An update on the regulation of adipogenesis

Drug Discovery Today: Disease Mechanisms

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Vol. 10, No. 1–2 2013

Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – University of Rochester Medical Center, Rochester, NY.

DISEASE Mechanisms of Obesity MECHANISMS

An update on the regulation of adipogenesis Miao-Hsueh Chen*, Qiang Tong* USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, United States

Obesity, a major risk factor for the development of type II diabetes, cardiovascular diseases, and cancer, is rising at an alarming rate worldwide. Obesity is caused

Section editor: Haiming Cao – NHLBI, National Institutes of Health, Bethesda, MD, USA.

by a chronic imbalance between energy expenditure and energy storage by adipose tissue. Adipogenesis is the process governing the formation and function of adipose tissue. This review article will discuss the most recent advances in understanding the regulation of adipogenesis, including adipose tissue lineage determination, the identity of the adipocyte progenitor cells, novel regulators controlling energy storage and expenditure and lastly the newly identified beige/brite cells. Introduction Adipogenesis, the formation of adipose tissue and adipocytes, has been under intensive study because of the health problems associated with obesity, a state of excess adiposity. Obesity is a major risk factor for metabolic syndrome, a combination of metabolic defects including systemic inflammation, dyslipidemia and insulin resistance, that ultimately leads to type II diabetes, cardiovascular diseases, cancer and other health problems [1]. Therefore, the study of adipogenesis not only provides fundamental knowledge about the development of obesity but also about developing therapeutic strategies to treat obesity-associated morbidities. There are two types of adipose tissue in mammals, white adipose tissue (WAT) and brown adipose tissue (BAT). WAT stores extra energy as triglycerides and secretes hormones and cytokines (adipokines) that regulate the function of many other tissues, *Corresponding authors.: : M.-H. Chen ([email protected]), Q. Tong ([email protected]) 1740-6765/$ ß 2013 Elsevier Ltd. All rights reserved.

while BAT dissipates energy to generate heat [2,3]. Traditionally, WAT or BAT was believed to consist mainly of white or brown adipocytes, respectively. Recently, a third type of adipocyte, called the beige/brite cell, that can convert to a highly thermogenic fat cell upon stimulation has been found within WAT [4]. The white adipocyte contains a single large lipid droplet (unilocular), which consists mainly of neutral lipids such as triglycerides. Conversely, the brown adipocyte contains many small lipid droplets (multilocular) and a large number of mitochondria that can convert chemical energy into heat through uncoupling protein 1 (UCP1), which is highly expressed in mature brown adipocytes. The beige cell is morphologically similar to the brown adipocyte. Adipose tissue is actually composed of multiple cell types in addition to adipocytes, including preadipocytes, fibroblasts, endothelial cells, lymphocytes and macrophages. Even WAT depots at different anatomical locations (visceral or subcutaneous) have different compositions and properties. In this review article, we will summarize recent advances in understanding the origins of and the molecules that regulate different adipose tissue types and adipocytes.

Adipose tissue origin Currently, the origin and developmental timing of WAT is not clear, but WAT development probably starts during late embryonic stages and continues after birth. The earliest visible WAT, the subcutaneous WAT located beneath the skin, can be seen at embryonic day 18.5 in mice [5]. By

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contrast, BAT can be detected as early in development as mouse embryonic day 14.5. Recent studies also suggest that BAT and skeletal muscle share the same developmental origin [6,7]. Recently, lineage-tracing studies have made significant progress toward identifying the adipocyte progenitor cells residing within adipose depots. Most notably, adipocyte progenitor cells were found to localize close to or within the adipose vasculature. In the first definitive study, Tang et al. traced the expression of peroxisome proliferator activated receptor g (PPARg), a master regulator of adipocyte differentiation. They found that PPARg expression localizes to mural cells, the pericytes surrounding the vasculature [8]. Later, Tran et al. found VE-cadherin-expressing endothelial cells in WAT and BAT can also give rise to lipid producing adipocytes in mice and in humans [9]. Furthermore, Zfp423, a critical regulator of preadipocyte commitment, was recently found to be expressed in both pericytes and endothelial cells in the vasculature of WAT and BAT [10]. Adipogenic progenitor cells were also isolated using fluorescence-activated cell sorting (FACS) based on surface markers. Rodeheffer et al. first reported that Lin :CD29+:CD34+:Sca-1+:CD24+ cell populations from adult WAT contain adipocyte progenitor cells. These cells were able to proliferate and differentiate into mature adipocytes in vitro (cell culture) and to form adipose tissue in vivo when transplanted subcutaneously into mice [11]. Schulz et al. also isolated from subcutaneous WAT, BAT and skeletal muscle Sca-1+:CD45 :Mac1 cells that were capable of differentiating into brown adipocyte-like cells in vivo [12]. Another method for studying early stages of adipogenesis, in vitro differentiation of pluripotent stem cells into white or brown adipocytes [13], demonstrated that S6 kinase 1 (S6K1) is required for the preadipocyte commitment but not for terminal differentiation of adipocytes [14].

