MicroRNAs-361-5p and miR-574-5p associate with human adipose morphology and regulate EBF1 expression in white adipose tissue

Accepted Manuscript MicroRNAs-361-5p and miR-574-5p associate with human adipose morphology and regulate EBF1 expression in white adipose tissue Yasmina Belarbi, Niklas Mejhert, Hui Gao, Peter Arner, Mikael Rydén, Agné Kulyté PII:

S0303-7207(17)30604-4

DOI:

10.1016/j.mce.2017.11.018

Reference:

MCE 10132

To appear in:

Molecular and Cellular Endocrinology

Received Date: 29 June 2017 Revised Date:

24 September 2017

Accepted Date: 23 November 2017

Please cite this article as: Belarbi, Y., Mejhert, N., Gao, H., Arner, P., Rydén, M., Kulyté, Agné., MicroRNAs-361-5p and miR-574-5p associate with human adipose morphology and regulate EBF1 expression in white adipose tissue, Molecular and Cellular Endocrinology (2017), doi: 10.1016/ j.mce.2017.11.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT miR-361-5p (miR-361-5p + miR-574-5p)

miR-574-5p (?)

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Adipose hypertrophy Insulin resistance Altered lipolysis

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EBF1

EBF1

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Metabolic and adipogenic genes

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Proposed pathway of EBF1 regulation by miR-361-5p and -574-5p in hypertrophic human WAT. T-bars denote inhibition, vertical arrows indicates up/downregulation of expression levels.

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MicroRNAs-361-5p and miR-574-5p associate with human adipose

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morphology and regulate EBF1 expression in white adipose tissue

Yasmina Belarbi1, Niklas Mejhert1, Hui Gao1,2, Peter Arner1, Mikael Rydén1 and Agné Kulyté1,*

Lipid Laboratory, Department of Medicine Huddinge, Karolinska Institutet, SE-14186 Stockholm,

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Sweden.

Department of Biosciences & Nutrition, Karolinska Institutet, SE-141 Stockholm, Sweden.

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*Corresponding author: Lipid Laboratory, Department of Medicine Huddinge, Karolinska Institutet, SE-141 86 Stockholm, Sweden. Phone: +46 8 58580623, fax: +46 8 58585470, e-mail:

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[email protected].

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Abbreviated title: MicroRNAs regulate EBF1 in human WAT Key terms: EBF1, microRNA, adipocyte morphology, white adipose tissue

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Number of figures: 4 Number of tables: 1

Number of supplemental figures: 1 figure and 1 table

Disclosure statement: the authors have nothing to disclose.

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Abstract Reduced adipose expression of the transcription factor Early B cell factor 1 (EBF1) is linked to white adipose tissue (WAT) hypertrophy. We aimed to identify microRNAs (miRNAs) associated with WAT hypertrophy and EBF1 regulation. We mapped WAT miRNA expression from 26 non-obese

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women discordant in WAT morphology and determined EBF1 activity in the non-obese and 30 obese women. Expression of 15 miRNAs was higher in hypertrophy and 10 were predicted to target EBF1. Binding of miR-365-5p/miR-574-5p were validated with 3'-UTR assay. Overexpression of miR-365-

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5p or miR-574-5p reduced EBF1 while inhibition of miR-574 increased EBF1 expression in human adipocytes in vitro. Additive effects on EBF1 were observed when concomitantly overexpressing both

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miRNAs. EBF1 targets were affected by over expression/inhibition of either miRNAs. Finally, miR365-5p/miR-574-5p expression in 56 individuals correlated significantly with EBF1 activity. Our results suggest that miR-365-5p and miR-574-5p may be linked to WAT hypertrophy via effects on EBF1 expression.

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Words: 150

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1. Introduction White adipocyte tissue (WAT) dysfunction is a central risk factor for the development of insulin resistance (IR) and type 2 diabetes mellitus (TD2M). Although many pathophysiological processes may contribute, it is well established that differences in fat cell size and number are important (Arner,

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Westermark et al. 2010). A phenotype characterized by few but large fat cells (hypertrophy) is associated with increased adipose inflammation, lipolysis, IR and risk of developing T2DM, while many small adipocytes (hyperplasia) are protective (Weyer, Foley et al. 2000; Hoffstedt, Arner et al.

