Adipose Tissue MicroRNAs as Regulators of CCL2 Production in Human Obesity

Erik Arner; Niklas Mejhert; Agné Kulyté; Piotr J. Balwierz; Mikhail Pachkov; Mireille Cormont; Silvia Lorente-Cebrián; Anna Ehrlund; Jurga Laurencikiene; Per Hedén; Karin Dahlman-Wright; Jean-François Tanti; Yoshihide Hayashizaki; Mikael Rydén; Ingrid Dahlman; Erik van Nimwegen; Carsten O. Daub; Peter Arner

Disclosures

Diabetes. 2012;61(8):1986-1993.

Abstract and Introduction

Abstract

In obesity, white adipose tissue (WAT) inflammation is linked to insulin resistance. Increased adipocyte chemokine (C-C motif) ligand 2 (CCL2) secretion may initiate adipose inflammation by attracting the migration of inflammatory cells into the tissue. Using an unbiased approach, we identified adipose microRNAs (miRNAs) that are dysregulated in human obesity and assessed their possible role in controlling CCL2 production. In subcutaneous WAT obtained from 56 subjects, 11 miRNAs were present in all subjects and downregulated in obesity. Of these, 10 affected adipocyte CCL2 secretion in vitro and for 2 miRNAs (miR-126 and miR-193b), regulatory circuits were defined. While miR-126 bound directly to the 3'-untranslated region of CCL2 mRNA, miR-193b regulated CCL2 production indirectly through a network of transcription factors, many of which have been identified in other inflammatory conditions. In addition, overexpression of miR-193b and miR-126 in a human monocyte/macrophage cell line attenuated CCL2 production. The levels of the two miRNAs in subcutaneous WAT were significantly associated with CCL2 secretion (miR-193b) and expression of integrin, α-X, an inflammatory macrophage marker (miR-193b and miR-126). Taken together, our data suggest that miRNAs may be important regulators of adipose inflammation through their effects on CCL2 release from human adipocytes and macrophages.

Introduction

Obesity is associated with a low-grade inflammatory state in white adipose tissue (WAT), which influences fat cell function and may promote insulin resistance and type 2 diabetes.^[1,2] Adipocytes and infiltrating inflammatory cells (primarily macrophages) present within the tissue secrete key inflammatory proteins, such as tumor necrosis factor-α, interleukin (IL)-6, and chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemoattractant protein, and their gene expression and release are markedly increased in obesity.^[3,4] CCL2 may be particularly important since it has been proposed to initiate adipose inflammation by attracting inflammatory cells from the blood stream into WAT.^[5,6] Studies in mice show that CCL2 production and signaling are essential for the development of WAT inflammation.^[7] Although a number of different cell types in WAT may produce CCL2, the fat cells are of particular interest since adipocyte-derived CCL2 may promote local inflammation independent of the presence of macrophages/leukocytes in human WAT.^[8] However, the mechanisms controlling WAT CCL2 production in obesity are not clear.

The pathogenesis of obesity involves a complex interplay between various types of factors. At the molecular level, such interdependencies can be conceptualized as transcriptional regulatory networks with regulatory proteins and different classes of RNA molecules as nodes and their interactions as edges.^[9] Gene transcription is controlled at several different levels of which some have been elucidated only in recent years. MicroRNAs (miRNAs) have emerged as important factors regulating gene expression through binding to complementary sequences of target mRNAs resulting in decreased mRNA levels.^[10] Alterations in the levels of miRNAs have been shown to affect gene expression and thereby cell function in several pathophysiological conditions, including inflammation.^[11] The miRNAs may act directly on the target genes or indirectly by first regulating transcription factors (TFs), which, in turn, control the expression of genes.^[11] The role of miRNAs in adipose inflammation and obesity is, however, not known.

In this study, we aimed to define adipose miRNAs dysregulated in human obesity and their possible role in controlling CCL2 production. Through a systematic and unbiased approach, we were able to identify 10 obesity-regulated miRNAs that affected adipocyte CCL2 secretion in vitro. For two of these (miR-126 and miR-193b), we could define their mechanism of action, which involved direct or indirect (through TFs) regulation of CCL2 production in human adipocytes and macrophages.

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Table 1. Clinical characteristics of cohort 1
Clinical parameter	Obese (n = 30)	Nonobese (n = 26)	t test P value
Age (years)	43 ± 2	43 ± 3	0.833
Weight (kg)	111.7 ± 4.1	67.5 ± 1.7	0.0001
BMI (kg/m²)	40.9 ± 1.3	24.1 ± 1.8	0.0001
Waist circumference (cm)	121.7 ± 13.9	85.2 ± 9.2	0.0001
Body fat (%)	56.5 ± 1.6	33.7 ± 1.1	0.0001
Adipose CCL2 secretion*	32.5 ± 3.1	15.4 ± 3.0	0.0001
Fat cell volume (pL)	897.8 ± 38.5	503.5 ± 30.3	0.0001
Log10 HOMAIR	0.458 ± 0.060	−0.01 ± 0.049	0.0001
Log10 insulin-stimulated adipocyte lipogenesis†	0.346 ± 0.077	0.763 ± 0.089	0.0009

Cohort 1 comprised 30 obese otherwise healthy and 26 nonobese healthy women who were investigated in the morning after an overnight fast. Height, weight, waist circumference, fat cell volume, HOMAIR (an index of overall insulin sensitivity), and insulin-stimulated glucose uptake (measured as incorporation of [³H]glucose into lipids) were determined and evaluated. Results were analyzed using t test and are shown as mean ± SEM. *Related to the number of fat cells incubated (i.e., ng per 10⁷ cells). †Expressed as nmol per 2 h and 107 cells.

