Regulatory networks of non-coding RNAs in brown/beige adipogenesis

BAT (brown adipose tissue), a specialized mammalian organ, is distinguished from WAT (white adipose tissue) by its location, morphology and physiological function [1]. BAT promotes energy expenditure via uncoupled respiration mediated by mitochondrial UCP1 (uncoupling protein 1) [2]. Another type of adipocyte expressing UCP1 is referred as beige or brite (brown in white) adipocyte and regarded as a non-classical/inducible brown adipocyte [3,4]. Ablation of BAT can induce obesity and metabolic diseases [5]; in contrast, enhancing BAT activity and function through different strategies leads to increased energy expenditure against obesity [6]. Therefore manipulation of BAT mass and function is an attractive therapeutic approach for obesity-associated metabolic syndrome and its complications.

There has been an upsurge of interest in understanding the mechanism underlying brown/beige adipogenesis. The master regulator of brown fat development is PRDM16 (PR domain-containing 16) which specifies the brown fat lineage from white fat and muscle [7]. It drives brown adipocyte development by binding and activating the transcriptional cofactors PGC-1α [PPAR (peroxisome-proliferator-activated receptor) γ co-activator 1α], PGC-1β, PPARα and PPARγ to induce brown-fat-selective genes, and by interacting with transcriptional co-repressors such as CtBP1 (C-terminal binding protein 1) and CtBP2 to repress white-fat-cell-selective genes [8,9]. In addition, a variety of extrinsic or intrinsic protein factors have been identified as regulators for BAT adipogenesis and physiological function (Figure 1). These studies have been reviewed in other excellent articles [6,10–13] and so we will not discuss these extensively in the present review.

In the present review, we focus on the function of ncRNA (non-coding RNA) in brown/beige fat development and consider two distinct subsets of ncRNAs: the 20–24-nt miRNA, and the lncRNA (long non-coding RNA), which is arbitrarily classified as RNA longer than 200 nt [14]. miRNA is evolutionarily conserved and recognizes its target mRNA by imperfect pairing between its seed sequence and mRNA. Paired miRNA–mRNA together with Argonaute protein form a RNA–protein complex, miRISC (miRNA-induced silencing complex), in which miRNA induces target mRNA translational repression and/or AGO2 (Argonaute 2)-mediated mRNA cleavage [15,16]. In contrast, lncRNA has poor sequence conservation, and through its primary, secondary and tertiary structures, interacts with RBPs (RNA-binding proteins) to regulate gene expression at transcriptional or post-transcriptional levels [14,17]. Transcriptome studies have revealed distinctive expression patterns of ncRNA in different adipose depots, a dynamic change in ncRNA expression during BAT and WAT adipogenesis and functional importance of these ncRNAs in these biological processes [14,18–20]. In the present review, we aim to cover the recent advances in understanding the role of ncRNAs in brown/beige adipocyte biology (Table 1 and Figure 1) and point out that manipulating ncRNAs could be a novel therapeutic approach for combating metabolic diseases. Because of limitations of space, it is impossible to cover all articles published in this rapid growing field, so we only focus on a few seminal pioneering studies and some recent studies with physiological evidence. We apologize for many excellent studies that are not discussed in the present review.

To determine the role of miRNA, as a whole, in adipose tissue, several mouse strains of adipose-specific knockout of DGCR8 (DiGeorge syndrome critical region 8) or Dicer, two key regulators of miRNA biogenesis pathways, have been generated [18,21]; in addition, a tamoxifen-inducible Dicer-knockout strain has also been generated [21]. Both DGCR8- and Dicer-knockout mice display enlarged but pale interscapular brown fat with reduced BAT marker expression, accompanied by an impairment of insulin sensitivity. Although deletion of DGCR8 or Dicer results in different defects in WAT, both of them impair WAT browning upon exposure to cold [18,21]. These results demonstrate that miRNA plays a critical role in BAT development, WAT browning and brown/beige fat physiology.

