Intrinsic depot-specific differences in the secretome of adipose tissue, preadipocytes, and adipose tissue-derived microvascular endothelial cells

Abstract

Objective: Visceral adipose tissue (VAT) is more closely linked to insulin resistance than subcutaneous adipose tissue (SAT). We conducted a quantitative analysis of the secretomes of VAT and SAT to identify differences in adipokine secretion that account for the adverse metabolic consequences of VAT.

Research design and methods: We used lectin affinity chromatography followed by comparison of isotope-labeled amino acid incorporation rates to quantitate relative differences in the secretomes of VAT and SAT explants. Because adipose tissue is composed of multiple cell types, which may contribute to depot-specific differences in secretion, we isolated preadipocytes and microvascular endothelial cells (MVECs) and compared their secretomes to those from whole adipose tissue.

Results: Although there were no discrete depot-specific differences in the secretomes from whole adipose tissue, preadipocytes, or MVECS, VAT exhibited an overall higher level of protein secretion than SAT. More proteins were secreted in twofold greater abundance from VAT explants compared with SAT explants (59% versus 21%), preadipocytes (68% versus 0%), and MVECs (62% versus 15%). The number of proteins in the whole adipose tissue secretome was greater than the sum of its cellular constituents. Finally, almost 50% of the adipose tissue secretome was composed of factors with a role in angiogenesis.

Conclusions: VAT has a higher secretory capacity than SAT, and this difference is an intrinsic feature of its cellular components. In view of the number of angiogenic factors in the adipose tissue secretome, we propose that VAT represents a more readily expandable tissue depot.

FIG. 1.

LAC–CILAIR workflow for comparing protein synthesis and secretion from two different samples. Paired cell cultures or tissues are incubated in media containing different isotopes of arginine and lysine, which are subsequently incorporated into newly synthesized and secreted proteins. Paired conditioned media samples are mixed in equal proportions, and proteins are subjected to mass spectrometry. Incorporation of amino acid isotopes results in a shift in molecular weight and mass to charge (m/z) ratio. Peak heights are measured, and their ratios are used to determine relative abundance of proteins from samples a and b.

FIG. 2.

A: White adipose tissue (WAT) explants secretory proteins are secreted in greater amounts from VAT. B: Preadipocyte secreted proteins are secreted at greater amounts from visceral-derived preadipocytes. C: Endothelial cell secretory proteins are secreted at greater amounts from visceral-derived endothelial cells. WAT explants, preadipocytes, and microvascular endothelial cells from visceral and subcutaneous WAT depots were cultured in media containing two different isotopes of arginine and lysine. LAC–CILAIR mass spectrometry was performed, and isotopic ratios were calculated. The histogram shows distribution of visceral-to-subcutaneous ratios for proteins detected. Note the use of the natural log scale.

FIG. 3.

Venn diagram showing overlapping proteins detected from samples. A total of 116 secretory proteins were detected across all samples. PEDF, pigment epithelial derived factor; TIMP, tissue inhibitor of metalloproteinase 1. The diagram was composed using 3-Venn applet (48). (A high-quality color representation of this figure is available in the online issue.)

FIG. 4.

Venn diagram showing overlap of protein secretion from WAT explants, 3T3–L1 adipocytes, and isolated rat adipocytes (from ref 27). The 12 proteins detected in all three samples were adiponectin, adipsin, angiotensinogen (SerpinA8), cathepsin B, cathepsin D, collagen α-1(IV), collagen α-2(IV), complement C1s, haptoglobin, laminin subunit β-2, osteonectin, and thrombospondin-1. The diagram was composed using 3-Venn applet (48). (A high-quality color representation of this figure is available in the online issue.)