human preadipocyte cell strain with high capacity for adipose differentiation. International journal of obesity and related metabolic disorders: journal of the International Association for the Study of Obesity 25, 8–15 (2001). 3. Fischer-Posovszky, P., Newell, F. S., Wabitsch, M. & Tornqvist, H. E. Human SGBS cells - a unique tool for studies of human fat cell biology. Obesity facts 1, 184–189, https://doi.org/10.1159/000145784 (2008). 4. McGillicuddy, F. C. et al. Interferon gamma attenuates insulin signaling, lipid storage, and differentiation in human adipocytes via activation of the JAK/STAT pathway. The Journal of biological chemistry 284, 31936–31944, https://doi.org/10.1074/jbc.M109.061655 (2009). 5. Tews, D. et al. Comparative gene array analysis of progenitor cells from human paired deep neck and subcutaneous adipose tissue. Molecular and cellular endocrinology 395, 41–50, https://doi.org/10.1016/j.mce.2014.07.011 (2014). 6. Sacks, H. & Symonds, M. E. Anatomical Locations of Human Brown Adipose Tissue. Functional Relevance and Implications in Obesity and Type 2 Diabetes 62, 1783–1790, https://doi.org/10.2337/db12-1430 (2013). 7. Pandzic Jaksic, V. et al. Neck adipose tissue - tying ties in metabolic disorders. Hormone molecular biology and clinical investigation 33, https://doi.org/10.1515/hmbci-2017-0075 (2018). 8. Cypess, A. M. et al. Identification and importance of brown adipose tissue in adult humans. The New England journal of medicine 360, 1509–1517, https://doi.org/10.1056/NEJMoa0810780 (2009). 9. Hovey, R. C. & Aimo, L. Diverse and active roles for adipocytes during mammary gland growth and function. Journal of mammary gland biology and neoplasia 15, 279–290, https://doi.org/10.1007/s10911-010-9187-8 (2010).Figure 2. UCP1 protein expression in SGBS and PHWSC (upper panel). In an independent experiment, SGBS were differentiated in T3 and rosiglitazone depleted medium (lower panel).4Scientific RepoRtS |         (2020) 10:9634  | https://doi.org/10.1038/s41598-020-64706-wwww.nature.com/scientificreportswww.nature.com/scientificreports/ 10. Giordano, A., Smorlesi, A., Frontini, A., Barbatelli, G. & Cinti, S. White, brown and pink adipocytes: the extraordinary plasticity of the adipose organ. European journal of endocrinology 170, R159–171, https://doi.org/10.1530/eje-13-0945 (2014). 11. van Harmelen, V. et al. Effect of BMI and age on adipose tissue cellularity and differentiation capacity in women. International journal of obesity and related metabolic disorders: journal of the International Association for the Study of Obesity 27, 889–895, https://doi.org/10.1038/sj.ijo.0802314 (2003). 12. Carey, A. L. et al. Reduced UCP-1 Content in In Vitro Differentiated Beige/Brite Adipocytes Derived from Preadipocytes of Human Subcutaneous White Adipose Tissues in Obesity. PLOS ONE 9, e91997, https://doi.org/10.1371/journal.pone.0091997 (2014). 13. van Marken Lichtenbelt, W. D. et al. Cold-activated brown adipose tissue in healthy men. The New England journal of medicine 360, 1500–1508, https://doi.org/10.1056/NEJMoa0808718 (2009). 14. Knopp, C. et al. Twenty-one years to the right diagnosis - clinical overlap of Simpson-Golabi-Behmel and Beckwith-Wiedemann syndrome. Am J Med Genet A 167A, 151–155, https://doi.org/10.1002/ajmg.a.36825 (2015). 15. Raspotnig, G. et al. Colorimetric determination of cell numbers by Janus green staining. Anal Biochem 275, 74–83, https://doi.org/10.1006/abio.1999.4309 (1999).Author contributionsC.R.Y. and M.A. study conception and experimental design, collection and assembly of data, data analysis and interpretation, manuscript writing and revisions; S.H. RNA-seq data analysis and interpretation, manuscript writing; A.S. subject recruitment for bariatric surgery and adipose tissue biopsies, review of manuscript; M.K.S. collection and assembly of data, manuscript revisions; C.M.K. data analysis and interpretation; S.H., V.C. and M.S. collection and assembly of data; E.S.T. and A.V. data analysis and manuscript revisions; S.T. financial support, study conception and design, data analysis and interpretation, manuscript writing and revisions, final approval of manuscript. All authors have reviewed and approved the manuscript.competing interestsThe authors declare no competing interests.Additional informationCorrespondence and requests for materials should be addressed to S.-A.T.Reprints and permissions information is available at www.nature.com/reprints.Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per-mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020

English

Reply to: 'Browning capabilities of human primary adipose-derived stromal cells compared to SGBS cells'.

https://www.repository.cam.ac.uk/bitstream/handle/1810/306795/41598_2020_Article_64706.pdf?sequence=2&isAllowed=y

Reply to: ‘Browning capabilities of human primary adipose-derived stromal cells compared to SGBS cells’

Abstract

Similar works

Full text

Available Versions

ScholarBank@NUS

Apollo (Cambridge)

Apollo (Cambridge)

Apollo (Cambridge)