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2010). Recent findings suggest that the transcription factor Early B cell factor 1 (EBF1) plays a causal role in determining WAT morphology. Decreased EBF1 expression in mice results in WAT

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hypertrophy (Gao, Mejhert et al. 2014) and attenuated EBF1 activity and protein expression in subcutaneous WAT (scWAT) is associated with increased fat cell size and several parameters of the metabolic syndrome (MS) (Petrus, Mejhert et al. 2015). So far, only TNFα has been shown to act as an upstream regulator of EBF1 (Gao, Mejhert et al. 2014) but the primary mechanisms that induce

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EBF1 downregulation remain to be elucidated.

MicroRNAs are small non-coding RNAs that bind to complementary sequences in the 3'-untranslated region (UTR) of target mRNAs, either blocking translation or causing mRNA degradation (Bartel

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2009). Several miRNAs are dysregulated in obese WAT and have been implicated in the development of the MS (Xie, Sun et al. 2009). However, only a minor set of the effects proposed to mediated by

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miRNA have been confirmed experimentally (Arner and Kulyte 2015). Herein we aimed to identify miRNAs associated with differences in WAT morphology and determine whether they could affect the expression of EBF1.

2. Matherials and Methods 2.1. Clinical material Cohort 1 comprised 30 obese (BMI ≥30 kg/m2) and 26 non-obese (BMI ≤30 kg/m2) healthy women, none of which were on any continuous medication. The cohort has been described in detail before 3

ACCEPTED MANUSCRIPT (Table 1 and Table S1 in the publications (Arner, Mejhert et al. 2012; Gao, Mejhert et al. 2014) ) together with the procedures for tissue collection. The differentiation cohort comprised 12 lean/overweight (BMI ≤30 kg/m2) healthy women and has been described before in Table 1 in the publication (Bambace, Dahlman et al. 2013). Mature adipocytes from biopsies were prepared using

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the collagenase procedure as described (Rodbell 1964; van Harmelen, Dicker et al. 2002). The regional board of ethics approved the study, and written informed consent was obtained from all participants.

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2.2. Affymetrix GeneChip Human Gene 1.0 ST and microRNA array protocols

Transcriptional profiles have been published previously (Arner, Mejhert et al. 2012) and are available

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via GEO Profiles (http://www.ncbi.nlm.nih.gov/geoprofiles), accession number GSE25402.

2.3. Global Gene Expression Analysis, Motif Activity Response Analysis, and Network Construction

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Motif activity response analysis has been described in detail (Suzuki, Forrest et al. 2009; Arner, Mejhert et al. 2012). Predicted targets of miRNAs were retrieved from the miRWalk database (release

2.4. Cell culture

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April 2015) (Dweep and Gretz 2015).

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Primary adipocyte cultures for in vitro studies were plated using cells obtained from scWAT from healthy lean and overweight men and women (BMI ≤30 kg/m2) undergoing cosmetic liposuction. In this group, there was no selection for age, sex, or BMI. In brief, stroma vascular fraction (SVF) cells were isolated and differentiated as described (van Harmelen, Skurk et al. 2005; Pettersson, Stenson et al. 2013). Cells obtained from different individuals were not mixed. 3T3-L1 cells were handled as recommended in the protocols from ATCC (Manassas, VA).

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ACCEPTED MANUSCRIPT 2.5. MicroRNA transfection For overexpression of miRNA activity, in vitro differentiated adipocytes (day 10-12 post-induction) were transfected with various concentrations (5-40 nM) of miRIDIAN miRNA mimics or inhibitor in 24-, 12-, or 6-well plates and HiPerFect Transfection Reagent, respectively 4,5, 9 or 18 µl (Qiagen,

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Hilden, Germany) according to the manufacturer’s protocol. Control cells were transfected with miRIDIAN miRNA Mimic/Inhibitor Non-Targeting Negative Control (Dharmacon/Thermo Fisher

proteins were collected.

