- Discovering metabolic disease gene interactions by correlated effects on cellular morphology
Yang Jiao, Umer Ahmed, M.F. Michelle Sim, Andrea Bejar, Xiaolan Zhang, M. Mesbah Uddin Talukder, Robert Rice, Jason Flannick, Anna I. Podgornaia, Dermot F. Reilly, Jesse M. Engreitz, Maria Kost-Alimova, Kate Hartland, Josep-Maria Mercader, Sara Georges, Vilas Wagh, Marija Tadin-Strapps, John G. Doench, J. Michael Edwardson, Justin J. Rochford, Evan D. Rosen, Amit R. Majithia
Objective: Impaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to
identify novel diseaseegene interactions during adipocyte differentiation.
Methods: Genes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes.
The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte
differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and
used to construct morphologic profiles for each gene.
Results: Over 107 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14
genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and
AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel proteine
protein and gene regulatory interactions.
Conclusions: A morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach
enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown.
Mol Metab. 2019 Jun; 24: 108–119. https://doi.org/10.1016/j.molmet.2019.03.001. Epub 2019 Mar 13.
- Exposure of adipocytes to bisphenol-A in vitro interferes with insulin action without enhancing adipogenesis
De Filippis E, Li T, Rosen ED
Bisphenol-A (BPA) is a lipophilic compound widely used in the manufacture of plastic items and thought to play a role in the growing obesity epidemic. Recent publications suggest that BPA may have a pro-adipogenic effect. Here we explore the effect of low, but environmentally relevant, concentrations of BPA on adipogenesis using a variety of cellular models. Mouse 3T3-L1, C3H10T1/2 and human adipose-derived stromal cells (hADSCs) were cultured with BPA concentrations ranging from 0.1nM to 100μM. We failed to observe positive effects on differentiation at any dose or in any model. 3T3-L1 adipocytes differentiated with high concentrations of BPA showed decreased mRNA expression of several adipocyte markers. Mature adipocytes differentiated in the presence of BPA were insulin resistant, with an approximate 25% reduction in insulin-stimulated glucose uptake. This was accompanied by a significant decrease in insulin-stimulated Akt phosphorylation, and an increase in mRNA levels of inflammatory markers (i.e. IL-6, TNFα). In conclusion, low, but environmentally relevant, doses of BPA may contribute to the development of a chronic, low-grade inflammatory state in exposed adipocytes, which in turn may affect adipose tissue insulin sensitivity, independent of adipogenesis. These studies suggest an alternative mechanism by which BPA may contribute to the development of obesity.
PLoS One. 2018 Aug 22;13(8):e0201122. doi: 10.1371/journal.pone.0201122. eCollection 2018
- Brown Adipose Tissue Controls Skeletal Muscle Function via the Secretion of Myostatin
Kong X, Yao T, Zhou P, Kazak L, Tenen D, Lyubetskaya A, Dawes BA, Tsai L, Kahn BB, Spiegelman BM, Liu T, Rosen ED
Skeletal muscle and brown adipose tissue (BAT) are functionally linked, as exercise increases browning via secretion of myokines. It is unknown whether BAT affects muscle function. Here, we find that loss of the transcription factor IRF4 in BAT (BATI4KO) reduces exercise capacity, mitochondrial function, ribosomal protein synthesis, and mTOR signaling in muscle and causes tubular aggregate formation. Loss of IRF4 induces myogenic gene expression in BAT, including the secreted factor myostatin, a known inhibitor of muscle function. Reducing myostatin via neutralizing antibodies or soluble receptor rescues the exercise capacity of BATI4KO mice. In addition, overexpression of IRF4 in brown adipocytes reduces serum myostatin and increases exercise capacity in muscle. Finally, mice housed at thermoneutrality have reduced IRF4 in BAT, lower exercise capacity, and elevated serum myostatin; these abnormalities are corrected by excising BAT. Collectively, our data point to an unsuspected level of BAT-muscle crosstalk driven by IRF4 and myostatin.
Cell Metab. 2018 Oct 2;28(4):631-643.e3. doi: 10.1016/j.cmet.2018.07.004. Epub 2018 Aug 2
- Warming Induces Significant Reprogramming of Beige, but Not Brown, Adipocyte Cellular Identity
Roh HC, Tsai LTY, Shao M, Tenen D, Shen Y, Kumari M, Lyubetskaya A, Jacobs C, Dawes B, Gupta RK, Rosen ED
Beige and brown adipocytes generate heat in response to reductions in ambient temperature. When warmed, both beige and brown adipocytes exhibit morphological “whitening,” but it is unknown whether or to what extent this represents a true shift in cellular identity. Using cell-type-specific profiling in vivo, we uncover a unique paradigm of temperature-dependent epigenomic plasticity of beige, but not brown, adipocytes, with conversion from a brown to a white chromatin state. Despite this profound shift in cellular identity, warm whitened beige adipocytes retain an epigenomic memory of prior cold exposure defined by an array of poised enhancers that prime thermogenic genes for rapid response during a second bout of cold exposure. We further show that a transcriptional cascade involving glucocorticoid receptor and Zfp423 can drive warm-induced whitening of beige adipocytes. These studies identify the epigenomic and transcriptional bases of an extraordinary example of cellular plasticity in response to environmental signals.
