ABSTRACT
The key pathological link between obesity and type 2 diabetes is insulin resistance, but the molecular mechanisms are not entirely identified. micro-RNAs (miRNA) are dysregulated in obesity and may contribute to insulin resistance. Our objective was to detect and functionally investigate miRNAs linked to insulin sensitivity in human subcutaneous white adipose tissue (scWAT). Subjects were selected based on the insulin-stimulated lipogenesis response of subcutaneous adipocytes. Global miRNA profiling was performed in abdominal scWAT of 18 obese insulin-resistance (OIR), 21 obese insulin-sensitive (OIS), and 9 lean women. miRNAs demonstrating differential expression between OIR and OIS women were overexpressed in human in vitro-differentiated adipocytes followed by assessment of lipogenesis and identification of miRNA targets by measuring mRNA/protein expression and 3'-untranslated region analysis. Eleven miRNAs displayed differential expression between OIR and OIS states. Overexpression of miR-143-3p and miR-652-3p increased insulin-stimulated lipogenesis in human in vitro differentiated adipocytes and directly or indirectly affected several genes/proteins involved in insulin signaling at transcriptional or posttranscriptional levels. Adipose expression of miR-143-3p and miR-652-3p was positively associated with insulin-stimulated lipogenesis in scWAT independent of body mass index. In conclusion, miR-143-3p and miR-652-3p are linked to scWAT insulin resistance independent of obesity and influence insulin-stimulated lipogenesis by interacting at different steps with insulin-signaling pathways.
Subject(s)
Gene Expression Regulation , Insulin Resistance , MicroRNAs/metabolism , Obesity, Morbid/metabolism , Obesity/metabolism , Subcutaneous Fat, Abdominal/metabolism , 3' Untranslated Regions , Adult , Biopsy , Body Mass Index , Cells, Cultured , Cohort Studies , Female , Gene Expression Profiling , Humans , Lipogenesis , Male , MicroRNAs/agonists , Middle Aged , Obesity/pathology , Obesity, Morbid/pathology , RNA/metabolism , Subcutaneous Fat, Abdominal/pathologyABSTRACT
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 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-365-5p 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 miRNAs. EBF1 targets were affected by over expression/inhibition of either miRNAs. Finally, miR-365-5p/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.
Subject(s)
Adipose Tissue, White/anatomy & histology , Adipose Tissue, White/metabolism , Gene Expression Regulation , MicroRNAs/metabolism , Trans-Activators/genetics , Adipocytes/metabolism , Adipocytes/pathology , Cell Differentiation/genetics , Female , Humans , Hypertrophy , MicroRNAs/genetics , Reproducibility of Results , Trans-Activators/metabolismABSTRACT
CONTEXT: MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression. In white adipose tissue (WAT), recent studies suggest that miRNA levels are altered in various metabolic diseases, including obesity. OBJECTIVE: The objective of the study was to determine whether adipocyte-expressed miRNAs altered by obesity can regulate adiponectin expression/secretion in fat cells. DESIGN: Eleven miRNAs previously shown to be altered in obese human WAT were overexpressed in human in vitro-differentiated adipocytes followed by assessments of adiponectin levels in conditioned media. SETTING: This was cohort study (n = 56) in an academic hospital. PATIENTS: Subcutaneous WAT was obtained from nonobese and obese individuals. INTERVENTIONS: There were no interventions in this study. MAIN OUTCOME MEASURE(S): Protein and mRNA levels of adiponectin were measured. RESULTS: Of the 11 investigated miRNAs, three (miR-193b/-126/-26a) increased adiponectin secretion when overexpressed in human adipocytes. However, in human WAT only miR-193b expression correlated with adiponectin gene expression and homeostasis model assessment of insulin resistance. Moreover, quantitative PCR of miR-193b in both WAT and isolated adipocytes showed a significant association with serum adiponectin levels. Overexpression of miR-193b altered the gene expression of seven known adiponectin regulators. 3'-untranslated region reporter assays confirmed binding to cAMP-responsive element binding protein 5, nuclear receptor interacting protein 1, and nuclear transcription factor Yα. The effects of miR-193b on nuclear transcription factor Yα expression were confirmed at the protein level. Transfection with individual miRNA target protectors selective for nuclear transcription factor Yα and nuclear receptor interacting protein 1 abolished the stimulatory effect of miR-193b on adiponectin secretion. CONCLUSIONS: In human adipocytes, miR-193b controls adiponectin production via pathways involving nuclear transcription factor Yα and possibly nuclear receptor interacting protein 1.
Subject(s)
Adiponectin/genetics , Adipose Tissue, White/metabolism , MicroRNAs/physiology , 3T3-L1 Cells , Adaptor Proteins, Signal Transducing/physiology , Adiponectin/metabolism , Animals , CCAAT-Binding Factor/physiology , Case-Control Studies , Cells, Cultured , Cohort Studies , Female , Gene Expression Regulation , Humans , Male , Mice , Nuclear Proteins/physiology , Nuclear Receptor Interacting Protein 1 , Obesity/genetics , Obesity/metabolismABSTRACT
Adipose tissue inflammation is present in insulin-resistant conditions. We recently proposed a network of microRNAs (miRNAs) and transcription factors (TFs) regulating the production of the proinflammatory chemokine (C-C motif) ligand-2 (CCL2) in adipose tissue. We presently extended and further validated this network and investigated if the circuits controlling CCL2 can interact in human adipocytes and macrophages. The updated subnetwork predicted that miR-126/-193b/-92a control CCL2 production by several TFs, including v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) (ETS1), MYC-associated factor X (MAX), and specificity protein 12 (SP1). This was confirmed in human adipocytes by the observation that gene silencing of ETS1, MAX, or SP1 attenuated CCL2 production. Combined gene silencing of ETS1 and MAX resulted in an additive reduction in CCL2 production. Moreover, overexpression of miR-126/-193b/-92a in different pairwise combinations reduced CCL2 secretion more efficiently than either miRNA alone. However, although effects on CCL2 secretion by co-overexpression of miR-92a/-193b and miR-92a/-126 were additive in adipocytes, the combination of miR-126/-193b was primarily additive in macrophages. Signals for miR-92a and -193b converged on the nuclear factor-κB pathway. In conclusion, TF and miRNA-mediated regulation of CCL2 production is additive and partly relayed by cell-specific networks in human adipose tissue that may be important for the development of insulin resistance/type 2 diabetes.