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White-to-brown metabolic conversion of human adipocytes by JAK inhibition

Nature Cell Biology volume 17, pages 57–67 (2015)Cite this article

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Abstract

The rising incidence of obesity and related disorders such as diabetes and heart disease has focused considerable attention on the discovery of new therapeutics. One promising approach has been to increase the number or activity of brown-like adipocytes in white adipose depots, as this has been shown to prevent diet-induced obesity and reduce the incidence and severity of type 2 diabetes. Thus, the conversion of fat-storing cells into metabolically active thermogenic cells has become an appealing therapeutic strategy to combat obesity. Here, we report a screening platform for the identification of small molecules capable of promoting a white-to-brown metabolic conversion in human adipocytes. We identified two inhibitors of Janus kinase (JAK) activity with no precedent in adipose tissue biology that stably confer brown-like metabolic activity to white adipocytes. Importantly, these metabolically converted adipocytes exhibit elevated UCP1 expression and increased mitochondrial activity. We further found that repression of interferon signalling and activation of hedgehog signalling in JAK-inactivated adipocytes contributes to the metabolic conversion observed in these cells. Our findings highlight a previously unknown role for the JAK–STAT pathway in the control of adipocyte function and establish a platform to identify compounds for the treatment of obesity.

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Figure 1: Browning screen in human stem cell-derived adipocytes.
Figure 2: Selected compounds modulate lipid morphology.
Figure 3: Validation of tofacitinib and R406 browning compounds in primary adipocytes.
Figure 4: Inhibition of STAT phosphorylation downstream of tofacitinib and R406.
Figure 5: JAK inhibition stably induces a brown-like profile in adipocytes.
Figure 6: JAK inhibition induces brown-like metabolic properties in adipocytes.
Figure 7: Gene signature and cellular identity of JAK-inactivated adipocytes.
Figure 8: IFN and SHH signalling contribute to adipocyte browning downstream of JAK inhibition.

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Acknowledgements

The authors thank I. Clausen, M. Kapps, R. Schmucki and A. Schuler for technical support, K. Christensen and M. Graf for stem cell support, L. Badi for preliminary data analysis, C. Solier, A. Schell-Steven and T. Bergauer for experimental planning and M. Pawlak (Natural and Medical Sciences Institute at the University of Tübingen) for RPPA analyses. A.M. was supported by the Roche Postdoctoral Fellowship (RPF) program (2011–2013). This research was supported in part by F. Hoffmann-La Roche; grant R01DK095384 (C.A.C. and Y.K.L.) and R01DK097768 (C.A.C.) from the United States Institutes of Health (NIH); and Harvard University.

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Authors and Affiliations

  1. Roche Pharma Research and Early Development, Roche Innovation Center Basel, 124 Grenzacherstrasse, Basel CH 4070, Switzerland,

    Annie Moisan, Jitao David Zhang, Claas A. Meyer, Michael Prummer, Sannah Zoffmann, Hoa Hue Truong, Martin Ebeling, Anna Kiialainen, Régine Gérard & Kurt E. Amrein

  2. Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University, Massachusetts 02138, USA

    Youn-Kyoung Lee, Carolyn S. Hudak, Fang Xia, Robert T. Schinzel & Chad A. Cowan

  3. Center for Regenerative Medicine, Massachusetts General Hospital, Boston Massachusetts 02114, USA,

    Youn-Kyoung Lee, Carolyn S. Hudak, Fang Xia, Robert T. Schinzel & Chad A. Cowan

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  1. Annie Moisan
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Contributions

A.M. designed and performed experiments, analysed data and wrote the manuscript; Y-K.L. performed experiments, analysed data and edited the manuscript; R.G., C.S.H. and F.X. performed experiments; J.D.Z. and M.E. performed bioinformatics analyses and contributed to the main text related to Fig. 7; H.H.T., S.Z. and M.P. performed high-content imaging analysis; A.K. performed RNA-seq; C.A.M. and R.T.S. supervised stem cell activities; K.E.A. supervised the project and C.A.C. supervised the project and wrote the manuscript. A.M., Y-K.L., R.G., C.S.H., M.P., J.D.Z., H.H.T., S.Z. and A.K. contributed to description of Methods.