White adipogenesis Generally, adipogenesis can be divided into two steps. The first step is determination, through which pluripotent stem cells develop into mesenchymal stem cells with a limited capacity to form adipocytes, muscle cells, chondrocytes (cartilage cells), or osteocytes. In the second step, mesenchymal stem cells further commit to adipocyte lineage by forming preadipocytes, which can terminally differentiate into mature adipocytes. The molecular regulation of adipogenesis is tightly controlled by many signaling molecules and transcription factors. Most of our knowledge about adipocyte differentiation was obtained from studies of cultured cells, including C3H10T1/2 and 3T3-L1 cells [15,16]. C3H10T1/2 cells are mesenchymal stem cells that can be induced to differentiate into adipocytes, myocytes and osteocytes. 3T3-L1 cells are fibroblast-like preadipocytes that can be induced to differentiate into mature adipocytes upon e16

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stimulation. Studies using C3H10T1/2 cells revealed that the bone morphogenetic protein (BMP) family members BMP2 and BMP4 promoted commitment of mesenchymal progenitor cells to the adipocyte lineage [17]. Other extracellular signaling molecules, such as WNT factors, inhibit adipogenesis and promote osteogenesis [15,16]. Intracellular factors essential for the establishment of preadipocytes include the aforementioned Zfp423 and S6K1 as well as early B cell factor 1 (Ebf1). Mice lacking Ebf1 are deficient in adipocyte progenitor cells and adipose tissue and have increased bone formation [18]. The transcription repressor TCF7L1 also contributes to preadipocyte commitment [15]. A network of transcription factors that are sequentially upregulated and that work cooperatively with each other drive the terminal differentiation process. At the core of this network, CCAAT/ enhancer binding protein (C/EBP)b and C/EBPd are activated first and, in turn, activate C/EBPa and PPARg, which induce the transcription of many adipocyte related genes, including fatty acid synthase, lipoprotein lipase, fatty acid binding protein FABP4 and adiponectin [15]. A host of other factors also regulate adipogenesis through this core circuit (Fig. 1). For example, the GATA transcription factors GATA2 and GATA3 are expressed in preadipocytes and inhibit the expression and activity of C/EBPs and PPARg [15,16]. Therefore, downregulation of these factors is necessary for progression of adipocyte differentiation. On the contrary, the Krox20, KLF4 and CREB transcription factors promote adipogenesis by increasing C/EBPb levels [15,16]. Chromatin modifying enzymes, such as the histone methyltransferases SETD8 and MLL3, are required for priming the promoter regions of PPARg, C/EBPa and their target genes for transcription [15]. TLE3, a target gene of and also a cofactor for PPARg enhances adipogenesis by activating PPARg and suppressing WNT activity/expression. Interestingly, TLE3 also competes with the PR domain containing 16 (PRDM16) protein for binding to PPARg. As a result, TLE3 actually promotes white adipogenesis and inhibits brown adipocyte formation [19]. MicroRNAs can also regulate adipogenesis [20].

