MicroRNAs in brown and beige fat

Accepted Manuscript MicroRNAs in brown and beige fat

Deborah Goody, Alexander Pfeifer PII: DOI: Reference:

S1388-1981(18)30095-7 doi:10.1016/j.bbalip.2018.05.003 BBAMCB 58292

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26 October 2017 5 February 2018 4 May 2018

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ACCEPTED MANUSCRIPT MicroRNAs in brown and beige fat Deborah Goody and Alexander Pfeifer

Institute of Pharmacology and Toxicology, University Hospital Bonn, University of Bonn, 53127

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Bonn, Germany

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Correspondence:

Alexander Pfeifer, Institute of Pharmacology and Toxicology, University Hospital Bonn; University of

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Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany; (Tel) +49-(0)228-287 51300; (Fax) +49-

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(0)228-287 51301; email: [email protected]

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ACCEPTED MANUSCRIPT Highlights

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MicroRNA (miRNA) regulate brown and beige adipogenesis miRNA can promote or inhibit the brown adipogenesis We review the mechanisms behind miRNA-regulation of brown and beige adipose tissue Recent findings might lead to the development of miRNA-based treatments against obesity

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Abstract

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Brown adipose tissue (BAT) dissipates energy as heat and its activity correlates with leanness in human adults. Understanding the mechanisms behind the activation of BAT and the process of "browning", i.e. the appearance of inducible brown adipocytes called beige or brite (brown-in-white) cells in white adipose tissue (WAT), is of great interest for developing novel therapies to combat obesity. MicroRNAs (miRNAs) are small transcriptional regulators that control gene expression in a variety of tissues, including WAT and BAT. Recently, miRNAs were reported to regulate browning. Nevertheless, further studies are needed to fully understand the miRNA networks that are involved in the control of brown and beige/brite adipocytes. Particularly, most miRNA have so far been studied in mice, underlining the importance of additional human studies. In this review, we focus on the regulation of brown fat by miRNAs including their role in promoting or inhibiting the browning process. In recent years, RNA-based therapeutical approaches have entered clinical trials for treatment of other diseases, thus miRNAs could potentially be used to enhance brown and beige fat mass and activity as novel therapies against overweight and its complications.

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Keywords: Brown adipocyte, Beige adipocyte, MicroRNA, Obesity, Metabolism, Energy Homeostasis

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Abbreviations: ADAM17, ADAM metallopeptidase domain 17; AMPK, AMP-activated protein kinase; aP2, fatty-acid binding protein 4; AGO, Argonaute protein; ASO, anti-sense oligonucleotide; Bace, amyloid precursor protein-cleaving enzyme 1; BAT, brown adipose tissue; Blnc1, brown fat lncRNA 1; BMP7, bone morphogenetic protein 7; cAMP, cyclic AMP; C/EBP, CCAAT/enhancer-binding protein; cGMP, cyclic GMP; Cidea, cell death-inducing DNA fragmentation factor alpha-like effector A; Cox7, cytochrome c oxidase subunit 7; DIO, diet induced obesity; Dgcr8, microprocessor complex subunit; FABP4, fatty-acid binding protein 4; FGF21, fibroblast growth factor 21; Fndc5, fibronectin type III domain containing 5; HDAC3, histone deacetylase 3; HIF1, hypoxia-inducible factor 1; hMADS, human multipotent adipose tissue-derived stem cells; HoxC8, Homeobox C8; iWAT, inguinal white adipose tissue; Insig1, insulin induced gene 1; LNA, locked nucleic acid; lncRNAs, long non-coding RNAs; MAPK, p38 mitogen activated protein kinase; Mef2, myocyte enhancer factor-2; miRNA, microRNA; Mstn, myostatin; NE, norepinephrine; NPs, natriuretic peptides; Pde, phosphodiesterase; PGC-1α, peroxisome-proliferator-activated receptor γ-coactivator 1α; PHB, prohibitin; PKA, protein kinase A; PPAR, peroxisome-proliferator-activated receptor; PRDM16, protein PR domain containing 16; pri-miRNA, primary miRNA; RIP140, receptor-interacting protein 140; RNAi, RNA interference; Runx1t1, Runt-related transcription factor 1, translocated to, 1; scWAT, subcutaneous white adipose tissue; SIRT1, sirtuin 1; T2D, type 2 diabetes; UCP1, uncoupling protein 1; vWAT, visceral white adipose tissue; WAT, white adipose tissue.