It is interesting to introduce key miRNAs into DGCR8- or Dicer-knockout brown adipocytes and determine their sole contribution to brown adipocyte biology. Because global miRNA biogenesis has been blocked in DGCR8- or Dicer-knockout mice, these strains provide a clean background to identify the core set of miRNAs that are sufficient to support brown fat development. It has been reported that, in the Dicer-knockout brown adipocytes, reintroduction of miR-346 and miR-362 partially reverses the decrease in C/EBPβ (CCAAT/enhancer-binding protein β), whereas re-expression of miR-365 increases UCP1 mRNA expression 20-fold [21]. Another advantage of these platforms is to facilitate the identification of miRNA targets. Because mRNA targets are normally repressed by multiple miRNAs, the effect of a single miRNA is often masked by other miRNAs targeting the same mRNA. However, in the absence of other miRNAs, the effect of a given miRNA will be more evident when it is introduced back to DGCR8- or Dicer-knockout cells.

miRNAs execute their regulatory function by participating in signalling networks and fine-tuning or switching signalling output levels [22]. Recent studies have identified several miRNAs functioning in different stages of brown/beige fat adipogenesis [23]; these regulatory networks are summarized as follows (Figure 2).

Using microarray methods, researchers have identified a list of miRNAs enriched in BAT compared with WAT, among which miR-193b and miR-365 are a bicistronic transcript [24]. The level of miR-193b-365 is significantly up-regulated during brown adipogenesis. This miRNA cluster is needed for lineage-specific brown adipogenesis, as established by both overexpression and inhibition experiments. Blocking miR-193b-365 causes a remarkable reduction in lipid accumulation during adipogenesis, whereas ectopic expression of miR-193b decreases mRNA levels of myogenic markers on C2C12 myoblasts and up-regulates mRNA levels of two master adipogenic factors, PPARγ and C/EBPα, thus inducing a transition of myoblasts to brown adipocytes under adipogenic differentiation conditions. miR-193b executes these function at least by directly repressing Cdon (cell adhesion-associated, oncogene-regulated), Igfbp5 (insulin-like growth factor-binding protein 5) (two pro-myogenic factors) and Runx1t1 (runt-related transcription factor 1 translocated to 1) (an inhibitor of brown adipogenesis) [25]. This regulatory network is initiated by the brown lineage determination factor PRDM16, as PRDM16 induces miR-193b-365 expression at least partially by inducing expression of its transcription factor PPARα. Therefore miR-193b-365 represents a new part of the PRDM16-induced programme. However, blocking miR-193b-365 with miRNA inhibitors in mature brown adipocytes does not affect the expression of key brown adipocyte genes, indicating that this gene cluster carries out its primary function during development. Screening for miRNAs essential for maintenance of BAT features requires further investigation [24].

On the basis of profiling analysis of miRNAs in BAT before and after cold exposure, RNA sequencing for differentially expressed miRNAs between satellite cells and brown pre-adipocytes, and through bioinformatic analysis predicting PRDM16 targets, several groups have successively revealed miR-133 as a critical regulator of adipocyte browning [26–28].

miR-133 belongs to a group of myomiRs (muscle-specific miRNAs) and is a potent repressor of non-muscle genes, thereby playing a role in embryonic stem cell differentiation and muscle specification [29,30]. miR-133 serves as a major negative regulator of brown/beige adipogenesis. It has a distinct expression pattern among myogenic and brown adipogenic lineages, and their progenitors. The level of miR-133 is down-regulated during the induction of brown adipocyte differentiation and in BAT after cold exposure [26,27]. The down-regulation of miR-133 is inversely correlated with PRDM16 and UCP1 expression levels. Functional assays for miRNA target identification reveal that both miR-133a and miR-133b directly target the conserved 8-nt seed sequences in the 3′UTR of PRMD16 mRNA [26–28]. Moreover, overexpression of miR-133 in brown pre-adipocytes dramatically decreases the mRNA level of PRDM16, as well as other brown adipogenic markers and general adipogenic markers [27], but increases myogenic markers [26]. By contrast, inhibition of miR-133 causes an increase in BAT transcription factors PPARγ and PPARα, and a decrease in myogenic markers [26]. Therefore the brown lineage commitment is influenced directly by miR-133 through targeting PRDM16. miR-133 is regulated directly by MEF2 (myocyte enhancer factor 2) during adrenergic-stimuli-induced BAT development and differentiation. Adrenergic stimulation upon cold exposure leads to a decrease in MEF2, resulting in a reduced expression of miR-133 and an increased expression of PRDM16, which facilitates brown lineage commitment [26].

