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Adipocyte-mediated inflammatory signalling has been proposed to alter adipose physiology in obesity and Type 2 diabetes mellitus. Novel targets for alteration of inflammatory signalling are needed to improve obesity-related outcomes. The γ-secretase enzyme complex has been suggested to play a role both in adipocyte function as well as in immune regulation. We hypothesized that adipocyte-specific γ-secretase inhibition could alter the inflammatory makeup of adipose tissue. We found that genetic blockade of γ-secretase in adipocytes leads to a decrease in EMR1 (F4/80) expression, as a marker of macrophage presence, in adipose tissue without changes in expression of markers of other inflammatory cell types. To explore the mechanism by which adipocytes can alter macrophage function in vitro, fully differentiated 3T3-L1 adipocytes were treated with a γ-secretase inhibitor in the presence of lipopolysaccharide (LPS) and transcription of IL6 and ccl2 (MCP1) were quantified. IL-6 expression and secretion were significantly inhibited by γ-secretase blockade, with little effect on MCP1. Preconditioned media from 3T3-L1 adipocytes treated with a γ-secretase inhibitor also alters macrophage activation but did not affect macrophage translocation in vitro. Therefore, γ-secretase inhibition in fully differentiated adipocytes can alter IL-6 signalling to macrophages, consistent with our hypothesis that that γ-secretase is involved in adipocyte-initiated inflammatory signalling cascades. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to investigate cellular sources of microRNAs (miRNA) within adipose tissue and the impact of obesity on miRNA expression, as well as to examine targets of miRNAs.Methods: miRNA expression by quantitative polymerase chain reaction was examined in adipocytes, adipose tissue macrophages (ATM), and peripheral blood mononuclear cells from and individuals with normal weight and with obesity. Differentiated 3T3-L1 adipocytes were cocultured with macrophages, and 3T3-L1 and differentiated human mesenchymal stem cells were transfected with miR-155, with peroxisome proliferator-activated receptor gamma (PPAR-γ) and solute carrier family 2 member 4 (GLUT4) abundance measured via Western blot analysis.Results: Abundance of miR-155 and miR-210 was increased in ATM of participants with obesity by 6.7-fold and 2.9-fold (P = 0.002 and P = 0.013, respectively). miR-130b expression was increased 1.8-fold in ATM and 4.3-fold in adipocytes from participants with obesity (P = 0.007 and P = 0.02, respectively). PPARG mRNA expression decreased 32% (P = 0.044) in adipocytes from individuals with obesity. In 3T3-L1 cells exposed to macrophages, PPARG expression decreased 99.4% (P = 0.02). PPAR-γ protein content declined 75% (P = 0.001) in 3T3-L1 cells transfected with miR-155. GLUT4 protein levels were reduced by 55% (P = 0.021) in differentiated human mesenchymal stem cells exposed to miR-155.Conclusions: Adipose tissue miRNAs are influenced in a cell type-specific fashion by obesity, with macrophage miR-155 potentially impacting neighboring adipocytes. [ABSTRACT FROM AUTHOR]

The insulin-responsive glucose transporter, GLUT4, is regulated in various physiologic states at the transcriptional level. When expressed in transgenic mice, the human GLUT4 promoter is governed by two cis-acting sequences: an MEF2 binding domain and Domain I, that function both as positive and negative regulators depending on the physiologic state. MEF2 proteins and GLUT4 enhancer factor (GEF) are known ligands for these cis-acting elements, but their mechanism of action is unclear. To begin to understand this important process, we have characterized GEF structural domains and its interactions with the MEF2A isoform. We find that the C terminus of GEF comprises its DNA-binding domain, but does not contribute to GEF homo-oligomerization. We also have found that GEF dimerizes with increased affinity to a hypophosphorylated form of MEF2A. Furthermore, we demonstrated that MEF2A binding to its cognate binding site can increase the DNA binding activity of GEF to Domain I, suggesting a novel mechanism for MEF2A transcriptional activation. Finally, we have demonstrated that the transcriptional co-repressor HDAC5 can interact with GEF in the absence of MEF2 proteins and specifically inhibit GLUT4 promoter activity. These findings lead to the hypothesis that GEF and the MEF2 proteins form a complex on the GLUT4 promoter that allows for recruitment of transcriptional co-regulators (repressors and/or activators) to control GLUT4 promoter activity. [ABSTRACT FROM AUTHOR]

GLUT4 promoter activity is regulated by hormonal, metabolic, and tissue-specific controls. This complicates the study of GLUT4 gene transcription, as no cell culture model adequately recapitulates these extracellular regulators. While investigating cultured primary adipocytes as a model system for GLUT4 transcription, we observed that GLUT4 mRNA was specifically and rapidly downregulated upon tissue dispersal. Downregulation of GLUT4 mRNA was mediated in part by loss of regulatory control by the trans-acting factors that control GLUT4 transcriptional activity [the myocyte enhancer factor 2 (MEF2) transcription factor family and the GLUT4 enhancer factor] and their cognate DNA binding sites in transgenic mice. The differences in GLUT4 transcription when whole adipose tissue and cell culture model systems are compared can be correlated to a posttranslational phosphorylation of the transcription factor MEF2A. The difference in the MEF2A phosphorylation state in whole tissue vs. isolated cells may provide a further basis for the development of an in vitro system that could recapitulate fully regulated GLUT4 promoter activity. Development of an in vitro system to reconstitute GLUT4 transcriptional regulation will further efforts to discern the molecular mechanisms that underlie GLUT4 expression. [ABSTRACT FROM AUTHOR]