Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors
2018; Springer Nature; Volume: 10; Issue: 8 Linguagem: Inglês
10.15252/emmm.201708613
ISSN1757-4684
AutoresIrem Bayindir‐Buchhalter, Gretchen Wolff, Sarah Lerch, Tjeerd Sijmonsma, Maximilian Schuster, Jan Gronych, Adrian T. Billeter, Rohollah Babaeikelishomi, Damir Krunic, Lars Ketscher, Nadine Spielmann, Martin Hrabĕ de Angelis, Jorge L. Ruas, Beat P. Müller‐Stich, Mathias Heikenwälder, Peter Lichter, Stephan Herzig, Alexandros Vegiopoulos,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoResearch Article4 July 2018Open Access Source DataTransparent process Cited4 is a sex-biased mediator of the antidiabetic glitazone response in adipocyte progenitors Irem Bayindir-Buchhalter Irem Bayindir-Buchhalter orcid.org/0000-0003-2532-109X DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Gretchen Wolff Gretchen Wolff DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Sarah Lerch Sarah Lerch DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Tjeerd Sijmonsma Tjeerd Sijmonsma orcid.org/0000-0001-8950-2987 Division Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Maximilian Schuster Maximilian Schuster orcid.org/0000-0001-9030-2210 DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Jan Gronych Jan Gronych Division of Molecular Genetics, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Heidelberg, Germany Search for more papers by this author Adrian T Billeter Adrian T Billeter Department of General, Visceral, and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Rohollah Babaei Rohollah Babaei DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Damir Krunic Damir Krunic Light Microscopy Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Lars Ketscher Lars Ketscher Department of Physiology and Pharmacology, Molecular and Cellular Exercise Physiology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Nadine Spielmann Nadine Spielmann German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany Search for more papers by this author Martin Hrabe de Angelis Martin Hrabe de Angelis German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany Chair of Experimental Genetics, School of Life Science Weihenstephan, Technische Universität München, Freising, Germany German Center for Diabetes Research (DZD), Neuherberg, Germany Search for more papers by this author Jorge L Ruas Jorge L Ruas orcid.org/0000-0002-1110-2606 Department of Physiology and Pharmacology, Molecular and Cellular Exercise Physiology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Beat P Müller-Stich Beat P Müller-Stich Department of General, Visceral, and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Mathias Heikenwalder Mathias Heikenwalder Division Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Peter Lichter Peter Lichter Division of Molecular Genetics, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Heidelberg, Germany Search for more papers by this author Stephan Herzig Stephan Herzig orcid.org/0000-0003-3950-3652 Helmholtz Center Munich, Institute for Diabetes and Cancer IDC, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Search for more papers by this author Alexandros Vegiopoulos Corresponding Author Alexandros Vegiopoulos [email protected] orcid.org/0000-0001-6300-316X DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Irem Bayindir-Buchhalter Irem Bayindir-Buchhalter orcid.org/0000-0003-2532-109X DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Gretchen Wolff Gretchen Wolff DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Sarah Lerch Sarah Lerch DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Tjeerd Sijmonsma Tjeerd Sijmonsma orcid.org/0000-0001-8950-2987 Division Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Maximilian Schuster Maximilian Schuster orcid.org/0000-0001-9030-2210 DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Jan Gronych Jan Gronych Division of Molecular Genetics, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Heidelberg, Germany Search for more papers by this author Adrian T Billeter Adrian T Billeter Department of General, Visceral, and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Rohollah Babaei Rohollah Babaei DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Damir Krunic Damir Krunic Light Microscopy Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Lars Ketscher Lars Ketscher Department of Physiology and