Comparative Secretome Analyses of Primary Murine White and Brown Adipocytes Reveal Novel Adipokines

Abstract

The adipose organ, including white and brown adipose tissues, is an important player in systemic energy homeostasis, storing excess energy in form of lipids while releasing energy upon various energy demands. Recent studies have demonstrated that white and brown adipocytes also function as endocrine cells and regulate systemic metabolism by secreting factors that act locally and systemically. However, a comparative proteomic analysis of secreted factors from white and brown adipocytes and their responsiveness to adrenergic stimulation has not been reported yet. Therefore, we studied and compared the secretome of white and brown adipocytes, with and without norepinephrine (NE) stimulation. Our results reveal that carbohydrate-metabolism-regulating proteins are preferably secreted from white adipocytes, while brown adipocytes predominantly secrete a large variety of proteins. Upon NE stimulation, an increased secretion of known adipokines is favored by white adipocytes while brown adipocytes secreted higher amounts of novel adipokines. Furthermore, the secretory response between NE-stimulated and basal state was multifaceted addressing lipid and glucose metabolism, adipogenesis, and antioxidative reactions. Intriguingly, NE stimulation drastically changed the secretome in brown adipocytes. In conclusion, our study provides a comprehensive catalogue of novel adipokine candidates secreted from white and brown adipocytes with many of them responsive to NE. Given the beneficial effects of brown adipose tissue activation on its endocrine function and systemic metabolism, this study provides an archive of novel batokine candidates and biomarkers for activated brown adipose tissue.

Keywords: Adrenergic stimulation; Brown adipocytes; Cell secretion; Mass spectrometry; Molecular biology; Murine SVF cells; Omics; SILAC; Secretome; White adipocytes.

Graphical abstract

Fig. 1.

Principle and experimental design of the secretome analysis. The secretome analysis workflow started with incubation of primary white or brown adipocytes with media lacking methionine, lysine, and arginine for 30 min followed by media supplemented with AHA and either intermediate or heavy isotope labeled amino acids (lysine and arginine) for 24 h. In addition, 0.5 μm NE was added in selected analyses. After incubation, the supernatants were collected and the secreted (AHA-containing) proteins were enriched via click-chemistry, washed, digested, fractionated, and analyzed by LC-MS/MS.

Fig. 2.

Comparative secretome analysis between primary murine white and brown adipocytes. A. Volcano plot depicting 141 significantly (p.adj < 0.05, nominal p value cutoff = 0.023) differentially secreted proteins including 10 known adipokines (in green). B. GO enrichment analysis of the 141 proteins based on their higher secretion in white or brown adipocytes, with significantly enriched GO terms in color (p value threshold of 0.01) and nonsignificant ones in gray (p value threshold of 0.1). C. Volcano plot as in panel A. Proteins belonging to the enriched GO terms are named and colored. Additionally, the top differentially secreted proteins that are discussed in the results are labeled in black. WA: white adipocytes, BA: brown adipocytes.

Fig. 3.

Comparative secretome analysis between primary murine white and brown adipocytes upon NE stimulation. A. Volcano plot depicting 186 significantly (p.adj < 0.05, nominal p value cutoff = 0.023) differentially secreted proteins including 14 known adipokines (in green). B. GO enrichment analysis of the 186 proteins based on their higher secretion in white or brown adipocytes, with significantly enriched GO terms in color (p value threshold of 0.01) and nonsignificant ones in gray (p value threshold of 0.1). C. Volcano plot as in panel A. Proteins belonging to the enriched GO terms are named and colored. Additional top differentially secreted and discussed proteins are named in black. WA: white adipocytes, BA: brown adipocytes.

Fig. 4.

The secretory response of primary murine white and brown adipocytes between the NE-stimulated and basal state. Scatterplot representing the fold changes of the significantly differentially secreted proteins from Figs. 2 and 3, i.e. between white and brown adipocytes in the basal state and upon NE-stimulation. Proteins in Q1 and Q2 represent white adipocyte-enriched adipokines in the unstimulated state (y axis > 0), while Q3 and Q4 display brown adipocyte-enriched adipokines in the basal state (y axis < 0). Proteins in Q1 and Q4 are more secreted from white than brown adipocytes upon NE (x axis > 0), while Q2 and Q3 represent proteins higher secreted from brown than white adipocytes upon NE (x axis < 0). Only proteins that were detected in both analyses are represented. Proteins with preferential NE-responsive secretion in brown or white adipocytes are labeled in black. WA: white adipocytes, BA: brown adipocytes, Q1–4: quadrant 1–4.

Fig. 5.

Comparative secretome analysis of primary murine brown adipocytes with and without NE. A. Volcano plot depicting 280 significantly (p.adj < 0.05, nominal p value cutoff = 0.023) differentially secreted proteins and 11 known adipokines (in green). B. GO enrichment analysis (p value threshold of 0.01) of the 280 proteins based on their higher secretion with or without NE. Significantly enriched GO terms are in color and non-significant ones in gray (p value threshold of 0.1). C. Volcano plot as in panel A. Proteins belonging to the enriched GO terms are colored, respectively. Additional top differentially secreted and discussed proteins are named in black. WA: white adipocytes, BA: brown adipocytes.