Cell Metab. 2018 May 1;27(5):1121-1137.e5. doi: 10.1016/j.cmet.2018.03.005. Epub 2018 Apr 12
- The Synergy between Palmitate and TNF-α for CCL2 Production Is Dependent on the TRIF/IRF3 Pathway: Implications for Metabolic Inflammation
Ahmad R, Al-Roub A, Kochumon S, Akther N, Thomas R, Kumari M, Koshy MS, Tiss A, Hannun YA, Tuomilehto J, Sindhu S, Rosen ED.
The chemokine CCL2 (also known as MCP-1) is a key regulator of monocyte infiltration into adipose tissue, which plays a central role in the pathophysiology of obesity-associated inflammation and insulin resistance. It remains unclear how CCL2 production is upregulated in obese humans and rodents. Because elevated levels of the free fatty acid (FFA) palmitate and TNF-α have been reported in obesity, we studied whether these agents interact to trigger CCL2 production. Our data show that treatment of THP-1 and primary human monocytic cells with palmitate and TNF-α led to a marked increase in CCL2 production compared with either treatment alone. Mechanistically, we found that cooperative production of CCL2 by palmitate and TNF-α did not require MyD88, but it was attenuated by blocking TLR4 or TRIF. IRF3-deficient cells did not show synergistic CCL2 production in response to palmitate/TNF-α. Moreover, IRF3 activation by polyinosinic-polycytidylic acid augmented TNF-α-induced CCL2 secretion. Interestingly, elevated NF-κB/AP-1 activity resulting from palmitate/TNF-α costimulation was attenuated by TRIF/IRF3 inhibition. Diet-induced C57BL/6 obese mice with high FFAs levels showed a strong correlation between TNF-α and CCL2 in plasma and adipose tissue and, as expected, also showed increased adipose tissue macrophage accumulation compared with lean mice. Similar results were observed in the adipose tissue samples from obese humans. Overall, our findings support a model in which elevated FFAs in obesity create a milieu for TNF-α to trigger CCL2 production via the TLR4/TRIF/IRF3 signaling cascade, representing a potential contribution of FFAs to metabolic inflammation.
J Immunol. 2018 May 15;200(10):3599-3611. doi: 10.4049/jimmunol.1701552. Epub 2018 Apr 9
- Adipocyte glucocorticoid receptor is important in lipolysis and insulin resistance due to exogenous steroids, but not insulin resistance caused by high fat feeding
Shen Y, Roh HC, Kumari M, Rosen ED.
OBJECTIVE: The critical role of adipose tissue in energy and nutrient homeostasis is influenced by many external factors, including overnutrition, inflammation, and exogenous hormones. Prior studies have suggested that glucocorticoids (GCs) in particular are major drivers of physiological and pathophysiological changes in adipocytes. In order to determine whether these effects directly require the glucocorticoid receptor (GR) within adipocytes, we generated adipocyte-specific GR knockout (AGRKO) mice.
METHODS: AGRKO and control mice were fed chow or high fat diet (HFD) for 14 weeks. Alternatively, AGRKO and control mice were injected with dexamethasone for two months. Glucose tolerance, insulin sensitivity, adiposity, lipolysis, thermogenesis, and insulin signaling were assessed.
RESULTS: We find that obesity, insulin resistance, and dysglycemia associated with high fat feeding do not require an intact GR in the adipocyte. However, exogenous dexamethasone (Dex) promotes metabolic dysfunction in mice, and this effect is reduced in mice lacking GR in adipocytes. The ability of Dex to promote “whitening” of brown fat is also reduced in these animals. We also show that GR is required for β-adrenergic and cold stimulation-mediated lipolysis via expression of the key lipolytic enzyme ATGL.
CONCLUSIONS: Our data suggest that the GR plays a role in normal adipose physiology via effects on lipolysis and mediates at least some of the adverse effects of exogenous steroids on metabolic function. The data also indicate that intra-adipocyte GR plays less of a role than previously believed in the local and systemic pathology associated with overnutrition.
Mol Metab. 2017 Oct;6(10):1150-1160. doi: 10.1016/j.molmet.2017.06.013. Epub 2017 Jun 29
- Simultaneous Transcriptional and Epigenomic Profiling from Specific Cell Types within Heterogeneous Tissues In Vivo
Roh HC, Tsai LT, Lyubetskaya A, Tenen D, Kumari M, Rosen ED
Epigenomic mechanisms direct distinct gene expression programs for different cell types. Various in vivo tissues have been subjected to epigenomic analysis; however, these studies have been limited by cellular heterogeneity, resulting in composite gene expression and epigenomic profiles. Here, we introduce “NuTRAP,” a transgenic mouse that allows simultaneous isolation of cell-type-specific translating mRNA and chromatin from complex tissues. Using NuTRAP, we successfully characterize gene expression and epigenomic states of various adipocyte populations in vivo, revealing significant differences compared to either whole adipose tissue or in vitro adipocyte cell lines. We find that chromatin immunoprecipitation sequencing (ChIP-seq) using NuTRAP is highly efficient, scalable, and robust with even limited cell input. We further demonstrate the general utility of NuTRAP by analyzing hepatocyte-specific epigenomic states. The NuTRAP mouse is a resource that provides a powerful system for cell-type-specific gene expression and epigenomic profiling.