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ACCEPTED MANUSCRIPT 1. Introduction

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Over the past decades the worldwide population suffering from obesity has severely increased [1]. According to the world health organization (WHO) 1.9 billion human adults are overweight and more people die of overweight and obesity than of underweight and famine (WHO, 2014). Excessive fat accumulation is a result of an imbalance in energy homeostasis caused by an increased calorie intake versus calories expenditure. Various studies show that obesity and overweight are major health problems as they increase the risk of developing other diseases such as type 2 diabetes, cardiovascular disease and certain types of cancer [2]. Therapeutic options to treat overweight and obesity are lacking and understanding the underlying molecular mechanisms would help develop new, more efficient anti-obesity therapies. Current therapeutic options include dietetic and exercise treatment as well as bariatric surgery. Available pharmacological therapy to date, such as Orlistat and Liraglutide, are only indicated in special cases and can cause severe side-effects [3, 4]. In previous studies, we and others have established microRNAs (miRNAs) as relevant biomarkers of metabolism [5, 6]. Furthermore, recent research underlines the critical role of miRNAs in the regulation of adipose tissue and metabolism [7]. miRNAs have been reported to directly or indirectly regulate signaling pathways essential for the development and differentiation as well as function of adipose tissue. 1.1. Brown and beige fat tissue

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There are two kinds of adipose tissue in mammals: white adipose tissue (WAT) and brown adipose tissue (BAT) [8]. The main tissue responsible for storing energy in form of triglycerides is WAT. WAT accumulates excessive energy in form of fat and is distributed throughout the whole body. The major WAT depots are classified based on their location as visceral or gonadal WAT (vWAT, gWAT) versus subcutaneous or inguinal WAT (scWAT, iWAT). White adipocytes feature unilocular lipid droplets, and considerably vary in size depending on the lipid load [9]. Contrary to WAT, BAT utilizes stored chemical energy for the production of heat in a process called non-shivering thermogenesis (NST) [10]. The involvement of BAT in heat production has long been known [11], but it was believed to play a major role only in newborns. Although interscapular BAT is the major thermogenic depot in infants, BAT appears to be widely distributed throughout the body during the first decade of life [12]. Anatomical studies showed that interscapular BAT disappears with advancing years [12], however, brown adipocytes can be found also in adults in the deeper regions of the body [12]. It has only been in the last decade that metabolically active BAT was identified in adult humans using positron emission tomography (PET) coupled with computer tomography (CT) imaging of radioactive glucose uptake (FDG-PET/CT) [13-16]. FDG-PET/CT clearly detected active BAT in the supraclavicular and neck regions as well as in the mediastinum [11, 13, 14, 16-18]. Brown adipocytes contain a high abundance of mitochondria and small multilocular lipid droplets. The protein mainly responsible for NST is the uncoupling protein 1 (UCP1), which is located in the membrane of mitochondria and uncouples the proton gradient to generate heat [19, 20]. BAT is highly innervated by the sympathetic nervous system, which releases 3

ACCEPTED MANUSCRIPT norepinephrine (NE) and the cotransmitter ATP which is rapidly converted to adenosine upon cold stimulation [21]. NE activates G protein-coupled receptors, thereby inducing the production of cyclic AMP (cAMP) and activation of protein kinase A (PKA) in brown adipocytes [17]. Regarding the action of adenosine in BAT, there are important species differences: adenosine activates human and murine brown adipocytes, but inhibits activation of BAT from hamster or rat [17, 22-25].

