Norepinephrine triggers an immediate-early regulatory network response in primary human white adipocytes

Abstract

Background: Norepinephrine (NE) signaling has a key role in white adipose tissue (WAT) functions, including lipolysis, free fatty acid liberation and, under certain conditions, conversion of white into brite (brown-in-white) adipocytes. However, acute effects of NE stimulation have not been described at the transcriptional network level.

Results: We used RNA-seq to uncover a broad transcriptional response. The inference of protein-protein and protein-DNA interaction networks allowed us to identify a set of immediate-early genes (IEGs) with high betweenness, validating our approach and suggesting a hierarchical control of transcriptional regulation. In addition, we identified a transcriptional regulatory network with IEGs as master regulators, including HSF1 and NFIL3 as novel NE-induced IEG candidates. Moreover, a functional enrichment analysis and gene clustering into functional modules suggest a crosstalk between metabolic, signaling, and immune responses.

Conclusions: Altogether, our network biology approach explores for the first time the immediate-early systems level response of human adipocytes to acute sympathetic activation, thereby providing a first network basis of early cell fate programs and crosstalks between metabolic and transcriptional networks required for proper WAT function.

Keywords: Early cell fate; Immediate-early gene; Network biology; Norepinephrine stimulation; Transcriptional regulatory network; White adipocyte.

Fig. 1

NE stimulation triggers an acute and broad transcriptional response in human adipocytes. a Workflow diagram for sample preparation. Adipocyte precursor cells were isolated from donors (n = 4), differentiated and treated with NE or vehicle for 3 h, harvested and subsequently analyzed by RNA-seq (The female human silhouette was modified from a clipart with a public domain license creative commons CC0). b Principal component analysis (PCA) and unsupervised hierarchical clustering (c) showed clustering between stimulated (orange) and unstimulated (blue) adipocytes in two distinct groups, suggesting that NE stimulation causes deep changes in the transcriptome. Columns are biological replicates and rows represent differentially expressed genes. d Reactome pathway enrichment for up- and e down-regulated DEGs after acute NE stimulation

Fig. 2

Acute NE responsive network has immediate-early genes as nodes with high betweenness. a The interaction network reconstructed from DEGs shows nodes with high betweenness. Edges represent experimentally verified protein-protein or protein-DNA interactions. Up-regulated DEGs are in red and down-regulated DEGs in green. Size node is related to betweenness value. b Known immediate-early genes were found to be up-regulated after NE stimulation. Expression values are shown in log₂ counts per million (CPM). Expression differences between conditions were analyzed with a Kruskall-Wallis Test, P < 0.0001, Dunn’s Multiple Comparison Test P < 0.05; ET Test, P < 0.05 (*** p < 0.0001, ** p < 0.001)

Fig. 3

Regulatory network analysis reveals a set of immediate-early genes that are acutely responsive to NE stimulation. Integrated view of relationships between master (1) and local (2) regulators, their expression (3) and their predicted targets (4) mapped to the chromosomal context (5). Regulatory edges link the master regulators to their targets, which were colored as indicated in the figure. Up-regulated (a) and down-regulated (b) regulatory networks, based on acute responses to NE stimulation, revealed master regulators with different targets, as shown in histograms

Fig. 4

Functional enrichment analysis suggests a complex changes in metabolic and signaling pathways upon acute NE stimulation. Functional gene network analysis of up-regulated genes showing metagroup enrichment in distinct cellular functions as represented by colored nodes and clusters. White nodes represent genes that connect two or more functional modules. Metagroup compositions are depicted in Table 1