Publications

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.

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.

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.

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.

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.

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.

Adipocytes have been studied with increasing intensity as a result of the emergence of obesity as a serious public health problem and the realization that adipose tissue serves as an integrator of various physiological pathways. In particular, their role in calorie storage makes adipocytes well suited to the regulation of energy balance. Adipose tissue also serves as a crucial integrator of glucose homeostasis. Knowledge of adipocyte biology is therefore crucial for understanding the pathophysiological basis of obesity and metabolic diseases such as type 2 diabetes. Furthermore, the rational manipulation of adipose physiology is a promising avenue for therapy of these conditions.

Improved knowledge of all aspects of adipose biology will be required to counter the burgeoning epidemic of obesity. Interest in adipogenesis has increased markedly over the past few years with emphasis on the intersection between extracellular signals and the transcriptional cascade that regulates adipocyte differentiation. Many different events contribute to the commitment of a mesenchymal stem cell to the adipocyte lineage including the coordination of a complex network of transcription factors, cofactors and signalling intermediates from numerous pathways.

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.

PPARgamma and C/EBPalpha are critical transcription factors in adipogenesis, but the precise role of these proteins has been difficult to ascertain because they positively regulate each other's expression. Questions remain about whether these factors operate independently in separate, parallel pathways of differentiation, or whether a single pathway exists. PPARgamma can promote adipogenesis in C/EBPalpha-deficient cells, but the converse has not been tested. We have created an immortalized line of fibroblasts lacking PPARgamma, which we use to show that C/EBPalpha has no ability to promote adipogenesis in the absence of PPARgamma. These results indicate that C/EBPalpha and PPARgamma participate in a single pathway of fat cell development with PPARgamma being the proximal effector of adipogenesis.