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In addition to WAT and BAT, inducible brown adipocytes also called beige or brite (“brownin-white”) adipocytes exist in WAT depots, mainly iWAT/scWAT [26]. These cells are induced upon cold acclimatization and can dissipate energy as heat, a process also known as “browning” [27, 28]. Morphologically, beige adipocytes share major characteristics with classical brown adipocytes, including multilocular fat droplets, a high mitochondrial content and expression of brown fat-specific genes, including UCP1, cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea), peroxisome-proliferator-activated receptor γ-coactivator 1α (PGC-1α), protein PR domain containing 16 (PRDM16) and CCAAT/enhancer-binding protein β (C/EBPβ) [28]. Several studies indicate that brown adipocytes have a different origin than white and beige adipocytes (Seale et al., 2008). Nonetheless, it is still under debate whether mature white adipocytes have the ability to transdifferentiate, or "un-mask" into beige adipocytes, or whether beige cells derive from a separate precursor cell line, which shares the same origin as white adipocytes, by de-novo differentiation [29-31]. Interestingly, Long and colleagues showed that the origin of beige adipocytes is very heterogenous depending on the location of WAT depots [32].

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2. Brown fat: Function of miRNAs in brown adipogenesis

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The development and function of brown adipocytes is tightly regulated by various hormones and proteins factors [28, 33, 34]. We and others have previously demonstrated that miRNAs also play a critical role in brown adipose tissue as transcriptional regulators and biomarkers [5, 7]. Extracellular miRNAs can be found in fluids, including blood and urine, packaged in extracellular vesicles or in microparticle-free form [35-38]. Their level has been linked to the status and progression of various diseases, including cancer [36, 39, 40]. Circulating miR-122 has been shown to be associated with type 2 diabetes and exosomal miR-92a inversely correlates with murine and human BAT activity [5, 41]. MiRNAs belong to the family of small non-coding RNAs, which comprise approximately 22 nucleotides and mediate RNA interference (RNAi) [42]. They are produced from short hairpin structures by two RNase III proteins, Drosha and Dicer, and associate with Argonaute (AGO) proteins [43]. These small RNAs decrease protein synthesis via interaction with partially complementary sites in the 3′untranslated region (3´UTR) of target messenger RNAs (mRNAs) [44]. MiRNA exerts its function as a guide by recognizing mRNAs, whereas AGO proteins recruit factors that induce translational repression and mRNA destabilization [45]. In this way, miRNAs may positively or negatively regulate classical white, brown and beige fat development. Although the involvement of miRNA in brown/beige adipogenesis is still not completely understood [4648], several studies have identified miRNAs that have specific functions in brown (Fig. 1) and/or beige (Fig. 2) adipocytes. 4

ACCEPTED MANUSCRIPT 2.1. Positive regulators of brown fat miR-328

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Recent studies demonstrated the coincidence of ageing- and obesity-associated dysfunction of brown fat with impaired expression of the miRNA processing protein Dicer [49, 50]. Analysis of miRNA-expression identified miR-328 as a potential regulator of BAT differentiation [51]. MiR-328 was upregulated during brown adipocyte differentiation and decreased in BAT of DIO mice [51]. Inhibition of miR-328 in primary immortalized brown adipocytes resulted in decreased expression of thermogenic genes such as Cidea, Ucp1, Pgc-1α and Prdm16 (Fig. 1) [51]. Additionally, overexpression of miR-328 resulted in increased levels of C/EBPβ, UCP1 as well as oxygen consumption rates in brown adipocytes. Interestingly, there is a link between miR-328 and amyloid precursor protein-cleaving enzyme 1 (Bace1) [52]. Luciferase reporter assay revealed the direct downregulation of Bace1 by miR-328. Overexpression of miR-328 in brown adipocytes reduced Bace expression, while inhibition of miR-328 increased Bace protein levels. Moreover, Bace1 knockout mice demonstrate increased energy expenditure, elevated UCP1 expression in brown fat and are resistant against DIO [53]. miR-182/miR-203