Cell Rep. 2017 Jan 24;18(4):1048-1061. doi: 10.1016/j.celrep.2016.12.087
- Dnmt3a is an epigenetic mediator of adipose insulin resistance
You D, Nilsson E, Tenen DE, Lyubetskaya A, Lo JC, Jiang R, Deng J, Dawes BA, Vaag A, Ling C, Rosen ED, Kang S.
Insulin resistance results from an intricate interaction between genetic make-up and environment, and thus may be orchestrated by epigenetic mechanisms like DNA methylation. Here, we demonstrate that DNA methyltransferase 3a (Dnmt3a) is both necessary and sufficient to mediate insulin resistance in cultured mouse and human adipocytes. Furthermore, adipose-specific Dnmt3a knock-out mice are protected from diet-induced insulin resistance and glucose intolerance without accompanying changes in adiposity. Unbiased gene profiling studies revealed Fgf21 as a key negatively regulated Dnmt3a target gene in adipocytes with concordant changes in DNA methylation at the Fgf21 promoter region. Consistent with this, Fgf21 can rescue Dnmt3a-mediated insulin resistance, and DNA methylation at the FGF21 locus was elevated in human subjects with diabetes and correlated negatively with expression of FGF21 in human adipose tissue. Taken together, our data demonstrate that adipose Dnmt3a is a novel epigenetic mediator of insulin resistance in vitro and in vivo.
Elife. 2017 Nov 1;6. pii: e30766. doi: 10.7554/eLife.30766.
- Epigenomic and transcriptional control of insulin resistance
Insulin resistance is one of the defining features of type 2 diabetes and the metabolic syndrome and accompanies many other clinical conditions, ranging from obesity to lipodystrophy to glucocorticoid excess. Extraordinary efforts have gone into defining the mechanisms that underlie insulin resistance, with most attention focused on altered signalling as well as mitochondrial and endoplasmic reticulum stress. Here, nuclear mechanisms of insulin resistance, including transcriptional and epigenomic effects, will be discussed. Three levels of control involving transcription factors, transcriptional cofactors, and chromatin-modifying enzymes will be considered. Well-studied examples of the first include PPAR-γ in adipose tissue and the glucocorticoid receptor and FoxO1 in a variety of insulin-sensitive tissues. These proteins work in concert with cofactors such as PGC-1α and CRTC2, and chromatin-modifying enzymes including DNA methyltransferases and histone acetyltransferases, to regulate key genes that promote insulin-stimulated glucose uptake, gluconeogenesis or other pathways that affect systemic insulin action. Furthermore, genetic variation associated with increased risk of type 2 diabetes is often related to altered transcription factor binding, either by affecting the transcription factor itself, or more commonly by changing the binding affinity of a noncoding regulatory region. Finally, several avenues for therapeutic exploitation in the battle against metabolic disease will be discussed, including small-molecule inhibitors and activators of these factors and their related pathways.
J Intern Med. 2016 Nov;280(5):443-456. doi: 10.1111/joim.12547. Epub 2016 Oct 14
- A molecular census of arcuate hypothalamus and median eminence cell types
Campbell JN, Macosko EZ, Fenselau H, Pers TH, Lyubetskaya A, Tenen D, Goldman M, Verstegen AM, Resch JM, McCarroll SA, Rosen ED, Lowell BB, Tsai LT.
The hypothalamic arcuate-median eminence complex (Arc-ME) controls energy balance, fertility and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a new leptin-sensing neuron population, multiple agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) subtypes, and an orexigenic somatostatin neuron population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type-specific responses to energy status, including distinct responses in AgRP and POMC neuron subtypes. Finally, integrating our data with human genome-wide association study data implicates two previously unknown neuron populations in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred.
Nat Neurosci. 2017 Mar;20(3):484-496. doi: 10.1038/nn.4495. Epub 2017 Feb 6.