Brown adipogenesis Recently, several lineage-tracing studies revealed that BAT arises from the same developmental precursors that produce skeletal muscle. Atit et al. found that interscapular BAT, the dermis and skeletal muscle all arise from the En-1 expressing central dermomyotome [21]. Later lineage-tracing studies with the early skeletal muscle markers myogenic factor 5 (Myf5) and paired-box 7 (Pax7) confirmed that BAT and skeletal muscle indeed originate from skeletal muscle progenitor cells [22,23]. PRDM16 appears to be a key regulator for commitment to the brown adipocyte lineage. Knocking down expression of PRDM16 in primary brown preadipocytes increased expression of skeletal muscle marker genes. Conversely, overexpression of PRDM16 converted muscle

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White adipocyte progenitor cells

Brown adipocyte progenitor cells

Transcription factors & co-factors: Zfp423 S6K1 Ebf1 TCF7L1

Transcription factors & co-factors: PRDM16 Zfp423

?

Signaling molecules: BMP7

Signaling molecules: BMP2 BMP4 WNT

White preadipocyte CD29+:CD34+:Sca1+:C24+

miRNAs

Beige preadipocyte Sca-1+:CD45-:Mac-1-

Transcription factors & co-factors: PPARγ C/EBPs GATAs Krox20 KLF4 CREB TLE3

White adipocyte

Transcription factors & co-factors:

Transcription factors & co-factors:

PRDM16 FoxC2 PGC-1α COUP-TFII

PRDM16 PPARγ C/EBPβ PGC-1α

Hormone peptide: FNDC5

Signaling molecules: WNT miRNAs

Brown preadipocyte Sca-1+:CD45-:Mac-1-

miRNAs

Beige cells

Signaling molecules: BMP7 BMP8B miRNAs

Brown adipocyte Drug Discovery Today: Mechanisms

Figure 1. Current model for white, beige and brown adipogenesis. Adipogenesis begins when pluripotent progenitor cells commit to the adipocyte lineage. Currently, BAT is known to arise from the same lineage that produces skeletal muscle, while the origin of WAT and beige cells is not clear. After preadipocytes form, these cells further differentiate into mature adipocytes with the ability to store lipids, generate adipokines and/or become thermoactive.

progenitor cells into brown adipocytes [23]. At the molecular level, PRDM16 functions as a transcriptional co-activator for C/EBPb and directs this BAT/skeletal muscle fate switch toward the brown adipocyte lineage [24]. After the brown adipose lineage is specified, brown preadipocytes further differentiate into mature brown adipocytes. Most of the key regulators of white adipogenesis, such as the PPARg and C/EBP families of transcription factors, are also the core

regulators of brown adipocyte differentiation. However, additional factors are required to regulate expression of the genes in the thermogenic program of the mature brown adipocyte. Brown adipocytes are activated by norepinephrine released by the sympathetic nervous system innervating BAT in response to cold exposure. The hypothalamus is also involved in BAT activation [25]. Leptin secreted by white adipocytes acts on the hypothalamus to regulate food intake www.drugdiscoverytoday.com

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and energy expenditure, the latter through sympathetic activation of BAT [25]. Thyroid hormone also activates brown cell function and thermogenesis through the hypothalamus [26]. Environmental cues or social contact can also activate BAT via hypothalamic expression of brainderived neurotrophic factor (BDNF) [27]. One of the key regulators of expression of genes in the thermogenic program in brown adipocytes is PGC-1a, although it is not required for brown adipogenesis [28]. Cold exposure or adrenergic signaling induces elevation of cAMP levels in brown adipocytes, which activates the CREB transcription factor to increase PGC-1a expression. cAMP also activates p38 mitogen-activated protein (MAP) kinase that phosphorylates and stabilizes the PGC-1a protein. PGC-1a functions as a co-activator for nuclear receptors such as PPARg, PPARa and ERRa to drive transcription of genes involved in thermogenesis, such as UCP1 and other mitochondrial proteins [28]. Many important regulators that control BAT formation and function were recently identified, including two BMP factors, BMP7 and BMP8B. Mesenchymal progenitor cells (10T1/2 cells) commit to the brown cell fate after treatment with recombinant human BMP7 protein. Moreover, BAT is significantly reduced in BMP7 null mice, while overexpression of BMP7 in mice increases energy expenditure [29]. BMP8B is expressed in mature BAT and in the hypothalamus and can regulate thermogenesis in mice by enhancing both the brown adipocyte’s response to noradrenaline and central control of sympathetic activation of BAT. Mice lacking BMP8B show defects in energy expenditure and increased body weight despite having reduced food intake [30]. Additionally, cold exposure increases circulating levels of cardiac natriuretic peptides, which stimulate expression of thermogenic genes, including PGC-1a and UCP1, through the p38 MAPK pathway [31]. Several miRNAs also regulate brown adipogenesis. Sun et al. reported that a brown-fat-enriched miRNA cluster, miR193b–365, can be induced by PRDM16. miR-193b and/or miR-365 blocks expression of myogenic marker genes and induces differentiation into brown adipocytes [32]. MyomiR133, a skeletal muscle-enriched miRNA, negatively regulates brown adipogenesis by inhibiting PRDM16 expression [33].