Inhibition of miR-133 during BAT differentiation causes enhanced mitochondrial activity and BAT programme, leading to an increase in the basal oxygen consumption rate [26]. Similar modulations are observed in the progression of frozen injury-induced muscle regeneration in which miR-133 antagonism induces an increase in uncoupled respiration, glucose uptake and thermogenesis in intramuscular brown adipocytes [27]. The bioenergetic effects of miR-133 antagonism on muscle cells make it a critical modulator of whole-body energy expenditure: antagonism of miR-133 in mice fed with either a normal diet or an HFD (high-fat diet) display a significant increase in total energy expenditure, without much change in physical activity and food intake. Moreover, antagonism of miR-133 leads to improved glucose tolerance and decreased infiltration of inflammatory cells in eWAT (epididymal WAT) [27]. Consistent with this, knockout of miR-133 in mice leads to SAT (subcutaneous adipose tissue) browning and increased insulin sensitivity [28]. All of these studies indicate that miR-133 can serve as a therapeutic target to defend against diet-induced obesity.

miR-196a expression is induced in SVF (stromal-vascular fraction) cells during brown adipogenesis by an adenylate cyclase activator, forskolin, or cold exposure [31]. The miR-196a gene is located within the gene clusters encoding the Hox (homeobox) family, which plays a role in system differentiation including adipogenesis [31,32]. One of the Hox genes, Hoxc8, is categorized as a white-fat gene and a repressor of brown adipogenesis [33]. Hoxc8 targets the C/EBPβ regulatory sequence and represses C/EBPβ, a positive regulator of the BAT programme. During the brown adipogenesis of white fat progenitors, the expression of Hoxc8 is suppressed directly by miR-196a, which facilitates the browning progression. In iBAT (interscapular BAT)-SVF cells, miR-196a is barely expressed, and its target Hoxc8 is even absent, which implies that miR-196a does not play a role in conventional brown adipogenesis [31]. Fat-specific miR-196a transgenic mice generate metabolically functional beige cells in WAT, and exhibit enhanced energy expenditure, improved glucose metabolism and resistance to obesity. Therefore manipulation of miR-196a–Hoxc8–C/EBPβ signalling in WAT progenitors to initiate browning can be a therapeutic strategy for obesity-associated metabolic consequences.

TGFβ₁ (transforming growth factor β1) potently inhibits adipogenesis, and knockout of Smad3 induces the WAT to BAT phenotypic transition [34,35]. A recent study demonstrated that the inhibitory effect of TGFβ₁/Smad signalling on brown fat adiposeness is partly exerted through induction of miR-155 [36]. miR-155 is highly expressed in pre-adipocytes, but decreases dramatically after induction of differentiation. It forms a bistable feedback loop with its target C/EBPβ to maintain the balance between proliferation and differentiation of brown pre-adipocytes. miR-155 transgenic mice display reduced BAT mass, whereas miR-155-knockout mice show a better adaption to cold exposure by recruiting brite fat cells (inducible beige cells) and enhancing differentiation of BAT [36]. These data demonstrate that miR-155 serves as a negative regulator for brown fat differentiation.