Pharmacology, Molecular and Cellular Exercise Physiology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Nadine Spielmann Nadine Spielmann German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany Search for more papers by this author Martin Hrabe de Angelis Martin Hrabe de Angelis German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany Chair of Experimental Genetics, School of Life Science Weihenstephan, Technische Universität München, Freising, Germany German Center for Diabetes Research (DZD), Neuherberg, Germany Search for more papers by this author Jorge L Ruas Jorge L Ruas orcid.org/0000-0002-1110-2606 Department of Physiology and Pharmacology, Molecular and Cellular Exercise Physiology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Beat P Müller-Stich Beat P Müller-Stich Department of General, Visceral, and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Mathias Heikenwalder Mathias Heikenwalder Division Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Peter Lichter Peter Lichter Division of Molecular Genetics, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Heidelberg, Germany Search for more papers by this author Stephan Herzig Stephan Herzig orcid.org/0000-0003-3950-3652 Helmholtz Center Munich, Institute for Diabetes and Cancer IDC, Neuherberg, Germany Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany Search for more papers by this author Alexandros Vegiopoulos Corresponding Author Alexandros Vegiopoulos [email protected] orcid.org/0000-0001-6300-316X DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Author Information Irem Bayindir-Buchhalter1,12, Gretchen Wolff1, Sarah Lerch1, Tjeerd Sijmonsma2,13, Maximilian Schuster1, Jan Gronych3, Adrian T Billeter4, Rohollah Babaei1, Damir Krunic5, Lars Ketscher6, Nadine Spielmann7, Martin Hrabe de Angelis7,8,9, Jorge L Ruas6, Beat P Müller-Stich4, Mathias Heikenwalder2, Peter Lichter3, Stephan Herzig10,11 and Alexandros Vegiopoulos *,1 1DKFZ Junior Group Metabolism and Stem Cell Plasticity, German Cancer Research Center, Heidelberg, Germany 2Division Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany 3Division of Molecular Genetics, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Heidelberg, Germany 4Department of General, Visceral, and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany 5Light Microscopy Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany 6Department of Physiology and Pharmacology, Molecular and Cellular Exercise Physiology, Karolinska Institutet, Stockholm, Sweden 7German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany 8Chair of Experimental Genetics, School of Life Science Weihenstephan, Technische Universität München, Freising, Germany 9German Center for Diabetes Research (DZD), Neuherberg, Germany 10Helmholtz Center Munich, Institute for Diabetes and Cancer IDC, Neuherberg, Germany 11Joint Heidelberg-IDC Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Germany 12Present address: Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany 13Present address: LOEWE Center for Cell and Gene Therapy Frankfurt, Department of Medicine, Hematology/Oncology, Goethe University Frankfurt, Frankfurt am Main, Germany *Corresponding author. Tel: +49 6221 423585; E-mail: [email protected] EMBO Mol Med (2018)10:e8613https://doi.org/10.15252/emmm.201708613 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Most antidiabetic drugs treat disease symptoms rather than adipose tissue dysfunction as a key pathogenic cause in the metabolic syndrome and type 2 diabetes. Pharmacological targeting of adipose tissue through the nuclear receptor PPARg, as exemplified by glitazone treatments, mediates efficacious insulin sensitization. However, a better understanding of the context-specific PPARg responses is required for the development of novel approaches with reduced side effects. Here, we identified the transcriptional cofactor Cited4 as a target and mediator of rosiglitazone in human and murine adipocyte progenitor cells, where it promoted specific sets of the rosiglitazone-dependent transcriptional program. In mice, Cited4 was required for the proper induction of thermogenic expression by Rosi specifically in subcutaneous fat. This phenotype had high penetrance in females only and was not evident in beta-adrenergically stimulated browning. Intriguingly, this specific defect was associated with reduced capacity for systemic