2.6. RNA isolation, cDNA synthesis and real-time PCR

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Scientific, Lafayette, CO). The cells were incubated for 48-72 h at which time RNA, medium and

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Total RNA was extracted from in vitro differentiated adipocytes using miRNeasy kit (Qiagen). Synthesis of cDNA was performed using miScript II RT-Kit and miScript HiFlex Buffer (Qiagen) enabling detection of multiple miRNAs and mRNAs from a single cDNA preparation. RT-qPCR of coding genes or miRNAs was performed using commercial Taqman probes (Applied Biosystems,

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Foster City, CA) or miScript Primer Assays (Qiagen), respectively. Relative gene expression was calculated using the 2(-Delta Delta C(T)) method (Livak and Schmittgen 2001). LRP10 and SNORD68 or miR-103 were used as internal controls for the normalization of, respectively, coding

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genes and miRNAs. The expression of the references did not differ between groups.

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2.6. Luciferase Reporter Assay

Empty luciferase reporter vector and vector containing 3'-UTR of EBF1 were obtained from GeneCopoeia (Rockville, MD). The luciferase reporter assay in 3T3-L1 cells was performed as described in details (Arner, Mejhert et al. 2012).

2.7. Isolation of protein and Western blot In vitro differentiated adipocytes were transfected with mimics of miR-361-5p or miR-574-5p for 4872 h and nuclear extracts were prepared as described (Lorente-Cebrian, Mejhert et al. 2014). From samples treated with inhibitor of miR-574-5p nuclei were isolated 72 h postransfection. Adipocyte 5

ACCEPTED MANUSCRIPT nuclei obtained from approximately 500.000 cells were suspended in 150 µL of radioimmunoprecipitation assay buffer (Stenson, Ryden et al. 2009) supplemented with 5 mM NaF, 1 mM Na3VO4, protease inhibitor cocktail set V (Calbiochem), and benzonase (Sigma). The nuclear proteins (5-10 µg) were separated by SDS-PAGE and Western blot was performed according to

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standard protocols. Antibodies against EBF1 (clone EPR4183) were obtained from LSBio (Seattle, WA) and used at 1:1000 dilution. β-actin (Sigma-Aldrich) was used as a loading control. Secondary rabbit IgG-horseradish peroxidase antibodies were from Sigma-Aldrich. Antibody-antigen complexes

Healthcare).

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2.8 Enzyme-linked immunosorbent assay

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were detected by chemiluminescence using ECL™ Select Western Blotting Detection Kit (GE

Total adiponectin levels in conditioned media from in vitro differentiated adipocytes were analyzed using an ELISA assay from Mercodia.

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2.9. Statistical analyses

Data presented are mean ± SEM. When appropriate, the data was log-transformed to obtain normal distribution. Results were analyzed with unpaired t-test, linear regression or significant analysis of

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3. Results

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microarrays (Tusher, Tibshirani et al. 2001).

3.1. Identification of miRNAs differentially expressed in hypertrophic adipose tissue To identify miRNAs associated with adipose morphology, we analyzed previously published miRNA expression microarrays from scWAT of 26 non-obese individuals subdivided into hypertrophy (n=13) or hyperplasia (n=13) from cohort 1 (Arner, Mejhert et al. 2012; Gao, Mejhert et al. 2014). As reported previously, there were no significant differences in age, BMI, total body fat or WHR between the two groups (Gao, Mejhert et al. 2014). The reason for focusing on non-obese subjects was based on the fact that obesity per se induces profound changes in gene and miRNA expression

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ACCEPTED MANUSCRIPT and the metabolic impact of different morphologies is therefore less evident in the obese state (Gao, Mejhert et al. 2014). Thus, we identified 15 miRNAs that were differentially regulated (miR-574-5p, 361-5p, -143, -222, -221, -125b, -106a, -26a, -23a, -16, -17, let-7a, -7c, -7d and -7i), all of which were significantly higher in hypertrophy (Figure 1A). Out of these, ten (let-7a, -7c, -7d, -7i and miR-106-

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5p, -143, -221, -23a, -361-5p and -574-5p) were predicted to target EBF1 (Supplemental Table 1).

