1248 Diabetes Volume 63, April 2014 Agné Kulyté,1 Yasmina Belarbi,1 Silvia Lorente-Cebrián,1 Clara Bambace,1 Erik Arner,1,2 Carsten O. Daub,3 Per Hedén,4 Mikael Rydén,1 Niklas Mejhert,1 and Peter Arner1 Additive Effects of MicroRNAs and Transcription Factors on CCL2 Production in Human White Adipose Tissue Adipose tissue inflammation is present in insulin- converged on the nuclear factor-kB pathway. In resistant conditions. We recently proposed conclusion, TF and miRNA-mediated regulation of a network of microRNAs (miRNAs) and transcription CCL2 production is additive and partly relayed by factors (TFs) regulating the production of the cell-specific networks in human adipose tissue that proinflammatory chemokine (C-C motif) ligand-2 may be important for the development of insulin (CCL2) in adipose tissue. We presently extended and resistance/type 2 diabetes. further validated this network and investigated if the Diabetes 2014;63:1248–1258 | DOI: 10.2337/db13-0702 METABOLISM circuits controlling CCL2 can interact in human adipocytes and macrophages. The updated subnetwork predicted that miR-126/-193b/-92a White adipose tissue (WAT) function plays an important control CCL2 production by several TFs, including role in the development of insulin resistance/type 2 di- v-ets erythroblastosis virus E26 oncogene homolog 1 abetes. Fat cells present in WAT secrete a number of (avian) (ETS1), MYC-associated factor X (MAX), molecules, collectively termed adipokines, which affect and specificity protein 12 (SP1). This was confirmed insulin sensitivity by autocrine and/or paracrine mecha- in human adipocytes by the observation that gene nisms (1,2). In insulin-resistant obese subjects, WAT silencing of ETS1, MAX, or SP1 attenuated CCL2 displays a chronic low-grade inflammation, which is production. Combined gene silencing of ETS1 and proposed to alter insulin signaling by increased fatty acid MAX resulted in an additive reduction in CCL2 release and altered adipokine production (3). production. Moreover, overexpression of miR-126/ Chemokine (C-C motif) ligand-2 (CCL2), also referred -193b/-92a in different pairwise combinations as monocyte chemoattractant protein-1, is an early reduced CCL2 secretion more efficiently than either proinflammatory signal in WAT that acts by promoting miRNA alone. However, although effects on CCL2 adipose infiltration of macrophages (4,5). After diet- secretion by co-overexpression of miR-92a/-193b induced obesity, Ccl2-/- mice are less insulin-resistant and and miR-92a/-126 were additive in adipocytes, the display a less pronounced inflammatory reaction in WAT combination of miR-126/-193b was primarily additive (6). Adipocytes and macrophages both contribute sig- in macrophages. Signals for miR-92a and -193b nificantly to CCL2 secretion from WAT. 1Department of Medicine, Huddinge, Lipid Laboratory, Karolinska Institutet, Corresponding authors: Peter Arner, [email protected], and Agné Kulyté, agne Stockholm, Sweden [email protected]. 2 RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Received 3 May 2013 and accepted 15 December 2013. Yokohama, Japan This article contains Supplementary Data online at http://diabetes 3Department of Biosciences and Nutrition, Huddinge, Karolinska Institutet, .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0702/-/DC1. Stockholm, Sweden 4Akademikliniken, Stockholm, Sweden © 2014 by the American Diabetes Association. See http://creativecommons .org/licenses/by-nc-nd/3.0/ for details. diabetes.diabetesjournals.org Kulyté and Associates 1249 Little is known about the mechanisms regulating WAT Another part of the tissue (at least 500 mg) was sub- inflammation. Results in recent years have demonstrated jected to collagenase treatment, and mean adipocyte that microRNAs (miRNAs) play important roles in con- volume and weight were determined as described (16). trolling inflammation in other conditions and non- Packed fat cells (200 mL) and intact WAT (400 mg) were adipose tissues, as reviewed (7). In mammals, miRNAs frozen at 270°C for future mRNA and miRNA mea- act by inhibiting the expression of target genes after surements as described (11). The Karolinska University binding to defined complementary sites in target 39- Hospital Ethical Committee (Stockholm, Sweden) ap- untranslated regions (UTRs) (8). proved this study. All subjects were informed in detail Cellular function is controlled by transcriptional about the studies, and written informed consent was (transcription factors [TFs]) and posttranscriptional obtained. mechanisms (miRNAs). Using global expression profiling For in vitro studies, subcutaneous WAT was obtained data and mathematical models, the transcriptome can be from healthy men and women undergoing cosmetic li- mapped into transcriptional regulatory