A single miRNA family can regulate a large number of targets, some of which may be involved in the same biological process or signalling pathway [16,23]. miR-27a/miR-27b is such a miRNA family, which serves as a upstream regulator of brown/beige adipogenesis by directly targeting several key transcription factors such as PRDM16, PPARα, CREB (cAMP-response-element-binding protein) and PGC-1β [37]. Moreover, antagonizing miR-27b function prevents dexamethasone (one type of glucocorticoid) induced inhibition of BAT differentiation in primary adipocytes, and improves glucocorticoid treatment-induced central fat accumulation and metabolic dysfunction in mice. Glucocorticoid induces the expression of miR-27b through glucocorticoid receptor-mediated transcriptional regulation [38]. Therefore miR-27 is a responder of glucocorticoid signal, as well as a central regulator of brown/beige adipogenesis.

miR-378 resides in the gene locus of PGC-1β and is induced during brown adipogenesis. miR-378 promotes BAT differentiation and expansion, but suppresses the formation of beige adipocytes in subcutaneous WAT. miR-378 directly targets Pde1b (phosphodiesterase 1b) that catalyses the turnover of cAMP [39]. This may be one of the regulators in BAT activity and expansion, as single nucleotide polymorphisms in the Pde1b gene are associated with cattle back-fat thickness, and cAMP can potentiate adipocyte differentiation [40,41]. Indeed, miR-378 transgenic mice display elevated cAMP levels which leads to increased lipolysis and activity in BAT, accompanied with enlarged organ mass and improved metabolic profile.

Upon cold exposure, 3T3-L1 fat cells display a marked decrease in miR-34a and an increase in browning-related genes, such as those encoding UCP1, PGC-1α and PRDM16. This inverse correlation is also observed in adipose tissue, which suggests that miR-34a inhibits fat browning [42]. Mechanistically, miR-34a directly targets FGFR1 (fibroblast growth factor receptor 1) to attenuate FGF21 (fibroblast growth factor 21)/SIRT1 (sirtuin 1)-dependent deacetylation of PGC-1α, which controls the browning transcriptional programme [42,43]. Circulating FGF21, an endocrine mitokine, enhances fatty acid β-oxidation in liver and adipose tissue, induces WAT browning, and promotes protection from diet-induced obesity and insulin resistance [44]. The integrated actions from multiple tissues induced by FGF21 signalling makes its upstream miR-34a a potent modulator in metabolic regulatory networks. Importantly, beneficial effects mediated by inhibition of miR-34a in HFD-fed mice include several aspects, including reduced adiposity and improved serum profiles [42]; miR-34a inhibition induces browning in all types of WAT, and promotes a more robust BAT programme [42]. These distinctive effects imply that miR-34a exerts roles in multiple tissues through targeting FGF signalling.

The function of lncRNAs in BAT is poorly understood despite existing reports on their expression pattern in BAT [45,46]. Using massively parallel sequencing of polyadenylation-selected RNAs, researchers have identified 175 lncRNAs which are regulated during adipogenesis. Further RNAi-mediated loss-of-function experiments have characterized ten lncRNAs that are required for adipogenesis and are referred to as lnc-RAP-n (lncRNA Regulated in AdiPogenesis) [47]. One of them, lnc-RAP-1 (or Firre, functional intergenic repeating RNA element) interacts with the nuclear matrix factor hnRNPU (heterogeneous nuclear ribonucleoprotein U) to mediate trans-chromosomal interactions, and brings loci encoding known regulators of adipogenesis into close proximity. This hnRNPU–Firre-mediated interaction may be required for proper function of multiple biological processes including adipogenesis [48].

This study is mainly based on the WAT system, but the role of lncRNA in brown adipocyte differentiation is not well understood. A recent research has identified Blnc1 (brown fat lncRNA 1) as a BAT-enriched lncRNA, which promotes brown and beige adipocyte differentiation through a ribonucleoprotein complex formed with EBF2 (early B-cell factor 2) to enhance expression of thermogenic genes such as UCP1. This regulation pattern is feedforward because Blnc1 itself is positively regulated by EBF2 [49].