- IRF3 promotes adipose inflammation and insulin resistance and represses browning
Kumari M, Wang X, Lantier L, Lyubetskaya A, Eguchi J, Kang S, Tenen D, Roh HC, Kong X, Kazak L, Ahmad R, Rosen ED
The chronic inflammatory state that accompanies obesity is a major contributor to insulin resistance and other dysfunctional adaptations in adipose tissue. Cellular and secreted factors promote the inflammatory milieu of obesity, but the transcriptional pathways that drive these processes are not well described. Although the canonical inflammatory transcription factor NF-κB is considered to be the major driver of adipocyte inflammation, members of the interferon regulatory factor (IRF) family may also play a role in this process. Here, we determined that IRF3 expression is upregulated in the adipocytes of obese mice and humans. Signaling through TLR3 and TLR4, which lie upstream of IRF3, induced insulin resistance in murine adipocytes, while IRF3 knockdown prevented insulin resistance. Furthermore, improved insulin sensitivity in IRF3-deficient mice was associated with reductions in intra-adipose and systemic inflammation in the high fat-fed state, enhanced browning of subcutaneous fat, and increased adipose expression of GLUT4. Taken together, the data indicate that IRF3 is a major transcriptional regulator of adipose inflammation and is involved in maintaining systemic glucose and energy homeostasis.
J Clin Invest. 2016 Aug 1;126(8):2839-54. doi: 10.1172/JCI86080. Epub 2016 Jul 11
- Burning Fat by Bugging the System
- Nuclear Mechanisms of Insulin Resistance
Kang S, Tsai LT, Rosen ED
Insulin resistance is a sine qua non of type 2 diabetes and is associated with many other clinical conditions. Decades of research into mechanisms underlying insulin resistance have mostly focused on problems in insulin signal transduction and other mitochondrial and cytosolic pathways. By contrast, relatively little attention has been focused on transcriptional and epigenetic contributors to insulin resistance, despite strong evidence that such nuclear mechanisms play a major role in the etiopathogenesis of this condition. In this review, we summarize the evidence for nuclear mechanisms of insulin resistance, focusing on three transcription factors with a major impact on insulin action in liver, muscle, and fat.
Trends Cell Biol. 2016 May;26(5):341-351. doi: 10.1016/j.tcb.2016.01.002. Epub 2016 Jan 25
- Making Biological Sense of GWAS Data: Lessons from the FTO Locus
Herman MA, Rosen ED
GWAS have yielded many candidate loci for complex diseases like obesity, but interpreting the biological context of these findings has been difficult. Claussnitzer et al. (2015) use a sophisticated combination of bioinformatic and experimental approaches to address this bottleneck for variants in the FTO locus that associate with obesity.
Cell Metab. 2015 Oct 6;22(4):538-9. doi: 10.1016/j.cmet.2015.09.018
- Two paths to fat
Excess caloric intake leads to both the growth of existing fat cells and the generation of new adipocytes. New findings show that PI3K-Akt2 signalling is involved in the differentiation of adult adipose precursor cells—a pathway not required for adipogenesis in the embryo.
Nat Cell Biol. 2015 Apr;17(4):360-1. doi: 10.1038/ncb3133
- Identification of nuclear hormone receptor pathways causing insulin resistance by transcriptional and epigenomic analysis
Kang S, Tsai LT, Zhou Y, Evertts A, Xu S, Griffin MJ, Issner R, Whitton HJ, Garcia BA, Epstein CB, Mikkelsen TS, Rosen ED.
Insulin resistance is a cardinal feature of Type 2 diabetes (T2D) and a frequent complication of multiple clinical conditions, including obesity, ageing and steroid use, among others. How such a panoply of insults can result in a common phenotype is incompletely understood. Furthermore, very little is known about the transcriptional and epigenetic basis of this disorder, despite evidence that such pathways are likely to play a fundamental role. Here, we compare cell autonomous models of insulin resistance induced by the cytokine tumour necrosis factor-α or by the steroid dexamethasone to construct detailed transcriptional and epigenomic maps associated with cellular insulin resistance. These data predict that the glucocorticoid receptor and vitamin D receptor are common mediators of insulin resistance, which we validate using gain- and loss-of-function studies. These studies define a common transcriptional and epigenomic signature in cellular insulin resistance enabling the identification of pathogenic mechanisms.
Nat Cell Biol. 2015 Jan;17(1):44-56. doi: 10.1038/ncb3080. Epub 2014 Dec 15
- IRF4 is a key thermogenic transcriptional partner of PGC-1α
Kong X, Banks A, Liu T, Kazak L, Rao RR, Cohen P, Wang X, Yu S, Lo JC, Tseng YH, Cypess AM, Xue R, Kleiner S, Kang S, Spiegelman BM, Rosen ED
Brown fat can reduce obesity through the dissipation of calories as heat. Control of thermogenic gene expression occurs via the induction of various coactivators, most notably PGC-1α. In contrast, the transcription factor partner(s) of these cofactors are poorly described. Here, we identify interferon regulatory factor 4 (IRF4) as a dominant transcriptional effector of thermogenesis. IRF4 is induced by cold and cAMP in adipocytes and is sufficient to promote increased thermogenic gene expression, energy expenditure, and cold tolerance. Conversely, knockout of IRF4 in UCP1(+) cells causes reduced thermogenic gene expression and energy expenditure, obesity, and cold intolerance. IRF4 also induces the expression of PGC-1α and PRDM16 and interacts with PGC-1α, driving Ucp1 expression. Finally, cold, β-agonists, or forced expression of PGC-1α are unable to cause thermogenic gene expression in the absence of IRF4. These studies establish IRF4 as a transcriptional driver of a program of thermogenic gene expression and energy expenditure.