thermogenesis and compromised insulin sensitization upon therapeutic rosiglitazone treatment in female but not male mice. Our findings on Cited4 function reveal novel unexpected aspects of the pharmacological targeting of PPARg. Synopsis The identification of the Cited4 cofactor as a sex-, tissue- and signal-specific mediator of transcriptional responses to glitazones in adipocyte progenitors reveals unexpected aspects of therapeutic PPARg targeting for insulin sensitization in type 2 diabetes and prediabetes. Cited4 is a glitazone target in human and murine adipocyte progenitors promoting the induction of beige adipocyte differentiation. Cited4 is required for rosiglitazone-mediated but not beta-adrenergic induction of thermogenic expression in subcutaneous fat in a sex-biased manner. Systemic energy expenditure and maximal beta-adrenergic adipocyte respiration are reduced in Cited4-deficient female mice under rosiglitazone treatment. Reduced thermogenic expression in subcutaneous fat is associated with compromised insulin sensitization upon therapeutic rosiglitazone treatment specifically in female mice. Introduction The functional status of adipose tissue has emerged as a key determinant for systemic metabolic homeostasis and disease. Obesity and in particular excess visceral fat are prominent risk factors for type 2 diabetes and cardiovascular disease (Cornier et al, 2008; Lee et al, 2013). The dissociation of obesity and metabolic dysfunction in the paradigms of lipodystrophy and metabolically healthy obesity indicates that it is not the quantity of fat per se but the impaired function which underlies the pathogenic process (Vegiopoulos et al, 2017). Inadequate lipid metabolism in adipocytes results in increased circulating and ectopically deposited lipids and consequently in lipotoxicity and malfunction of metabolism in multiple organs (Samuel & Shulman, 2012). Chronically, this contributes to the dysregulation of endocrine circuits, insulin resistance, and essentially to the development of type 2 diabetes. However, most of the current treatment options in type 2 diabetes and prediabetes target mainly the symptoms rather than insulin sensitivity and adipose tissue metabolism as causative factors (Soccio et al, 2014; Chatterjee et al, 2017). The glitazone drugs of the thiazolidinedione (TZD) class, agonists of the peroxisome proliferator-activated receptor gamma (PPARg), represent an exception in this regard. TZDs act as potent insulin sensitizers, and this action seems to be mediated predominantly by PPARg in adipose tissue (Soccio et al, 2014). Although the use of TZDs has strongly declined due to their side effects, PPARg remains a promising target in the prevention and treatment of type 2 diabetes as highlighted by ongoing basic and clinical research (Ahmadian et al, 2013; Soccio et al, 2014; Banks et al, 2015; Chatterjee et al, 2017). Thiazolidinediones have pleiotropic effects on adipose tissue, essentially resulting in improved uptake and metabolism of fatty acids and glucose as well as endocrine function (Rangwala & Lazar, 2004; Ye et al, 2004; Boden et al, 2005; Festuccia et al, 2009). Beyond their ability to enhance adipocyte formation and turnover, TZDs promote mitochondrial biogenesis and fatty acid oxidation in human and rodent white adipose tissue and increase its thermogenic potential (“browning”; Okuno et al, 1998; Fukui et al, 2000; Yamauchi et al, 2001; Wilson-Fritch et al, 2004; Boden et al, 2005; Bogacka et al, 2005; Tang et al, 2011). Interestingly, the enrichment of pathways of mitochondrial oxidation and lipid metabolism in subcutaneous fat was recently shown to be the most prominent effect of the TZD rosiglitazone on the transcriptome across adipose tissues (Soccio et al, 2017). Increased capacity for adipose tissue thermogenesis is generally accepted to be protective against insulin resistance and dyslipidemia but to which extent the regulation of browning by TZDs mediates insulin sensitization remains unclear (Sidossis & Kajimura, 2015). Thiazolidinediones are potent stimulators of adipocyte progenitor differentiation in human and murine cell culture and promote the formation of beige/brite thermogenic adipocytes (Digby et al, 1998; Elabd et al, 2009; Petrovic et al, 2010; Ohno et al, 2012; Ahmadian et al, 2013). In the adult organism, adipocyte progenitors mediate the recruitment of new white and beige adipocytes as it