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To address the question on how miRNAs contribute to maintenance of brown adipocytes, Kim and colleagues specifically inhibited Dgcr8 in mice [54]. Drosha, the key regulator of miRNA biogenesis, together with its key cofactor Dgcr8 (also known as Pasha in D. melanogaster) form the microprocessor complex, which further processes primary miRNA (pri-miRNA) in pre-miRNA in the cell nucleus [55]. Adipose tissue-specific Dgcr8 knockout mice showed "whitened" interscapular BAT with decreased expression of thermogenic genes as well as enlarged scWAT and epididymal WAT [54]. Furthermore, miRNA expression profiling identified miR-182 and miR-203 as new regulators of brown adipocyte development. The treatment of brown adipocytes with miR-182 and miR-203 inhibitors in vitro caused a significant reduction of brown fat markers, including UCP1, PGC-1α and Cidea, as well as mitochondrial markers (e.g. cytochrome c oxidase subunit 7, Cox7) (Fig. 1). Interestingly, the mRNA levels of classical adipogenic markers, such as peroxisome proliferator-activated receptor gamma (Pparγ) and fatty acid binding protein 4 (Fabp4) did not change upon inhibition of these miRNAs. A potential mechanism that might explain the effects of miR-182 and miR-203 in brown adipocytes are their targets, including the plateletderived growth factor alpha (PDGFRα) and insulin-induced gene 1 (INSIG1), which have been described as disruptors of adipocyte differentiation [56, 57]. miR-193b-365 In 2011, Sun and colleagues demonstrated that miR-193b and miR-365 form a cluster (miRNA-193b-365), which is highly expressed in BAT [52]. Moreover, they showed that the miRNA-193b-365 cluster significantly promotes murine brown adipocyte adipogenesis by inhibiting runt-related transcription factor 1 translocated to 1 (Runx1t1) expression - an inhibitor of white and brown adipogenesis (see also miR-455) [52]. Contradictory to these 5

ACCEPTED MANUSCRIPT findings, later in vivo studies showed that blocking miR-193b-365 cluster does not change the morphology of BAT and the expression of brown markers (UCP1, PRDM16) [58]. Therefore, further studies are necessary to understand the potential role of the miR-193b-365 cluster in differentiation and function of BAT. 2.2. Negative regulators of brown fat miR-106b-93

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Various members of the miR-17 family, including miR-106 and miR-93, control differentiation of stem cells in developing mouse embryos [59]. High levels of miR-106b and miR-93 were found in brown adipose tissue of obese mice [60]. In brown adipocytes, knockdown of miR-106b and miR-93 using locked nucleic acid (LNA) miRNA inhibitors markedly increased the expression of brown fat-specific genes, including Ucp1, Prdm16 and Cidea, while overexpression of miR-106b and miR-93 suppressed UCP1 expression (Fig. 1) [60]. Based on these findings, the miR-106b-93 cluster might play a significant role in energy homeostasis through negative regulation of brown adipocytes. Based on preliminary in silico target-analysis PPARα may be a target of miR-106b/-93 [60]. Nevertheless, further in vitro and in vivo studies are required to identify the exact mechanism of this miRNA regulator.

3. Beige fat-specific miRNAs: Role of miRNAs in browning

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In addition to being critical regulators of brown adipocyte development and function, recent studies revealed that miRNAs potentially play a central role in beige adipocytes [61]. Understanding the regulatory mechanism of miRNAs during this process may help developing new therapeutic approaches to increase energy expenditure and combat the obesity. Thus, we present here the current research on miRNAs specifically involved in the browning process (Fig. 2).

miR-196a

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3.1. Regulator that specifically enhance browning

Homeobox genes (Hox) genes, which are important for differentiation (e.g. hematopoiesis, myogenesis) [62, 63], have been shown to be differentially regulated in WAT and BAT [64]. Hoxc8 has been characterized as a marker gene for white fat, which is targeted by miR-196a [64, 65]. In 2012, Mori et al. observed an upregulation of miR-196a in iWAT from mice exposed to cold or adrenergic stimulation [65]. Moreover, miR-196a induces brown adipogenesis in human WAT. Regarding the mechanism, Mori et al showed that HOXC8 represses the brown fat-regulator C/EBPβ in cooperation with histone deacetylase 3 (HDAC3) in subcutaneous white adipocytes (Fig. 2) [65]. In addition, mice overexpressing miR-196a showed resistance to obesity and improved glucose metabolism [65]. 3.2. Negative regulator of browning 6