Cell. 2014 Jul 3;158(1):69-83. doi: 10.1016/j.cell.2014.04.049
- Adipocyte-specific transgenic and knockout models
Kang S, Kong X, Rosen ED
Adipose tissue plays a major role in metabolic homeostasis, which it coordinates through a number of local and systemic effectors. The burgeoning epidemic of metabolic disease, especially obesity and type 2 diabetes, has focused attention on the adipocyte. In this chapter, we review strategies for genetic overexpression and knockout of specific genes in adipose tissue. We also discuss these strategies in the context of different types of adipocytes, including brown, beige, and white fat cells.
Methods Enzymol. 2014;537:1-16. doi: 10.1016/B978-0-12-411619-1.00001-X
- Early B-cell factor-1 (EBF1) is a key regulator of metabolic and inflammatory signaling pathways in mature adipocytes.
Griffin MJ, Zhou Y, Kang S, Zhang X, Mikkelsen TS, Rosen ED
EBF1 plays a crucial role in early adipogenesis; however, despite high expression in mature adipocytes, its function in these cells is currently unknown. To identify direct and indirect EBF1 targets in fat, we undertook a combination of transcriptional profiling of EBF1-deficient adipocytes and genome-wide EBF1 location analysis. Our results indicate that many components of metabolic and inflammatory pathways are positively and directly regulated by EBF1, including PI3K/AKT, MAPK, and STAT1 signaling. Accordingly, we observed significant reduction of multiple signaling events in EBF1 knockdown cells as well as a reduction in insulin-stimulated glucose uptake and lipogenesis. Inflammatory signaling, gene expression, and secretion of inflammatory cytokines were also significantly affected by loss of EBF1 in adipocytes, although ChIP-sequencing results suggest that these actions are indirect. We also found that EBF1 occupies some 35,000 sites in adipocytes, most of which occur in enhancers. Significantly, comparison with three other published EBF1 ChIP-sequencing data sets in B-cells reveals both gene- and cell type-specific patterns of EBF1 binding. These results advance our understanding of the transcriptional mechanisms regulating signaling pathways in mature fat cells and indicate that EBF1 functions as a key integrator of signal transduction, inflammation, and metabolism.
J Biol Chem. 2013 Dec 13;288(50):35925-39. doi: 10.1074/jbc.M113.491936. Epub 2013 Oct 30
- Interferon regulatory factor 4 regulates obesity-induced inflammation through regulation of adipose tissue macrophage polarization
Eguchi J, Kong X, Tenta M, Wang X, Kang S, Rosen ED
Interferon regulatory factors (IRFs) play functionally diverse roles in the transcriptional regulation of the immune system. We have previously shown that several IRFs are regulators of adipogenesis and that IRF4 is a critical transcriptional regulator of adipocyte lipid handling. However, the functional role of IRF4 in adipose tissue macrophages (ATMs) remains unclear, despite high expression there. Here we show that IRF4 expression is regulated in primary macrophages and in ATMs of high-fat diet-induced obese mice. Irf4(-/-) macrophages produce higher levels of proinflammatory cytokines, including interleukin-1β and tumor necrosis factor-α, in response to fatty acids. In coculture experiments, IRF4 deletion in macrophages leads to reduced insulin signaling and glucose uptake in 3T3-L1 adipocytes. To determine the macrophage-specific function of IRF4 in the context of obesity, we generated myeloid cell-specific IRF4 knockout mice, which develop significant insulin resistance on a high-fat diet, despite no difference in adiposity. This phenotype is associated with increased expression of inflammatory genes and decreased insulin signaling in adipose tissue, skeletal muscle, and liver. Furthermore, Irf4(-/-) ATMs express markers suggestive of enhanced M1 polarization. These findings indicate that IRF4 is a negative regulator of inflammation in diet-induced obesity, in part through regulation of macrophage polarization.
Diabetes. 2013 Oct;62(10):3394-403. doi: 10.2337/db12-1327. Epub 2013 Jul 8
- New insights into adipocyte-specific leptin gene expression
Wrann CD, Rosen ED
The adipocyte-derived hormone leptin is a critical regulator of many physiological functions, ranging from satiety to immunity. Surprisingly, very little is known about the transcriptional pathways that regulate adipocyte-specific expression of leptin. In a recent published study, we pursued a strategy integrating BAC transgenic reporter mice, in vitro reporter assays, and chromatin state mapping to locate an adipocyte-specific cis-element upstream of the LEP gene in human fat cells. Quantitative proteomics (stable isotope labeling by amino acids in cell culture, SILAC) with affinity enrichment of protein-DNA complexes identified the transcription factor FOSL2 as a specific binder to the identified region. We confirmed that FOSL2 is an important regulator of LEP gene expression in vitro and in vivo using cell culture models and genetic mouse models. In this commentary, we discuss the transcriptional regulation of LEP gene expression, our strategy to identify an adipocyte-specific cis-regulatory element and the transcription factor(s) responsible for LEP gene expression. We also discuss our data on FOSL2 and leptin levels in physiology and pathophysiology. We speculate on unanswered questions and future directions.