occurs upon tissue expansion, cold exposure, or TZD treatment (Tang et al, 2011; Hepler et al, 2017). Although the core transcriptional network driving adipogenesis downstream of PPARg activation is well established, the factors responsible for depot-, sex-, and stimulus-specific recruitment of progenitors remain to be determined. Moreover, how the context-dependent regulation of progenitors relates to tissue function and insulin sensitization is poorly understood. In this study, we searched for novel mediators of PPARg activation in defined adipocyte progenitor cells and identified the transcriptional cofactor Cited4 (CREB-binding protein/p300-interacting transactivator with ED-rich tail, [Braganca et al, 2002; Yahata et al, 2002)]. Little is known so far about the physiological function of Cited4, beyond its involvement in the regulation of cardiac hypertrophy (Bostrom et al, 2010; Bezzerides et al, 2016). We demonstrate that Cited4 promotes the transcriptional program induced by rosiglitazone in differentiating murine and human adipocyte progenitors and that Cited4 deficiency impairs TZD-dependent but not β-adrenergically stimulated browning specifically in subcutaneous fat. Remarkably, this defect also manifested upon therapeutic rosiglitazone treatment and was associated with reduced insulin sensitization in a sex-specific manner. Results Cited4 is a target of rosiglitazone in murine and human adipocyte progenitors promoting beige differentiation and Ucp1 expression We have previously dissected the global transcriptional response of defined immuno-selected Lin(Ter119/CD31/CD45)−Sca1+ adipocyte progenitors to carbaprostacyclin (cPGI2), the stable analogue of prostacyclin and PPARg agonist promoting beige adipocyte differentiation (Vegiopoulos et al, 2010; Bayindir et al, 2015; Ghandour et al, 2016; Babaei et al, 2017). To search for novel physiologically relevant factors mediating the effects of PPARg activation in progenitors, we mined time course expression profiles for cPGI2-regulated genes (Bayindir et al, 2015). We identified Cited4 due to a robust but transient induction by cPGI2 during progenitor differentiation (Fig 1A). Treatment with rosiglitazone (Rosi) in place of cPGI2 potentiated the transient induction of Cited4 (Fig 1B) and this was recapitulated in the mesenchymal progenitor cell line C3H10T1/2 but not in the preadipocyte cell model 3T3-L1 (Fig EV1A and B). In addition, pioglitazone, a TZD currently used as antidiabetic, transiently increased Cited4 mRNA expression in primary progenitor cells, albeit with overall lower potency compared to Rosi (Fig EV1C). These data demonstrate that Cited4 is a likely target of TZDs and PPARg in differentiating adipocyte progenitors. Figure 1. Cited4 is a target of rosiglitazone in murine and human adipocyte progenitors promoting beige differentiation A. mRNA expression in FACS-isolated Lin(Ter119/CD31/CD45)−Sca1+ progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 1 μM cPGI2 or vehicle for the indicated time, as determined by expression profiling (n = 3, E-MTAB-3693). ****P = 3 × 10−6 (Day 2), ****P = 4 × 10−7 (Day 4), ****P = 1 × 10−6 (Day 6) in 2 × 2 ANOVA with Bonferroni's posttests (cPGI2 vs. vehicle). B. mRNA expression in MACS-isolated Lin−Sca1+ progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT–PCR (n = 4). ****P = 1 × 10−10 (Days 1 and 2), **P = 0.001, in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). C. mRNA expression in female Lin−Sca1+ cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR (n = 3). t-test Cited4−/− vs. Cited4+/+ (Rosi) *P = 0.013 (Ucp1), **P = 0.004 (Cpt1b), *P = 0.026 (Dio2). D–F. mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi (D) or vehicle (D–F), as determined by qRT–PCR at the indicated time points (n = 5 patients). ♀/♂ represents individual data. (D) ****P = 3 × 10−5 (Day 2), ****P = 3 × 10−6 (Day 6), ****P = 4 × 10−9 (Day 10), ****P = 3 × 10−7 (Day 14), in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). (E, F) Pearson correlation coefficient (r) and P-value are shown. G. mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT–PCR (n = 3). ***P = 0.0002 (CITED4), **P = 0.002 (UCP1), *P = 0.02 (UCP1), *P = 0.035/0.026 (PPARG), ***P = 0.0006 (SLC2A4), **P = 0.002 (ADIPOQ) in one-way ANOVA with Tukey's posttests (vs. siCtrl). Data information: Data are presented as mean ± SEM except for (D) ♀/♂, (E and F) individual data. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Cited4 is a target of rosiglitazone in murine adipocyte progenitors promoting beige differentiation A. mRNA expression in C3H10T1/2 cells, differentiated in the presence of 1 μM Rosi or vehicle for the indicated time, as determined by qRT–PCR (day 0, n = 3; day 4, n = 4; day 6, Ctrl, n = 2, Rosi, n = 4; day 10, n = 4). ***P = 0.001, **P = 0.007 (Day 6), **P = 0.005 (Day 10), in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). B. mRNA expression in 3T3-L1 cells, differentiated in the presence of 1 μM Rosi or vehicle for the indicated time, as determined by qRT–PCR (n = 3). 2 × 2 ANOVA with Bonferroni's posttests, P > 0.05 (Rosi vs. vehicle). C. mRNA expression in female Lin−Sca1+ cells, differentiated in the presence of 0.1 or 1 μM pioglitazone (Pio) or vehicle for 8 days, as determined by qRT–PCR (n = 4 for Day 2, n = 4/2/6 for Day 8). ***P = 1*10−10, **P = 0.003, in 2 × 2 ANOVA with Bonferroni's posttests (Pio vs. vehicle). D. mRNA expression in female Lin−Sca1+ cells, differentiated in the presence of the indicated substances for 8 days, as determined by qRT–PCR (n = 3). **P = 0.0035, ***P = 4 × 10−10, ***P = 7 × 10−10 (Ucp1), *P = 0.036, ***P = 0.0002, ***P = 1 × 10−10, ***P = 1 × 10−10 (Cpt1b), ***P = 2 × 10−5 (Cidea), ***P = 1 × 10−6, ***P = 4 × 10−9, ***P = 1 × 10−8, (Elovl3), ***P = 9 × 10−8, ***P = 2 × 10−7 (Cox7a1), ***P = 0.0002, ***P = 1 × 10−8, ***P = 4 × 10−5, ***P = 0.0002 (Cox8b), *P = 0.032, **P = 0.006, ***P = 0.0007 (Dio2), ***P = 1 × 10−6, ***P = 2 × 10−8 (Cyc1), ***P = 8 × 10−5, ***P = 3 × 10−6 (Ndufb3) in 2 × 2 ANOVA with Holm–Sidak posttests (Cited4−/− vs. Cited4+/+). E. mRNA expression in male Lin−Sca1+ progenitor cells, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT–PCR (n = 3). ****P = 1 × 10−10 (Days 2 and 4), ****P = 4 × 10−8 (Day 8) in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). F. mRNA expression in male Lin−Sca1+ cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR (n = 3, t-test). Data information: Data are presented as mean ± SEM except for (C) (individual data). Download figure Download PowerPoint To determine whether the induction of Cited4 expression is of functional importance for Rosi-stimulated progenitor differentiation and in particular for the oxidative/thermogenic adipocyte phenotype, we examined primary Lin−Sca1+ cells isolated from female Cited4−/− knockout mice, lacking the complete Cited4 coding sequence and Cited4 protein (Appendix Fig S1A and B). Whereas only a trend of reduced mRNA expression of adipogenic marker genes could be detected in Cited4−/− cells after 8 days of differentiation, we observed a significant reduction of key thermogenic marker genes, i.e., Ucp1, Cpt1b, and Dio2, with Ucp1 mRNA decreased by more than threefold (Fig 1C). The effects of Cited4 knockout on thermogenic markers were comparable in a direct comparison between pioglitazone and Rosi, at least at a higher pioglitazone dose, which was required for the effective stimulation of thermogenic expression (Fig EV1D). Intriguingly, there was no effect of the Cited4 knockout on the differentiation of progenitors from male mice despite the induction of Cited4 by Rosi, indicating a sex-specific requirement (Fig EV1E and F). We next sought to validate these findings in the human system. Indeed, Rosi treatment during the differentiation of stromal vascular fraction (SVF) cells, freshly isolated from subcutaneous fat, induced CITED4 expression by maximally 15-fold independently of donor gender (Figs 1D and EV2A–C). Compared to mouse cells, maximal induction occurred late and declined only marginally at 14 days of differentiation. CITED4 upregulation paralleled the induction of the general differentiation marker ADIPOQ by Rosi (Fig EV2B). However, it is noteworthy that in the absence of Rosi, CITED4 expression tended to positively correlate with UCP1 rather than ADIPOQ mRNA (Fig 1E and F). Importantly, Rosi-mediated UCP1 mRNA was markedly diminished in female cells upon CITED4 knockdown using independent siRNAs and this was accompanied by a milder but significant reduction in CPT1B and PPARG (Fig 1G), whereas SLC2A4 and ADIPOQ were only affected by one siRNA. In contrast to mouse cells, CITED4 knockdown resulted in reduced UCP1 levels in male cells (Fig EV2D). Overall, the Rosi-dependent CITED4 expression and knockdown phenotype mirrored the murine data and