Beige/brite cells More recently, a new type of adipocyte, called the beige/brite cell, was found in subcutaneous and visceral WAT depots [4]. Increased amounts of UCP1-expressing beige cells can be induced in WAT by treating animals with b3-selective adrenergic agonists, exposure to cold, or the PPARg agonist rosiglitazone [34]. The origin of beige/brite cells is currently unknown but is probably distinct from that of both white and brown preadipocytes [23,35]. Transcription factors that were previously implicated in regulating BAT function were also found to regulate beige e18

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cells in WAT. Notably, PRDM16 may also mediate beige cell function as transgenic mice with adipose tissue specific overexpression of PRDM16 have subcutaneous WAT with high thermogenic activity [36]. Other transcription factors, such as FoxC2, COUP-TFII and PGC-1a, are able to activate the thermogenic program in WAT [4]. Furthermore, miR-196a induces beige cell formation in WAT. MiR-196a transgenic mice have increased energy expenditure and increased resistance to obesity [37]. Most recently, a secreted protein named fibronectin type III domain-containing 5 (FNDC5) was found to be expressed in skeletal muscle and can be induced by exercise or PGC-1a overexpression. A cleaved form of FNDC5 called irisin is secreted into the bloodstream after exercise in both mice and humans and can stimulate beige cell function [38].

Conclusion It is now clear that different adipose tissue cells arise from distinct lineages. BAT develops first during embryogenesis, followed by WAT. Recent studies of adipogenesis have provided new insights into the location and properties of adipocyte progenitor cells within different adipose depots. Novel genes such as Zfp423 and PRDM16 have been found to be critical regulators of adipocyte lineage establishment. Obesity is occurring at an alarming rate worldwide, indicating that new and more effective therapeutic methods for battling obesity are needed. Understanding the molecular mechanisms that regulate the formation and function of various adipose depots in animals and humans will facilitate the development of new therapeutic modalities against obesity. Simply inhibiting adipogenesis to reduce energy storage is not a very effective strategy to battle obesity. While this strategy would certainly prevent adiposity, the lack of proper storage for excess lipids caused by lipodystrophy will cause harm to other organs, such as the liver. Lack of adipose tissue would also reduce the production of beneficial adipokines, such as leptin or adiponectin, with adverse outcomes. A more viable approach to counteract obesity is to activate the thermogenic action of BAT and beige cells to increase energy expenditure. This option has become more attractive recently because BAT and beige cells have been found in the adult human [39]. Circulating molecules like natriuretic peptides or myokine (irisin) can be exploited for their thermogenesisstimulating potential. However, to utilize these thermogenic tissues and cells to fight against obesity, more studies need to be done. First, even though BAT and skeletal muscle fate switching can be induced in cultured cells, this switch has yet to be verified in animals or humans. Second, new factors that specify different stages of adipogenesis in different adipose depots, especially the early lineage specification stage, remain to be discovered. Third, recent studies have shown that beige cells can be isolated from WAT; however, the identity of these beige progenitor cells and their developmental lineage

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remain unknown. Lastly, new molecules that regulate the activation of these beige cells need to be identified and characterized.

Acknowledgements We thank Dr Eric Jaehnig for critical reading of the manuscript. This work was supported by a grant from US Department of Agriculture/Agricultural Research Service to M.-H. Chen and Q. Tong.

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