Abstract 139: Id3 Regulation of High-Fat-Diet--Induced Visceral Adipose Tissue Macrophages

Abstract

BACKGROUND: Global deletion of the helix-loop-helix (HLH) transcription factor Inhibitor of Differentiation 3 (Id3), attenuates HFD-induced obesity. Specifically, Id3 -/- mice have less visceral adipose tissue (VAT); an effect that may be linked to VAT inflammation. Adipose tissue macrophages (ATMs) play a key role in HFD-induced obesity. Monocyte chemoattractant protein-1 (MCP-1) is a key cytokine regulating VAT macrophage accumulation, yet the role of Id3 in regulating MCP-1 expression and ATM accumulation is unknown. RESULTS: Id3 -/- mice and C57Bl/6 litter-mate controls were fed 4 weeks of HFD. The epididymal adipose tissue (VAT) and sera were analyzed for MCP-1 expression. Consistent with published findings, results demonstrated that HFD induced MCP-1 mRNA (analyzed via qPCR) and protein expression (analyzed via ELISA) in VAT. Notably, there was marked attenuation of HFD-induced MCP-1 expression in Id3 -/- mice. Epididymal adipose tissue from both WT and knockout mice were cultured with and without IL-1β. MCP-1 protein levels were the same in both sets of mice, when fat from chow-fed mice was cultured in media alone. However, IL-1β induced MCP-1 protein expression in VAT, an effect significantly attenuated in mice null for Id3. Flow cytometry analysis revealed that at early time-points (4 weeks HFD), WT mice have a significant increase in M1 macrophage numbers in epididymal VAT in response to HFD. However, Id3 null mice had an attenuation of this response to HFD. In contrast, HFD induced a statistically significant increase in M2 macrophages in both WT and Id3 -/- mice. RAW 264.7 cells were stimulated with 100ng/ml of LPS for 0, 0.5, 3, 6 and 24 hours. LPS treatment led to a significant and rapid induction of Id3 mRNA expression, which preceded upregulation of MCP-1 mRNA and protein levels. NIH 3T3 cells were co-transfected with a 1-kb MCP-1 luciferase promoter construct, as well as an expression plasmid encoding E12, a member of the bHLH E-protein family of transcription factors. Increasing concentrations of E12 led to repression of MCP-1 promoter activity, seen as a decrease in RLU/s compared to transfection with the promoter alone.. CONCLUSION: The loss of Id3 decreases local and systemic MCP-1 expression, and decreases M1 macrophage numbers in visceral adipose tissue. Id3’s partner E12 represses MCP-1 promoter activity. Based on the Id3-E-protein paradigm, it is likely that Id3 would restore MCP-1 promoter activity. These data suggest that Id3 promotes HFD-induced MCP-1 expression and macrophage-mediated visceral adipose tissue expansion. Further studies are needed to confirm Id3 regulation of MCP-1 specifically in macrophages.