Adipocyte. 2012 Jul 1;1(3):168-172
- Regulation of early adipose commitment by Zfp521
Kang S, Akerblad P, Kiviranta R, Gupta RK, Kajimura S, Griffin MJ, Min J, Baron R, Rosen ED
While there has been significant progress in determining the transcriptional cascade involved in terminal adipocyte differentiation, less is known about early events leading to lineage commitment and cell fate choice. It has been recently discovered that zinc finger protein 423 (Zfp423) is an early actor in adipose determination. Here, we show that a close paralog of Zfp423, Zfp521, acts as a key regulator of adipose commitment and differentiation in vitro and in vivo. Zfp521 exerts its actions by binding to early B cell factor 1 (Ebf1), a transcription factor required for the generation of adipocyte progenitors, and inhibiting the expression of Zfp423. Overexpression of Zfp521 in cells greatly inhibits adipogenic potential, whereas RNAi-mediated knock-down or genetic ablation of Zfp521 enhances differentiation. In addition, Zfp521⁻/⁻ embryos exhibit increased mass of interscapular brown adipose tissue and subcutaneous white adipocytes, a cell autonomous effect. Finally, Ebf1 participates in a negative feedback loop to repress Zfp521 as differentiation proceeds. Because Zfp521 is known to promote bone development, our results suggest that it acts as a critical switch in the commitment decision between the adipogenic and osteogenic lineages.
PLoS Biol. 2012;10(11):e1001433. doi: 10.1371/journal.pbio.1001433. Epub 2012 Nov 27
- FOSL2 promotes leptin gene expression in human and mouse adipocytes
Wrann CD, Eguchi J, Bozec A, Xu Z, Mikkelsen T, Gimble J, Nave H, Wagner EF, Ong SE, Rosen ED
The adipocyte-derived hormone leptin is a critical regulator of many physiological functions, ranging from satiety to immunity. Surprisingly, very little is known about the transcriptional pathways that regulate adipocyte-specific expression of leptin. Here, we report studies in which we pursued a strategy integrating BAC transgenic reporter mice, reporter assays, and chromatin state mapping to locate an adipocyte-specific cis-element upstream of the leptin (LEP) gene in human fat cells. Quantitative proteomics with affinity enrichment of protein-DNA complexes identified the transcription factor FOS-like antigen 2 (FOSL2) as binding specifically to the identified region, a result that was confirmed by ChIP. Knockdown of FOSL2 in human adipocytes decreased LEP expression, and overexpression of Fosl2 increased Lep expression in mouse adipocytes. Moreover, the elevated LEP expression observed in obesity correlated well with increased FOSL2 levels in mice and humans, and adipocyte-specific genetic deletion of Fosl2 in mice reduced Lep expression. Taken together, these data identify FOSL2 as a critical regulator of leptin expression in adipocytes.
J Clin Invest. 2012 Mar;122(3):1010-21. doi: 10.1172/JCI58431. Epub 2012 Feb 13
- A minor role for lipocalin 2 in high-fat diet-induced glucose intolerance
Jun LS, Siddall CP, Rosen ED
Adipose tissue controls energy homeostasis and systemic insulin sensitivity through the elaboration of a series of cytokines and hormones, collectively termed “adipokines.” We and others have identified Lcn2 as a novel adipokine, but its exact role in obesity-induced insulin resistance remains controversial. The aim of this study was to examine the metabolic phenotype of Lcn2(-/-) mice to clarify the role of Lcn2 in metabolism. Male and female Lcn2(-/-) and wild-type (WT) littermates were placed on either chow or high-fat diet (HFD) to characterize their metabolic phenotype. Studies included body weight and body composition, glucose and insulin tolerance tests, and adipokine expression studies in serum and in white adipose tissue (WAT). Neither chow nor HFD cohorts showed any differences in body weight or body composition. Chow-fed Lcn2(-/-) mice did not exhibit any difference in glucose homeostasis compared with WT mice. Fasting serum glucose levels were lower in the chow-fed Lcn2(-/-) mice, but this finding was not seen in the HFD cohort. Serum adiponectin, leptin, resistin, and RBP4 levels were not different between WT and Lcn2(-/-) on chow diet. HFD-fed male Lcn2(-/-) mice did display a small improvement in glucose tolerance, but no difference in insulin sensitivity was seen in either male or female Lcn2(-/-) mice on HFD. We conclude that the global ablation of Lcn2 has a minimal effect on obesity-associated glucose intolerance but does not appear to affect either age- or obesity-mediated insulin resistance in vivo.