indicate a conserved function of CITED4 in adipocyte progenitors despite differences possibly attributable to the strong dependency of human adipocyte differentiation on PPARg agonists. Click here to expand this figure. Figure EV2. Cited4 is a target of rosiglitazone in murine and human adipocyte progenitors promoting beige differentiation A. Phase contrast microscopy of primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for 14 days (representative of n = 5 patients). Scale bar is 100 μm. B, C. mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle, as determined by qRT–PCR (n = 5 patients). (B) ****P = 1 × 10−10 (Day 2), ****P = 5 × 10−8 (Day 6), ****P = 1 × 10−9 (Days 10 and 14), (C) ****P = 7 × 10−7 (Day 6), ****P = 1 × 10−9 (Days 10 and 14) in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). D. mRNA expression in primary SVF cells from human male subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT–PCR (n = 3). ***P = 0.0002 (siCITED4.1 CITED4), ***P = 0.0003 (siCITED4.2 CITED4), **P = 0.005 (UCP1), *P = 0.01 (CIDEA), ***P = 0.0006 (CITED4.1 CPT1B), **P = 0.007 (CPT1B), ***P = 0.0006 (CITED4.1 SLC2A4),**P = 0.009 (ADIPOQ) in ANOVA with Tukey's posttests (vs. siCtrl). E. GFP fluorescence intensity distribution of Lin−Sca1+ progenitor cells 24 hours after transfection with GFP mRNA, determined by flow cytometry (compared to non-transfected cells). F. mRNA expression in female Cited4F/F Lin−Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR (n = 3). t-test Cre vs. Ctrl (Rosi), **P = 0.002 (Cited4), *P = 0.039 (Adipoq), *P = 0.015 (Ucp1), *P = 0.011 (Cidea), **P = 0.004 (Cpt1b), *P = 0.011 (Dio2). G. mRNA expression in female Cited4F/F Lin−Sca1+ progenitor cells transfected with Cre or control mRNA 3 days after induction of differentiation in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR (n = 4). t-test Cre vs. Ctrl, ***P = 0.0005 (Cited4), **P = 0.009 (Cpt1b). Data information: Data are presented as mean ± SEM except for (E) (individual data). Download figure Download PowerPoint We went on to interrogate the murine phenotype and tested whether it was due to a cell-autonomous function of Cited4 in progenitors. To this end, we transfected Lin−Sca1+ cells from Cited4F/F mice with Cre recombinase prior to differentiation induction, which resulted in the efficient disruption of the floxed Cited4 alleles and loss of expression (Fig EV2E and F). In resemblance with the constitutive knockout, Cre-transfected cells showed reduced expression of Ucp1, Cpt1b, and Dio2 upon differentiation, with no effect on general adipogenic markers (Fig EV2F). In contrast, transfection of Cited4F/F cells with Cre after differentiation induction did not have any considerable effects on differentiation markers (Fig EV2G), suggesting that Cited4 exerts its essential function in immature progenitors. We further analyzed the consequences of Cited4 deficiency in immature cells for differentiation by immunofluorescence. Whereas there was no difference between genotypes in the total cell number, we observed a reduction in the number of LipidTOX+ adipocytes in Cited4F/F + Cre cells by 1/3 (Fig 2A–C). This could be due to the preferential loss of beige adipocytes, given the lack of robust effects on general adipogenic markers. Accordingly, Western blotting revealed a robust reduction in Ucp1 protein in Cited4F/F + Cre cells (Fig 2D and E). Finally, we assessed whether Cited4 knockout could affect mitochondrial respiration as a key function of beige adipocyte metabolism. Basal and maximal mitochondrial respiration was indistinguishable between genotypes arguing against reduced mitochondrial content or a general defect in mitochondrial oxidation (Fig 2F). Treatment with the β3-adrenoreceptor agonist CL-316,243 (CL), which stimulates Ucp1 activity and mitochondrial uncoupling, increased oxygen consumption in wild-type cells. This was abolished in Cited4-knockout cells, suggesting a specific defect in uncoupled respiration (Fig 2F and G). Figure 2. Cited4 deficiency in progenitors affects Ucp1 protein expression and β3-adrenoreceptor mediated uncoupled respiration A–C. Quantitative fluorescence microscopy of LipidTOX- and DAPI-stained female Cited4F/F Lin−Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days (n = 5). **P = 0.002 in t-test (Cre vs. Ctrl). Scale bar is 10 μm. D, E. Ucp1 expression
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