Table 2. Mapping of miRNA expression in adipose tissue and fat cells
	Intact tissue, fold change obese/nonobese
Probeset name	Microarray data qRT-PCR data		t test P value	Isolated fat cells, fold change obese/nonobese	t test P value
hsa-miR-193a-5p_st	0.81	0.65	0.0003	0.56	0.0002
hsa-miR-30c_st	0.85	0.67	0.0021	0.36	0.0002
hsa-miR-26a_st	0.76	0.81	0.0158	0.48	0.0003
hsa-miR-143_st	0.63	0.79	0.0276	0.43	0.0005
hsa-miR-92a_st	0.81	0.72	0.0295	0.64	0.0014
hsa-miR-145_st	0.86	0.65	0.0003	0.49	0.0036
hsa-miR-652_st	0.68	0.58	0.0004	0.45	0.0051
hsa-let-7d_st	0.75	0.79	0.0056	0.53	0.0113
hsa-miR-193b_st	0.90	0.72	0.0063	0.59	0.0151
hsa-miR-126_st	0.65	0.80	0.0472	0.67	0.0181
hsa-let-7a_st	0.70	0.77	0.0132	0.44	0.0244
hsa-miR-378_st	0.78	0.71	0.0008	0.57	0.0651
hsa-miR-197_st	0.86	0.79	0.0168	0.75	0.1233
hsa-miR-139–5p_st	0.86	0.84	0.0499	0.95	0.3072
hsa-let-7i_st	0.66	0.81	0.0559	0.37	0.0352
hsa-miR-486–5p_st	0.73	0.68	0.0562	0.58	0.0353
hsa-miR-151–5p_st	0.74	0.84	0.0621	0.73	0.0228
hsa-miR-222_st	1.27	1.19	0.0632	1.41	0.0136
hsa-miR-342–3p_st	1.15	0.85	0.1008	0.56	0.0030
hsa-miR-16_st	0.57	0.81	0.1423	0.41	0.0001

Global miRNA expression profiling comparing intact adipose tissue from obese (n = 30) and nonobese (n = 26) subjects identified 20 miRNAs regulated by obesity. For confirmation and further studies of microarray results, miRNA levels were measured with qRT-PCR in paired samples of human intact tissue and isolated adipocytes (n = 44). Results were analyzed using t test and are presented as fold change relative to the nonobese.

Table 3. Validation of predicted miRNA-TF interactions
		qRT-PCR		Luciferase activity
miRNA	Predicted target	Fold change ĆmiRNA/control	t test P value	Fold change miRNA/control	t test P value
let-7a	MYC	0.914	0.2203
miR-126	TFDP1	0.988	0.9095
miR-193b	ARNT	0.862	0.0046	1.001	0.99108
miR-193b	ETS1	0.54	0.0001	0.493	0.00077
miR-193b	MAX	0.576	0.0001	0.482	0.00001
miR-652	NFATC3	1.098	0.5063
miR-652	NFKB1	1.054	0.6819
miR-92a	RELB	0.564	0.0001	0.917	0.11786
miR-92a	SP1	1.071	0.5431

Predicted interactions between miRNAs and TFs were investigated by overexpressing each miRNA and assessing the effects on TF mRNA expression in human in vitro differentiated adipocytes. Overexpression of miR-92a and miR-193b altered the expression of TF levels, and to assess whether this interaction was direct, 39UTR analyses (by reporter assays in 3T3-L1 cells) were performed. To rule out unspecific effects, control cells were transfected with a negative control miRNA. Luciferase activity and mRNA levels were measured 24/48 h after transfection. Results were analyzed using t test and are presented as fold change relative to the negative control.

Table 4. Association between miR-193b and miR-126 expression and adipose tissue inflammation
Parameter	CCL2	ITGAX
miR-193b	−0.286 (0.0248)	−0.303 (0.0174)
miR-126	−0.064 (0.6326)	−0.296 (0.0204)
CCL2		0.420 (0.0114)

Expression levels of miR-193b and miR-126 were correlated with adipose chemokine CCL2 secretion and ITGAX mRNA levels in hu-man adipose tissue obtained from 56 subjects. Results were evaluated using multiple regression analysis with BMI as covariate. Data are presented as partial r value (P value) for miRNAs, ITGAX,and CCL2.

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