Am J Physiol Endocrinol Metab. 2011 Nov;301(5):E825-35. doi: 10.1152/ajpendo.00147.2011. Epub 2011 Jul 19
- Transcriptional control of adipose lipid handling by IRF4
Eguchi J, Wang X, Yu S, Kershaw EE, Chiu PC, Dushay J, Estall JL, Klein U, Maratos-Flier E, Rosen ED
Adipocytes store triglyceride during periods of nutritional affluence and release free fatty acids during fasting through coordinated cycles of lipogenesis and lipolysis. While much is known about the acute regulation of these processes during fasting and feeding, less is understood about the transcriptional basis by which adipocytes control lipid handling. Here, we show that interferon regulatory factor 4 (IRF4) is a critical determinant of the transcriptional response to nutrient availability in adipocytes. Fasting induces IRF4 in an insulin- and FoxO1-dependent manner. IRF4 is required for lipolysis, at least in part due to direct effects on the expression of adipocyte triglyceride lipase and hormone-sensitive lipase. Conversely, reduction of IRF4 enhances lipid synthesis. Mice lacking adipocyte IRF4 exhibit increased adiposity and deficient lipolysis. These studies establish a link between IRF4 and the disposition of calories in adipose tissue, with consequences for systemic metabolic homeostasis.
Cell Metab. 2011 Mar 2;13(3):249-59. doi: 10.1016/j.cmet.2011.02.005
- Comparative epigenomic analysis of murine and human adipogenesis
Mikkelsen TS, Xu Z, Zhang X, Wang L, Gimble JM, Lander ES, Rosen ED
We report the generation and comparative analysis of genome-wide chromatin state maps, PPARγ and CTCF localization maps, and gene expression profiles from murine and human models of adipogenesis. The data provide high-resolution views of chromatin remodeling during cellular differentiation and allow identification of thousands of putative preadipocyte- and adipocyte-specific cis-regulatory elements based on dynamic chromatin signatures. We find that the specific locations of most such elements differ between the two models, including at orthologous loci with similar expression patterns. Based on sequence analysis and reporter assays, we show that these differences are determined, in part, by evolutionary turnover of transcription factor motifs in the genome sequences and that this turnover may be facilitated by the presence of multiple distal regulatory elements at adipogenesis-dependent loci. We also utilize the close relationship between open chromatin marks and transcription factor motifs to identify and validate PLZF and SRF as regulators of adipogenesis.
Cell. 2010 Oct 1;143(1):156-69. doi: 10.1016/j.cell.2010.09.006
- The orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II is a critical regulator of adipogenesis
Xu Z, Yu S, Hsu CH, Eguchi J, Rosen ED
The orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII; Nr2f2) is expressed in adipose tissue in vivo and declines during differentiation. Overexpression of COUP-TFII prevents adipogenesis, whereas shRNA-mediated reduction of COUP-TFII promotes differentiation, as shown by increased lipid accumulation and elevated expression of fat cell marker proteins. Furthermore, reduction of COUP-TFII allows uncommitted fibroblasts to be differentiated into fat cells. COUP-TFII represses the expression of a number of proadipogenic factors in adipocytes, with direct action noted at the CAAT enhancer-binding protein alpha promoter. We show that COUP-TFII acts downstream of hedgehog signaling and is required for the full antiadipogenic effect of this pathway. This effect is mediated in part by interaction with GATA factors. COUP-TFII and GATA2 are physically associated and repress target gene expression in an additive manner. Taken together, our data demonstrate that COUP-TFII represents an endogenous suppressor of adipogenesis, linking antiadipogenic extracellular signals to the core transcriptional cascade.
Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2421-6. doi: 10.1073/pnas.0707082105. Epub 2008 Feb 4
- Interferon regulatory factors are transcriptional regulators of adipogenesis
Eguchi J, Yan QW, Schones DE, Kamal M, Hsu CH, Zhang MQ, Crawford GE, Rosen ED
We have sought to identify transcriptional pathways in adipogenesis using an integrated experimental and computational approach. Here, we employ high-throughput DNase hypersensitivity analysis to find regions of altered chromatin structure surrounding key adipocyte genes. Regions that display differentiation-dependent changes in hypersensitivity were used to predict binding sites for proteins involved in adipogenesis. A high-scoring example was a binding motif for interferon regulatory factor (IRF) family members. Expression of all nine mammalian IRF mRNAs is regulated during adipogenesis, and several bind to the identified motifs in a differentiation-dependent manner. Furthermore, several IRF proteins repress differentiation. This analysis suggests an important role for IRF proteins in adipocyte biology and demonstrates the utility of this approach in identifying cis- and trans-acting factors not previously suspected to participate in adipogenesis.
Cell Metab. 2008 Jan;7(1):86-94. doi: 10.1016/j.cmet.2007.11.002
- The adipokine lipocalin 2 is regulated by obesity and promotes insulin resistance
Yan QW, Yang Q, Mody N, Graham TE, Hsu CH, Xu Z, Houstis NE, Kahn BB, Rosen ED.