A

Significant according to SAM (FDR 5%)

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miR-222

miR-361-5p

B

miR-574-5p

let-7c

miR-143

let-7a

miR-16

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miR-221

let-7i

0

Hyperplasia

0.3

let-7d

0.6

miR-26a

0.9

miR-23a

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1.2

miR-17

Relative miRNA expression (hypertrophy/hyperplasia)

1.5

*** **

miR-361-5p

miR-574-5p

miR-143-3p

let-7a

miR-221-3p

miR-23a

0

let-7d

0.5

miR-106-5p/-17-5p

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NegC

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Relative light units (miRNA/NegC)

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1.5

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ACCEPTED MANUSCRIPT Figure 1. Identification of altered miRNA expression in hyperplastic and hypertrophic WAT. A. Global miRNA expression profiling of intact adipose tissue from non-obese subjects (n= 26). Based on the WAT phenotype the individuals were divided in two subcategories, hypertrophic or hyperplastic WAT (n=13/13). Results were analyzed according to SAM with a 5 % FDR and are

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presented as fold change (hypertrophy/hyperplasia). B. Each candidate miRNA was transfected together with reporter constructs (3’UTR of EBF1 or

empty vector) in 3T3-L1 cells and changes of luciferase activity was measured. miR-106-5p and miR17-5p are presented in the same bar; because of their almost identical sequence differing with only

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one nucleotide. ***P <0.005, **P <0.01.

3.2. Functional validation of miRNAs and mapping of cellular expression In order to verify whether the candidate miRNAs bound directly to EBF1 mRNA, we performed a 3'UTR screen in 3T3-L1 cells where individual miRNAs were co-transfected together with the 3'-UTR

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of EBF1 fused to a luciferase reporter. Let-7i and -7c were excluded from the studies due to previously observed low expression in adipocytes (Arner, Mejhert et al. 2012). Out of the eight remaining miRNAs, overexpression of miR-361-5p and miR-574-5p lead to a significant

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(Figure 1B).

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downregulation of luciferase activity, indicating that EBF1 is indeed a true target for these miRNAs

To evaluate the cellular expression of miR-361-5p and miR-574 in human WAT, quantitative PCR was performed in isolated adipocytes and intact WAT. This revealed that both miR-361-5p and miR574-5p were enriched in isolated adipocytes (Figure 2A). The expression levels of miR-571-5p in both WAT and isolated adipocytes were significantly higher compared to miR-361-5p. Expression of miR-361-5p was unaltered during adipocyte differentiation as evidenced by the miRNA levels in primary adipocyte cultures at day 8 and 12 of differentiation relative to day 4 in differentiation cohort of 12 individuals (Figure 2B). Expression of miR-574-5p was increased by 20 % at day 8 of adipocyte differentiation but not at day 12. The expression levels of miR-571-5p in primary adipocytes were 8

ACCEPTED MANUSCRIPT about 20 fold higher than miR-361-5p. Validity of the adipogenesis in the given cohort is indicated by the increased expression of adipocyte specific genes adiponectin (ADIPOQ) and CCAAT/Enhancer Binding Protein Alpha (CEBPA) (Figure 2C).

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4.0

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3.0

0.2

2.0

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0.3

1.0

0.1

0.0

WAT

Cells

miR-361-5p

1.0 0.8 0.6 0.4 0.2 0.0

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miR-561-5p miR-361-5p

12 10

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***

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8 6 4 2 0

ADIPOQ CEBPA 4/5 8 12 Days of differentiation

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4/5 8 12 Days of differentiation

Cells

miR-574-5p

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Relative miRNA expression (day 4 vs. day 12)

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WAT

Relative mRNA expression (day 4 vs. day 12)

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Relative miRNA expression

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Relative miRNA expression (log10)

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ACCEPTED MANUSCRIPT Figure 2. Expression of miR-361-5p and miR-574-5p in WAT and isolated adipocytes and during adipocyte differentiation in vitro. A. Expression of miR-361-5p and miR-574-5p in intact scWAT and available isolated adipocytes of obese females (n=20) and non-obese females (n=23) from cohort 1 was measured by qRT-PCR.