ACCEPTED MANUSCRIPT miR-125-5p

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As described earlier, the precise origin of beige adipocytes has been controversial [31, 66]. Nevertheless, miR-125-5p was identified as a negative regulator of beige adipocyte formation and function [67]. This miRNA was shown to be expressed at lower levels in BAT than in WAT, and miR-125-5p levels decreased during browning, being inverse proportional to UCP1 expression (Fig. 2) [67]. Importantly, injection of miR-125-5p into iWAT inhibited beige adipocyte formation and mitochondrial biogenesis, whereas injection of LNA inhibitors resulted in increased browning [67]. However, the mechanism behind the effect of miR-125b5p on beige adipocytes remains unknown.

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4. Involvement of miRNAs in the regulation of both brown adipocytes and browning process

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Recent studies demonstrated that several miRNAs play important roles in both brown adipocytes and the browning process. Some miRNAs, such as miR-30 and miR-27, have identical effects in both brown and beige adipocytes, respectively [68-72]. In contrast miR378 remains the only characterized miRNA to have opposite functions in brown and beige adipogenesis [73]. Here, we present the current research on miRNAs involved in both brown adipocytes and the browning process (Fig. 3).

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4.1. Positive regulators of brown and beige adipocytes

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miR-30

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The miR-30 family was first identified in adipogenesis through deep sequencing of human multipotent adipose tissue-derived stem (hMADS) cells [68]. MiR-30 family members were upregulated during adipogenic differentiation and their inhibition impaired adipogenesis markedly, partially via regulation of the transcription factor RUNX2 [68]. Their role in the regulation of thermogenesis was later described in vitro and in vivo [69]. MiR-30b/c levels were induced by cold exposure, and positively regulated the expression of thermogenic genes, including UCP1 and Cidea, in brown adipocytes (Fig. 3). Moreover, overexpression of miR30b/c greatly increased thermogenic gene expression in primary subcutaneous white adipocytes [69]. The promoting effect of these miRNAs on the development of beige fat was further supported by the finding that miR-30b/c represses the receptor-interacting protein 140 (RIP140), which functions as a corepressor of thermogenic genes [74]. miR-26 The miR-26a and miR-26b were the first miRNAs identified that regulate both adipocyte development as well as the acquisition of brown adipocyte characteristics in humans [75]. Inhibition of both miRNAs in the human adipocyte cell line hMADS prevented lipid accumulation and the expression of adipogenic genes such as FABP4, while ectopic miRexpression increased the differentiation towards a brown phenotype (Fig. 3) [75]. The positive 7

ACCEPTED MANUSCRIPT effect of miR-26a/b on browning of white adipocytes is due to its repressor activity on the sheddase ADAM17. Mice lacking ADAM17 were previously shown to be resistant to dietinduced obesity, further underlining the physiological importance of the link between miR26a/b and ADAM17 [76, 77]. miR-455

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MiR-455 was first identified as a new key regulator of brown adipogenesis in multipotent mesenchymal cells after treatment with bone morphogenetic protein 7 (BMP7) [78]. BMP7 had previously been shown to promote brown differentiation of preadipocytes and increase brown fat mass [79, 80]. By analyzing rodent and human tissue samples, the authors established miR-455 as a genuine BAT marker for both species [78]. Further in vitro experiments characterized miR-455 as a downstream effector of BMP7- and cold-mediated browning [78]. Adipose-specific gain-of-function mice (transgenic mice expressing miR-455 under the control of the Fabp4 promoter) exhibited increased browning of iWAT upon cold exposure compared to control [78]. Mechanistically, miR-455 inhibits the adipogenic suppressors Runx1t1 and Necdin, thus activating the expression of adipogenic markers (e.g. PPARγ and PGC1α) (Fig. 3) [78]. Furthermore, miR-455 activates AMPK1 by targeting HIF1, promoting adipogenic differentiation and mitochondrial biogenesis [78, 81-83]. miR-32