networks (TRNs), posuction. This experimental group was not selected for with regulatory elements (e.g., TFs and miRNAs) as age, sex, or BMI. Isolation of human adipocyte progenitor nodes and their interactions as edges (9,10). We recently cells from subcutaneous WAT was performed as de- described and partly experimentally verified a miRNA-TF scribed (17). The progenitor cells obtained from separate network in the subcutaneous WAT of insulin-resistant individuals were not mixed. obese women that controls the expression of CCL2 (11). Affymetrix GeneChip Human Gene 1.0 ST and miRNA This TRN suggests that several miRNAs and TFs interact Array Protocols in different combinations to regulate CCL2 production. Although the reason for this complex regulation and re- Data obtained from gene and miRNA arrays from human dundancy is not entirely clear, it may allow for more exact subcutaneous WAT (Gene Expression Omnibus number fine-tuning of gene expression and/or signal enhancement GSE25402) and the protocols have been described (11). fi in response to speci c environmental cues. In line with Cell Culture this, recent studies in cancer cells have proposed that Culture and in vitro differentiation of human adipocyte partly overlapping regulatory circuits allow signal ampli- progenitor cells were performed as described (17). 3T3-L1 fication in an additive or synergistic manner (12,13). To andTHP1cellswerehandledasrecommendedinthepro- the best of our knowledge, it is not known whether these tocols from American Type Culture Collection (Manassas, types of mechanisms are present in WAT or whether they VA). For the induction of monocyte–macrophage differen- differ between specific cell types within the tissue. tiation, THP1 cells were stimulatedwith50ng/mLphorbol Through combinatorial gain-of-function/loss-of- 12-myristate 13-acetate (PMA). function studies focusing on TFs and miRNAs, we investigated how transcriptional regulators (TFs and miRNA and Small Interfering RNA Transfection miRNAs) can integrate effects on the expression of In vitro differentiated adipocytes (day 10–12 post- inflammatory factors in WAT. First, we extended and induction) were treated for 24–48 h with various con- further validated the proposed TRN (11). We sub- centrations (5–40 nmol/L) of miRIDIAN miRNA mimics sequently silenced potentially important TFs in the net- for overexpression of miRNA activity or 60 nmol/L of work, alone or in combination, and investigated how this miRIDIAN miRNA Hairpin Inhibitors for native miRNA altered CCL2 production in human adipocytes. Finally, inhibition (Thermo Fisher Scientific, Lafayette, CO) and we determined whether concomitant overexpression of HiPerFect Transfection Reagent (Qiagen, Hilden, miRNAs in human adipocytes or macrophages resulted in Germany), according to the manufacturers’ protocols. increased effects when compared with overexpressing Optimal transfection conditions were determined in each miRNA alone. separate titration experiments. Transfection efficiency was assessed with real-time quantitative PCR (qPCR) using RESEARCH DESIGN AND METHODS miRNA probes/assays (Applied Biosystems, Foster City, Clinical Material CA; and Qiagen, respectively). To rule out unspecific Cohort 1 comprised 30 obese (BMI .30 kg/m2) and effects, control cells were transfected with miRIDIAN 26 nonobese (BMI ,30 kg/m2) women who had no miRNA Mimic or Hairpin Inhibitor Non-Targeting Nega- chronic disease and were free of continuous medication. tive Controls (Thermo Fisher Scientific). This cohort has been described in detail previously (11). In some experiments, in vitro differentiated adipo- The subjects came to the laboratory in the morning after cytes were cotransfected with miScript Target Protector an overnight fast. Height, weight, and waist circumfer- for the miR-92a binding site on specificity protein 12 ence were determined. An abdominal subcutaneous WAT (SP1; 500 nmol/L; Qiagen) and miR-92a mimics (40 biopsy specimen (;1.0–1.5 g) was obtained by needle nmol/L) or appropriate control reagents (miScript Target aspiration as described (14). One part (300 mg) of the Protector Negative Control; Qiagen) and miRIDIAN tissue was used for measurement of CCL2 release and miRNA Mimic Non-Targeting Negative Control (Thermo expressed per number of fat cells as described (15). Fisher Scientific). THP1 cells were transfected with 1250 Additive Regulation of CCL2 by miRNAs and TFs Diabetes Volume 63, April 2014 miRNA mimics, as described above, for adipocytes 48 h Global Gene Expression Analysis, Motif Activity after incubation with PMA. Response Analysis, and Network Construction For RNA interference experiments, adipocytes were Motif activity response analysis and construction of the treated with various concentrations (10–40 nmol/L),