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Expression of miRNAs were normalized to the expression of miR-103. Values are presented as range (mean ±SEM) and results were analyzed using t-test. Relative expression values of miR-574-5p were log-transformed.

B. Expression of miR-361-5p and miR-574-5p at day 4, 8, and 12 of differentiation in primary

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adipocyte cultures from female (n=12) was measured by qRT-PCR. Expression of miRNAs were normalized to SNORD68. Results were analyzed using t-test and are expressed as relative fold change

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day 8 or day 12 vs. day 4.

C. Expression of ADIPOQ and CEBPA at day 4, 8, and 12 of differentiation in primary adipocyte cultures from female (n=12) was measured by qRT-PCR. Expression of mRNA were normalized to 18s. Results were analyzed using t-test and are expressed as relative fold change day 8 or day 12 vs.

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***P <0.005, * P <0.05.

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day 4.

3.3. miR-361-5p and miR-574-5p regulate EBF1 expression in human adipocytes

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To confirm that miR-361-5p and 574-5p affected EBF1 expression, we overexpressed each miRNA in in vitro differentiated human adipocytes. Analysis of mRNA expression by qRT-PCR demonstrated that EBF1 levels were reduced by 24 % and 15 %, respectively (Figure 3A). Decreased levels were also observed for Cell Death-Inducing DFFA-Like Effector A (CIDEA), a gene previously shown to be regulated by EBF1 (Gao, Mejhert et al. 2014) although not predicted to be a direct target of the studied miRNAs (Figure 3A). Inhibition of miR-574-5p resulted in reciprocal effects on EBF1 and CIDEA expression. (Figure 3B). Given its low expression in primary cultures, inhibition of miR-3745p was not performed Overexpression of miR-361-5p decreased EBF1 protein levels in the nuclear extracts, an effect that was not observed with miR-574-5p (Figure 3C). The validity of our 10

ACCEPTED MANUSCRIPT experimental setup was confirmed by demonstrating that established targets for miR-361-5p (VEGFA) and miR-574-5p (Sox12) (Kanitz, Imig et al. 2012; Zhang, Thevapriya et al. 2014) were downregulated following overexpression of their cognate miRNA (Supplemental Figure 1A).

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The observation that EBF1 expression levels were reduced by the individual miRNAs prompted us to test if they could exert additive effects. Co-transfection of miR-361-5p and -574-5p at low

concentrations of mimic reagents (10+10 nM) resulted in a more pronounced reduction of EBF1

expression (~15 %) compared with individual overexpression of either miRNA (10 nM; Figure 3D).

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Quantification of the overexpressed miRNA levels are presented in Supplemental Figure 1B.

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Figure 3 0,8

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0,2

Mimics

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EBF1

Lamin A/C

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miR-574-5p

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miR-574-5p

1,2 Neg C (inhibitor)

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CIDEA

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*** Relative gene expression (miR/NegC)

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miR-361-5p

Mimics miR-574-5p

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Relative EBF1 mRNA expression (miR/NegC)

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miR-574-5p

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NegC

0,0 miR-361-5p

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miR-574-5p

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miR-361-5p

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miR-361-5p

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Relative EBF1 protein (miR/NegC)

Neg C (mimics)

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miR-361-5p

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Relative adiponectin (miR/NegC)

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Relative gene expression (miR/NegC)

A ACCEPTED MANUSCRIPT *

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miRmiRNegC 574-5p 361-5p kDa -75 -50 -75 -50

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ACCEPTED MANUSCRIPT Figure 3. MicroRNA regulates EBF1 expression in human adipocytes. A. miR-361-5p and miR-574-5p were overexpressed at 40 nM in in vitro differentiated adipocytes and expression of EBF1 and CIDEA was assessed by RT-qPCR. Results are based on four biological/independent experiments.

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B. miR-574-5p was inhibited using 40 nM of miRNA inhibitor in in vitro differentiated adipocytes and expression of EBF1 and CIDEA was assessed by RT-qPCR. Results are based on four biological/independent experiments.