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MiR-32 has been showed to be involved in lipid metabolism, but its role in BAT function was not identified until recently [84]. Ng et al. employed chromatin immunoprecipitation sequencing (ChIP-seq) and identified miR-32 as a BAT-specific super-enhancer [85]. Moreover, miR-32 levels were significantly increased in BAT during cold exposure promoting BAT thermogenesis and iWAT browning. The in vivo inhibition of miR-32 via injection of miR-32 anti-sense oligonucleotide (ASO) led to impaired thermogenic response and lower core body temperatures after cold exposure [85]. The BAT of cold-exposed mice injected with miR-32-ASO showed no significant morphologically changes but markedly lower levels of UCP1 compared to control (Fig. 3). Interestingly, inhibition of miR-32 resulted in less browning of iWAT after cold exposure, including significantly fewer multilocular cells and downregulation of BAT markers: Cidea, PPAR, PGC1, and UCP1. miR-32 represses the tumor suppressor Tob1, thus activating the p38/MAPK pathway, which is stimulated during cold exposure and induces the expression of fibroblast growth factor 21 (FGF21) [86-88]. FGF21 is a secreted factor that has multiple physiological roles including enhanced browning of iWAT [89]. 4.2. Negative regulators of brown and beige adipocytes miR-27 miR-27 was first characterized as a negative regulator of adipogenesis in hMADS cells partially through suppression of PPARγ and C/EBPα [46, 70, 90]. The group of Dong Liu 8

ACCEPTED MANUSCRIPT later described a novel mechanism of miR-27 in the inhibition of adipogenesis by targeting prohibitin (PHB), which is highly expressed in cells that rely heavily on mitochondrial function [71, 91]. Furthermore, miR-27 loss-of-function experiments in iWAT revealed an upregulation of thermogenic markers including UCP1, PRDM16 and PGC1 (Fig. 3) [72]. Concomitantly, another group showed that miR-27b functions as a negative regulator of PRDM16 to control browning of WAT [92]. miR-155

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Previous studies identified adipogenic transcription factor C/EBPβ as a target gene of miR155 in inflammation and in vitro models of white adipogenesis [93-95]. Subsequently, miR155 was characterized by our group as a negative regulator of brown adipogenesis by targeting C/EBPβ resulting in impaired UCP1 and PGC1 expression (Fig. 3) [7]. Surprisingly, promoter sequence analysis revealed putative C/EBP-binding sequences in the miR-155 promoter region [7]. The authors propose a bistable loop of adipocyte commitment, where high levels of miR-155 inhibit C/EBPβ expression and premature differentiation, while C/EBPβ induction by pro-adipogenic hormones inhibits transcription of miR-155. At the same time, miRNA-155 is positively regulated by transforming growth factor-β1 (TGFβ1), which has been described as an inhibitor of adipogenesis [96]. Inhibition of miR-155 augmented brown adipocyte differentiation and promoted a browning phenotype in white adipocytes in vitro and in vivo [7]. MiRNA-155 deficient mice showed a higher cellular respiration and increased number of “brown-like” adipocytes in iWAT after cold exposure [7].

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miR-133

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MiR-133 was previously shown to be expressed in BAT and downregulated after cold exposure, accompanied by the upregulation of the key thermogenic regulator PRDM16 [52, 97, 98]. Furthermore, miR-133 demonstrates a putative binding motif within the 3´UTR of PRDM16 [98]. Ectopic expression of miR-133 greatly impaired PRDM16 expression and decreased expression of brown genes, including Ucp1, Ppar and Pparγ (Fig. 3). In contrast, inhibition of miR-133 or its transcriptional regulator myocyte enhancer factor-2 (Mef2) enhanced differentiation of brown adipocytes and browning of white adipocytes [98]. Remarkably, double knockout of miR-133a (a1 and a2) in mice increased the thermogenic gene program in iWAT and shifted the morphology of these cells to a beige phenotype [99]. miR-34a Fu et al. found that high levels of miR-34a in obesity inhibit fat browning and brown fat formation partially by suppressing FGF21 and sirtuin 1 (SIRT1) [100]. Mice with dietinduced obesity, which were systemically injected with lentivirus encoding antisense miR-34a showed an increased mitochondrial function and expression of thermogenic markers in all types of WATs [100]. Mechanistically, miR-34a inhibition promotes the expression of FGF21 and SIRT1, and consequently FGF21/SIRT1-dependent deacetylation of PGC1, resulting in the induction of the browning genes Ucp1, and Prdm16 (Fig. 3). Furthermore, miR-34a has 9