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Correspondence to Annie Moisan or Chad A. Cowan.

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Integrated supplementary information

Supplementary Figure 3

(a) UCP1 expression is increased in PSC-WA after treatment with indicated compounds. Rosiglitazone induced expression of both UCP1 and FABP4. Values represent the mean of two biological replicates. (b) Browning screen performance: Plate-wise control distribution and Z’ factors for UCP1 and FABP4. For UCP1, <Z’ >= 0.50, for FABP4, <Z’ >= 0.64. (c) Reproducibility of replicates. The correlation of the intra-plate replicates for UCP1 (left) and FABP4 (right) in log–log representation. Controls are plotted in red (negative) and green (positive). Compounds with large inter-run differences were excluded from further analysis. (d) Hit selection. Left panel: Quantile–quantile plot of the normalized UCP1 reads. The blue line indicates the expected profile for a Gaussian distribution without actives. The blue dashed lines limit the confidence band of a correlation test corroborating the hit selection from a different angle, as all the selected compounds (green dots) lie outside. The P-value distribution drawn in the inset, which is the basis of the hit selection, shows no irregular features. Right panel: Scatter plot of the normalized FABP4 and UCP1 signal on logarithmic axes. Black dots: inactive compounds. Color dots: active compounds, red: rosiglitazone, yellow: rosiglitazone-like compounds in screen, blue: potential browning hits in screen, green: rosiglitazone-like compounds and browning hits confirmed in validation run, grey : non-confirmed in validation run.

Supplementary Figure 4 PSC-WA were differentiated and treated as described in Fig. 1b.

At day 14, adipocytes were fixed, stained and imaged by confocal microscopy. Green: lipids, Red: nuclei. Scale bars: 50 mM.

Supplementary Figure 5 Chemical name and structure of the model browning compounds R406 and tofacitinib.

Supplementary Figure 6

(a) Uncropped bright field images showing that tofacitinib and R406 induce brown-like lipid morphology in human primary adipocytes more prominently than BMP7. ADSC: Adipose tissue-derived stem cells. (b) RT-PCR analysis of UCP1 expression in mouse visceral white adipose tissue explants following 7 days of treatment with tofacitinib (tofa.) or R406. Values are mean ± s.e.m. of n = three biological replicates of pooled tissue from 5 mice.

Supplementary Figure 7 Reverse phase protein analysis (RPPA) of tofacitinib and R406-treated PSC-WA.

Cells were collected 20’ after addition of compounds to PSC-WA and processed for RPPA. Data shown is relative to DMSO. All 51 antibodies recognized the phospho-isoforms of indicated proteins. 3 antibodies did not reach detection threshold and were excluded from graph. Data from one experiment (n = 3 biological replicates) representative of 2 independent experiments. Values are mean ± s.d. of three biological replicates.

Supplementary Figure 8

(a) R406 promoted mRNA expression of PGC1α, PGC1β and PPARG but not of PRDM16. (b) The transcriptional changes downstream of R406 include up-regulation of PPARG, SREBF and BMP target genes. (c) Gene expression regulation by tofacitinib (tofa., JAK3i) versus R406 (SYKi) in PSC-WA at day 7. 54 genes were up-regulated by both compounds, 17 of which were BA-specific, that is, low in PSC-WA and high in PSC-BA (yellow dots). (a–c) N = 3 biological replicates. Each independent biological replicate was pooled from two individual wells.

Supplementary Figure 9 The ratio of UCP1/FABP4 mRNA level was down-regulated in PSC-BA upon treatment with cyclopamine.

Values represent the mean of two biological replicates.

Supplementary Figure 10 Original scans of western blot analyses presented in Figs 3, 4 and 6.

Supplementary Table 1 Validated browning hits and PPAR agonists shown in Fig. 1d.
Full size table
Supplementary Table 2 Panomics QuantiGene2.0 Probes used for branched DNA analysis of gene expression.
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Moisan, A., Lee, YK., Zhang, J. et al. White-to-brown metabolic conversion of human adipocytes by JAK inhibition. Nat Cell Biol 17, 57–67 (2015). https://doi.org/10.1038/ncb3075

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