C. miR-361-5p and miR-561-5p were overexpressed in human differentiated adipocytes, cells were

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collected for the isolation of nuclei and thereafter nuclear lysates were analyzed by Western blot. Results are representative of seven biological/independent experiments, representative blots are

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shown.

D. miR-361-5p and miR-561-5p were overexpressed in human differentiated adipocytes and secreted levels of adiponectin were analyzed in conditional medium. Results are representative of six biological/independent experiments used for Western blot analysis.

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E. miR-361-5p and miR-574-5p was overexpressed alone at 10 nM or co-expressed (10+10 nM) in in vitro differentiated adipocytes, and mRNA levels of EBF1 were measured by RT-qPCR. Results are based on three biological/independent experiments.

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Expression of genes was normalized to the reference gene LRP10. Results were analyzed using t-test and presented in fold change ± SEM relative to negative control (Neg C). ***P <0.005, **P <0.01, *

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P <0.05.

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B

***

0.6 0.3

miR-361-5p

miR-574-5p

NegC

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VEGFA

Sox12

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NegC

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10+10

40

miR-361-5p (nM)

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miR-574-5p (nM)

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Supplemental Figure 1

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Relative miR expression (miR/NegC) log10

Relative gene expression (miR/NegC)

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Supplemental figure 1. Validation of experimental setup by quantification of miRNA expression and expression of known targets of each miRNA.

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A. miR-361-5p and -574-5p was overexpressed at 40 nM in in vitro differentiated adipocytes and expression of VEGFA and Sox12 was measured and normalized to the reference gene LRP10.

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B. MicroRNAs were overexpressed individually (10 nM and 40 nM) or co-expressed (10 nM+10 nM) in in vitro differentiated adipocytes and their expression of was assessed by RT-qPCR. Expression of miRNAs were normalized to the reference gene SNORD68 and relative expression values of were logtransformed.

Results were analysed using t-test and presented in fold change ± SEM relative to negative control (Neg C). ***P <0.005, **P <0.01, * P <0.05.

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ACCEPTED MANUSCRIPT 3.4. Association between expression of miR-365-5p and miR-574-5p and EBF1 activity We have previously performed a global analysis of transcription factor activity in cohort 1 using Motif Activity Response Analysis (MARA) (Arner, Mejhert et al. 2012). We utilized the data to evaluate association between motif activity of EBF1 and miRNAs-361-5p/-574-5p. A linear

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regression analysis revealed that EBF1 activity was significantly and negatively associated with the expression of either miRNA determined by qPCR (Table 1). These associations remained significant after adjustment for BMI.

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Table 1. Relationship between EBF1 motif activity and expression of miRNAs together with BMI in

Simple regression EBF1 Regressor r p-value miR-361-5p -0,34 0,03 miR-574-5p -0,31 0,04

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fat cells.

Multiple regression microRNA BMI partial r p-value partial r -0,27 0,03 -0,56 -0,29 0,02 -0,58

p-value <0,0001 <0,0001

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miRNA values were normalized to miR-103 and log transformed of available 43 lean and obese

4. Discussion

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individuals (Arner, Mejhert et al. 2012).

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Although reduced EBF1 levels were recently linked to the development of WAT hypertrophy (Gao, Mejhert et al. 2014; Petrus, Mejhert et al. 2015), the mechanisms controlling EBF1 expression are not known. Herein, we identified 15 miRNAs overexpressed in hypertrophic compared with hyperplastic human WAT. Out of these, ten were predicted to target EBF1 and we could confirm that miR-365-5p and miR-574-5p bound to EBF1 and regulated its’ expression in an additive manner.