ACCEPTED MANUSCRIPT been showed to be involved in myostatin (Mstn) mediated regulation of the protein fibronectin type III domain containing 5 (Fndc5) and browning of white adipocytes [101]. The cleaved version of Fndc5, a hormone called Irisin, was postulated as an inducer of WAT browning via activation of UCP1 expression [102]. 4.3. Regulator with opposing functions in brown and beige fat miR-378

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5. Summary and Perspectives

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The miR-378 has been described as the first and only miRNA to have opposite functions in brown and beige adipocytes [73]. Transgenic mice overexpressing miR-378 specifically in adipose tissue exhibit an increased brown adipogenesis and increased BAT mass, but not inguinal or gonadal WAT mass. The authors hypothesized that BAT expansion, rather than miR-378 per se, leads to inhibition of beige adipocytes in iWAT. Moreover, miR-378 overexpression in iWAT decreased UCP1 and PRDM16 levels, whereas concentration of classical adipogenic markers (PPARγ and aP2) remained normal [73]. Regarding the mechanism behind this dual regulation, miR-378 targets the phosphodiesterase Pde1b, which catalyses the turnover of cAMP and cGMP, in BAT but not in WAT [73, 103]. In this way, miRNA-378 selectively promotes brown adipogenesis, while impairing beige adipogenesis in WAT.

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In the last few years, BAT has been linked to amelioration of overweight and gained more interest as potential target of novel therapeutic tools for the fight of obesity [17]. Recently, inducible brown adipocytes in WAT depots, called beige or brite cells, have gained more interest due to their capability for energy expenditure and their positive effect on diet-induced obesity [104]. Besides hormones and protein factors, miRNAs are also key regulators of brown adipogenesis and the commitment of beige adipocytes to a brown-phenotype, in the “browning” process [5]. This regulation is based on the tight control of transcriptional regulators and important mediators of adipocyte development, including C/EBPβ and PGC1α/β, through miRNAs [7, 72]. MiRNA levels have been shown to be dysregulated in metabolic diseases, including obesity [105]. Because miRNA can be transported in body fluids, e.g. blood and urine, e.g. packaged in extracellular vesicles, they might play a central role in metabolic crosstalk between different cell-types in adipose tissues and/or between different organs. Interestingly, adipose tissue represents a major source of circulating exosomal miRNAs, and could therefore regulate gene expression in distant tissues [5, 106]. Consequently, circulating miRNAs may serve as useful biomarkers of brown/beige fat function, and may be potential therapeutic tool against overweight [5]. Although many studies have described circulating miRNAs as potential biomarkers of metabolic disorder [107], exosomal miRNAs were only recently described as indicators for BAT activity in the context of metabolism (Table 1) [5, 106]. Chen et al. demonstrated the inverse correlation of exosomal miR-92a with murine and human BAT activity [5]. In a recent study, Thomou et al. 10

ACCEPTED MANUSCRIPT showed that adipose tissue represents an important source of circulating exosomal miRNAs, which can function as regulators of secondary organs [106]. Table 1: Summary of circulating miRNAs involved in metabolism disorder.