These previous studies suffer from an incomplete annotation of lncRNA in adipose tissue. To address this obstacle, our group performed deep RNA-seq from BAT, inguinal WAT and eWAT, which provides us with approximately half a billion of reads for transcriptome reconstruction. We developed a computational pipeline and identified more than 1500 lncRNAs expressed in adipose tissue. Of these, 127 are BAT-restricted, and many of them are targeted by key adipogenic regulators such as PPARγ and C/EBPα [50]. Since they are BAT-enriched, we refer to them as lnc-BATEs. lnc-BATE1 is up-regulated 30-fold during adipogenesis and is required for brown adipogenesis and the maintenance of brown adipocyte features. lnc-BATE1 specifically binds the nuclear matrix factor hnRNPU; both of them are required for proper brown adipogenesis [48,50], but how this ribonucleotide complex exerts its functions is not well understood and requires further investigation.

Manipulation of BAT and WAT browning has been linked to a healthy metabolic phenotype and a defence against obesity and the metabolic syndrome. Several recent studies have identified new BAT-specific lncRNAs and revealed that some of them, such as lnc-BATE1 and Blnc1, promote brown and/or beige adipocyte differentiation and physiological function (Figure 3). Although the detailed regulatory networks and mechanisms are still not fully understood, lncRNAs are promising therapeutic targets for obesity and associated metabolic diseases.

BAT protects against obesity by promoting energy expenditure via uncoupled respiration, and is regarded as an attractive therapeutic target organ for obesity and its pathological consequences [1]. In the present review, we have summarized the functional role of two types of intrinsic factor, miRNA and lncRNA, in brown/beige adipogenesis and physiological function. Increased expression of miR-193b-365, miR-196a and miR-378 or inhibition of miR-133, miR-155, miR-27 and miR-34a can promote brown fat adipogenesis. Although it is still insufficient to construct a whole miRNA regulatory network in BAT adipogenesis, these studies display explicit correlations among miRNAs themselves and their targeted signalling pathways. On this basis, further investigations will probably reveal the core set of miRNAs responsible for reconstructing BAT with higher activities.

Comparing with the relatively unifying molecular mechanism of miRNAs, the mechanisms used by lncRNAs are more diverse. Firre and lnc-BATE1 interact with nuclear matrix factor hnRNPU to make a spatial reconstruction of chromosome to bring a few metabolic genes into closer proximity which may lead to co-regulation [48]. EBF2 and its cis-regulator Blnc1 form a feedforward loop to promote brown/beige adipogenesis [49]. More functional lncRNAs with different mechanisms are expected to emerge in the near future. These lncRNA studies, together with the miRNA study, will depict a new regulatory layer governing brown/beige fat plasticity under physiological and pathological conditions.

This work was supported by a Singapore National Research Foundation (NRF) fellowship [grant number NRF-2011NRF-NRFF 001-025 (to L.S.)]. This research is also supported by the Singapore National Research Foundation (NRF) under its Co-operative Basic Research Grant (CBRG) [grant number NMRC/CBRG/0070/2014] and administrated by the Singapore Ministry of Health's National Medical Research Council.

BAT

brown adipose tissue

Blnc1

brown fat lncRNA 1

C/EBP

CCAAT/enhancer-binding protein

CtBP

C-terminal binding protein

DGCR8

DiGeorge syndrome critical region 8

EBF2

early B-cell factor 2

eWAT

epididymal WAT

FGF

fibroblast growth factor

Firre

functional intergenic repeating RNA element

HFD

high-fat diet

hnRNPU

heterogeneous nuclear ribonucleoprotein U

Hox

homeobox

lnc-BATE

lncRNA BAT-enriched

lnc-RAP-n

lncRNA Regulated in AdiPogenesis

lncRNA

long non-coding RNA

MEF2

myocyte enhancer factor 2

ncRNA

non-coding RNA

PGC

PPARγ co-activator

PPAR

peroxisome-proliferator-activated receptor

PRDM16

PR domain-containing 16

SAT

subcutaneous adipose tissue

SVF

stromal-vascular fraction

TGFβ₁

transforming growth factor β1

UCP1

uncoupling protein 1

WAT

white adipose tissue