OBJECTIVE: We identified lipocalin 2 (Lcn2) as a gene induced by dexamethasone and tumor necrosis factor-alpha in cultured adipocytes. The purpose of this study was to determine how expression of Lcn2 is regulated in fat cells and to ascertain whether Lcn2 could be involved in metabolic dysregulation associated with obesity.
RESEARCH DESIGN AND METHODS: We examined Lcn2 expression in murine tissues and in 3T3-L1 adipocytes in the presence and absence of various stimuli. We used quantitative Western blotting to observe Lcn2 serum levels in lean and obese mouse models. To assess effects on insulin action, we used retroviral delivery of short hairpin RNA to reduce Lcn2 levels in 3T3-L1 adipocytes.
RESULTS: Lcn2 is highly expressed by fat cells in vivo and in vitro. Expression of Lcn2 is elevated by agents that promote insulin resistance and is reduced by thiazolidinediones. The expression of Lcn2 is induced during 3T3-L1 adipogenesis in a CCAAT/enhancer-binding protein-dependent manner. Lcn2 serum levels are elevated in multiple rodent models of obesity, and forced reduction of Lcn2 in 3T3-L1 adipocytes improves insulin action. Exogenous Lcn2 promotes insulin resistance in cultured hepatocytes.
CONCLUSIONS: Lcn2 is an adipokine with potential importance in insulin resistance associated with obesity.
Diabetes. 2007 Oct;56(10):2533-40. Epub 2007 Jul 16.
- Critical role for Ebf1 and Ebf2 in the adipogenic transcriptional cascade
Jimenez MA, Akerblad P, Sigvardsson M, Rosen ED
The Ebf (O/E) family of helix-loop-helix transcription factors plays a significant role in B lymphocyte and neuronal development. The three primary members of this family, Ebf1, 2, and 3, are all expressed in adipocytes, and Ebf1 promotes adipogenesis when overexpressed in NIH 3T3 fibroblasts. Here we report that these three proteins have adipogenic potential in multiple cellular models and that peroxisome proliferator-activated receptor gamma (PPARgamma) is required for this effect, at least in part due to direct activation of the PPARgamma1 promoter by Ebf1. Ebf1 also directly binds to and activates the C/EBPalpha promoter, which exerts positive feedback on C/EBPdelta expression. Despite this, C/EBPalpha is dispensable for the adipogenic action of Ebf proteins. Ebf1 itself is induced by C/EBPbeta and delta, which bind and activate its promoter. Reduction of Ebf1 and Ebf2 proteins by specific short hairpin RNA blocks differentiation of 3T3-L1 cells, suggesting a critical role for these factors and the absence of functional redundancy between members of this family. Altogether, these data place Ebf1 within the known transcriptional cascade of adipogenesis and suggest critical roles for Ebf1 and Ebf2.
Mol Cell Biol. 2007 Jan;27(2):743-57. Epub 2006 Oct 23.
- New drugs from fat bugs?
Authors: Rosen ED
Signal transduction cascades, such as Hedgehog (Hh) signaling, are potentially important targets for new drugs. A new study in this issue of Cell Metabolism identifies hedgehog signaling in the formation of the Drosophila fly body and in mammalian adipogenesis.
Cell Metab. 2006 Jan;3(1):1-2
- The transcriptional basis of adipocyte development
Authors: Rosen ED
Adipogenesis is the developmental process by which a multipotent mesenchymal stem cell differentiates into a mature adipocyte. This process involves a highly regulated and coordinated cascade of transcription factors that together lead to the establishment of the differentiated state. In the presence of the correct hormonal cues, committed pre-adipocytes express the bZIP factors C/EBPb and C/EBPd. These factors in turn induce the expression of C/EBPa and peroxisome proliferator-activated receptor g (PPARg). C/EBPa and PPARg together promote differentiation by activating adipose-specific gene expression and by maintaining each others expression at high levels. We have investigated the relative contributions of PPARg and C/EBPa to adipogenesis by selectively ablating these genes in mouse embryonic fibroblasts (MEFs). MEFs that lack C/EBPa are able to undergo adipogenesis, but only when PPARg is ectopically expressed. Interestingly, these cells are not sensitive to the metabolic actions of insulin. By way of contrast, cells that lack PPARg are utterly incapable of adipogenic conversion, even when supplemented with high levels of C/EBPa. Our current investigations are centered on the identification of novel adipogenic transcription factors, utilizing a variety of techniques, ranging from BAC transgenics to computational approaches. These approaches will be discussed, along with the roles of some new transcriptional players in adipogenesis, including the O/E family of proteins.
Prostaglandins Leukot Essent Fatty Acids. 2005 Jul;73(1):31-4.
- Energy balance: a new role for PPARalpha
- The molecular control of adipogenesis, with special reference to lymphatic pathology
- Molecular mechanisms of adipocyte differentiation
Authors: Rosen ED
Ann Endocrinol (Paris). 2002 Apr;63(2 Pt 1):79-82.