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Role in metabolism inverse correlation with BAT activity correlation with BAT and WAT abundance positive correlation with obesity positive correlation with obesity Inverse correlation with obesity and T2D inverse correlation with obesity inverse correlation with obesity positive correlation with obesity inverse correlation with obesity inverse correlation with obesity inverse correlation with obesity inverse correlation with obesity inverse correlation with obesity inverse correlation with obesity positive correlation with obesity positive correlation with obesity positive correlation with obesity inverse correlation with T2D and obesity inverse correlation with T2D and obesity positive correlation with T2D inverse correlation with T2D inverse correlation with T2D inverse correlation with T2D inverse correlation with T2D inverse correlation with T2D inverse correlation with T2D inverse correlation with T2D inverse correlation with T2D inverse correlation with T2D inverse correlation with T2D positive correlation with T2D positive correlation with T2D positive correlation with T2D positive correlation with T2D positive correlation with T2D positive correlation with T2D positive correlation with T2D

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Organism human and mouse human and mouse human human human human human human human human human human human human human human human human human human human human human human human human human human human human human human human human human human human

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miRNA exosomal miR-92a exosomal miR-99b miR-152 miR-17 miR-593 miR-138 miR-376a miR-15b miR-520c-3p miR-423-5p miR-532-5p miR-125b miR-130b miR-221 miR-140-5p miR-142-3p miR-222 miR-15a miR-503 miR-326 let-7a/f miR-20b miR-21 miR-24 miR-126 miR-191 miR-197 miR-223 miR-320 miR-486 miR-9 miR-29a miR-30d miR-34a miR-124a miR-146a miR-375

Reference [5] [106] [107] [107] [107] [38, 107] [38] [38] [108] [108] [108] [108] [108] [108] [108] [108] [108] [108-110] [38] [111] [111] [110] [110] [110] [110] [110] [110] [110] [110] [110] [112] [112] [112] [112] [112] [112] [112]

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In vitro and/or in vivo studies showed that miRNAs can positively and negatively impact brown/beige adipogenesis. In this review, we summarize positive regulators (brown-specific: miR-328, miR-182, miR-203, beige-specific: miR-196a) and negative regulators (brownspecific: miR-106b-93; beige-specific: miR-125-5p) of brown/beige adipocyte development. Interestingly, various miRNAs are involved in the regulation of both brown and beige adipocytes (miR-30, miR-26, miR-455, miR-32, miR-27, miR-155, miR-133, miR-34a, miR378). Another class of non-coding RNAs are long non-coding RNAs (lncRNAs), which are no longer than 200 nucleotides and play crucial biological roles [113]. Although their function in white adipocyte differentiation has been studied, their contribution to the browning process remains largely unknown [114, 115]. Recent studies showed that brown fat lncRNA 1 (Blnc1) promotes brown/beige adipogenesis and is required for the positive transcriptional regulation of thermogenic genes through recruitment of transcription factors of brown fat [116, 117]. 11

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Despite growing knowledge on the function of non-coding RNAs in fat, further studies are needed to fully discern the complex mechanisms behind miRNA-mediated adipose tissue regulation, in order to develop therapeutic approaches to treat overweight.

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ACCEPTED MANUSCRIPT References

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Figure 1: Molecular mechanisms of miRNAs that specifically regulate brown adipogenesis. miRNAs that have a positive effect on UCP1 in brown adipogenesis are depicted on the left; negative miRNA in brown adipocytes is shown on the right; exemplified here by their effect on UCP1 expression. Positive regulators block the inhibitory effect of theirs targets on thermogenic markers; the mechanism behind the negative regulator miR-106b-93 remains unknown.

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Figure 2: MiRNAs that regulate specifically the browning process. The positive miR-196a is shown on the left; negative miR-125-5p on the right; exemplified here by their effect on UCP1 expression. MiR-196a inhibits the negative effect of its target on thermogenic markers; the mechanism behind the negative regulator miR-125-5p is not clear.

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Figure 3: MiRNAs that are involved in the regulation of both brown adipocytes and browning process. Positive regulators are depicted on top (+); negative regulators are shown in the bottom (-). Positive regulators block the inhibitory effect of theirs targets on thermogenic markers; negative regulators target factors important for the browning process; exemplified here by their effect on UCP1 expression.

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MicroRNA (miRNA) regulate brown and beige adipogenesis miRNA can promote or inhibit the brown adipogenesis We review the mechanisms behind miRNA-regulation of brown and beige adipose tissue Recent findings might lead to the development of miRNA-based treatments against obesity

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