To our knowledge, this is the first study to investigate the expression of miRNAs in relation to human WAT morphology. MiR-365-5p and miR-574-5p have previously been described in association with coronary heart disease and cancer (Wang, Lo et al. 2014; Zhang, Thevapriya et al. 2014), but not in 15

ACCEPTED MANUSCRIPT the context of obesity and/or diabetes. Furthermore, while there is a hand-full of studies that have investigated the regulation of EBF1 by miRNAs, these have primarily focused on B-cells (Luo, Liu et al. 2015). Admittedly, the effects of miR-365-5p and miR-574-5p on EBF1 expression were relatively small and only the effects of the former could be confirmed at the protein level most likely due to the

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greater effects on EBF1 mRNA. However, this is in line with previous studies demonstrating that miRNAs are primarily involved in fine-tuning gene expression. It should also be pointed out that EBF1 protein displays a relatively slow turnover as it was recently shown that it takes seven days to observe attenuated EBF1 protein levels after shRNA treatment (Griffin, Zhou et al. 2013).

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Unfortunately, as EBF1 is also important for adipogenesis (Jimenez, Akerblad et al. 2007), it is not possible to perform long-term treatments of miRNAs/EBF1 without affecting cell differentiation.

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Nevertheless, the fact that that miR-365-5p and miR-574-5p exerted additive effects on EBF1 mRNA levels indicates that the consorted action of several miRNAs binding to different parts of 3’UTR may impact more substantially on gene expression. The observation that the miRNAs also affected the expression of the EBF1 target gene CIDEA, suggests that the effects on EBF1 gene expression do

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translate into reduced EBF1 activity.

The relevance of the other 13 dysregulated miRNAs in WAT hypertrophy remains to be evaluated.

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One can speculate that these miRNAs may regulate other factors involved in determining WAT morphology that have not yet been described or might be involved in various metabolic pathways that

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in turn affect WAT morphology. Some of these miRNAs have been reported to be dysregulated in obese WAT (miR-221, -17-5p, -26a, -125 as well as let-7a and -7d) or to be involved in adipose tissue inflammation (miR-221 and -222). Moreover, let -7d, miR-26a, -143, -26a regulate the secretion of pro-inflammatory factors such as CCL2 and/or TNFα as well as lipolysis and miR-143 may also influence differentiation, all reviewed in (Arner and Kulyte 2015).

In summary, the expression of several miRNAs is significantly increased in hypertrophic human scWAT. Expressional analysis in non-obese/obese women as well as experimental evaluations in vitro, confirm that miR-365-5p and miR-574-5p selectively target EBF1 and their expression correlate 16

ACCEPTED MANUSCRIPT with the activity of EBF1, a recently identified regulator of WAT morphology (Figure 4). These results suggest a link between miRNAs and WAT morphology which could be of pathophysiological

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relevance for the development of IR.

miR-361-5p (miR-361-5p + miR-574-5p)

miR-574-5p

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EBF1

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(?)

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Adipose hypertrophy Insulin resistance Altered lipolysis

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EBF1

Metabolic and adipogenic genes

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Figure 4

Figure 4. Proposed pathway of EBF1 regulation by miR-361-5p and -574-5p in hypertrophic human WAT. T-bars denote inhibition, vertical arrows indicates up/downregulation of expression levels.

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5. Acknowledgements The excellent technical assistance of Gaby Åström, Elisabeth Dungner, Kerstin Wåhlen and Eva Sjölin is highly appreciated. The authors thank Dr. Silvia Lorente-Cebrián for initial literature mining. This work was supported by several grants from the Swedish Research Council, the Swedish Diabetes

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Foundation, the Diabetes Program and CIMED Senior Investigator grants at Karolinska Institutet, Tore Nilsson foundation, Foundation for Gamla Tjänarinnor, Åke Wiberg foundation, EFSD/Lilly program and the Novo Nordisk foundationincluding the Tripartite Immuno-metabolism Consortium

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(TrIC) Grant Number NNF15CC0018486 and the MSAM consortium NNF15SA0018346.

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References

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MicroRNAs-361-5p and miR-574-5p associate with human adipose morphology and regulate EBF1 expression in white adipose tissue Highlights The expression of several miRNAs is significantly increased in hypertrophic human scWAT.

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miR-365-5p and miR-574-5p bind to 3’UTR of EBF1, recently identified regulator of WAT

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morphology.

The miRNAs regulate expression of EBF1 alone or in combinatorial with each other.

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miR-365-5p and miR-574-5p may be linked to WAT hypertrophy via effects on EBF1

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expression and it’s transcription activity.

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