Characteristic expression of extracellular matrix in subcutaneous adipose tissue development and adipogenesis; comparison with visceral adipose tissue

Abstract

Adipose tissue is a connective tissue specified for energy metabolism and endocrines, but functional differences between subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) have not been fully elucidated. To reveal the physiological role of SAT, we characterized in vivo tissue development and in vitro adipocyte differentiation. In a DNA microarray analysis of SAT and VAT in Wistar rats, functional annotation clusters of extracellular matrix (ECM)-related genes were found in SAT, and major ECM molecules expressed in adipose tissues were profiled. In a histological analysis and quantitative expression analysis, ECM expression patterns could be classified into two types: (i) a histogenesis-correlated type such as type IV and XV collagen, and laminin subunits, (ii) a high-SAT expression type such as type I, III, and V collagen and minor characteristic collagens. Type (i) was related to basal membrane and up-regulated in differentiated 3T3-L1 cells and in histogenesis at depot-specific timings. In contrast, type (ii) was related to fibrous forming and highly expressed in 3T3-L1 preadipocytes. Exceptionally, fibronectin was abundant in developed adipose tissue, although it was highly expressed in 3T3-L1 preadipocytes. The present study showed that adipose tissues site-specifically regulate molecular type and timing of ECM expression, and suggests that these characteristic ECM molecules provide a critical microenvironment, which may affect bioactivity of adipocyte itself and interacts with other tissues. It must be important to consider the depot-specific property for the treatment of obesity-related disorders, dermal dysfunction and for the tissue regeneration.

Keywords: adipocyte; differentiation.; extracellular matrix; subcutaneous; visceral.

Figure 1

Expression profiles of ECM and non-adipocyte markers in subcutaneous adipose tissue by DNA microarray. Signal strength was normalized and presented as the mean ± S.E.M. of four animals. Expression of CD45 (a stem cell marker), CD31 (an endothelial cell marker), Actn1 (a muscle marker) and F4/80 (a macrophage marker) were detected.

Figure 2

Typical histological image of rat skin. Skin of abdominal area was excised, fixed and immunohistochemically stained with anti-type I collagen (green) and counterstained with DAPI (blue), or stained with Masson's trichrome (right panel). A part of boundary between adipose tissue and neighboring tissue is presented by dashed line. Subcutaneous adipocytes exist just beneath the dermis and under panniculus carnosus (deep layer). ED: Epidermis, D: dermis, F: hair follicle, PC: panniculus carnosus, ASCT: areolar suprafascial connective tissue, AT: adipose tissue Scale bar: 200 μm.

Figure 3

Localization of major ECM in subcutaneous and visceral adipose tissue. A) Tissue specimens of abdominal skin (left panels) and epididymal fat (right panels) from 4 week-old rats were immunohistochemically stained with anti-type I collagen, anti-laminin, or anti-fibronectin antibody (green) and counterstained with DAPI (blue). Magnification: × 400 Scale bars: 50 μm. B) Images immunohistochemically stained with anti-type I collagen for 12 week-old rats. A part of boundary between adipose tissue and neighboring tissue is presented by dashed line. Magnification: × 100 Scale bars: 200 μm.

Figure 4

Adipose tissue weight ratio and gene expression of PPARγ, aFABP and major ECM molecules. Upper left panel is adipose tissue weight / body weight ratio (%) presented as the mean ± S.E.M. of five animals for each group. Other panels were quantified mRNA of interested gene normalized by 36B4. Relative values to VAT at 4 weeks of age are presented as the mean ± S.E.M. of five animals. *: p<0.05, compared with the value of the VAT

Figure 5

Differential expression of ECM proteins in adipose tissues by Western blotting. Quantified values were normalized by α-tubulin, and relative value to VAT in 4 week-old rats are presented as the mean ± S.E.M. of five animals. Each emphasized gel image corresponds to SAT and VAT at 4 weeks and at 12 weeks of age. *: p<0.05, compared with the value of the VAT.

Figure 6

Differential expression of ECM in 3T3-L1 cells by real-time PCR. Quantified mRNA in undifferentiated and differentiated 3T3-L1 cells was normalized by 36B4. Relative values to undifferentiated level are presented as the mean ± S.E.M. of four wells for each condition. *: p<0.05, compared between undifferentiated and differentiated cells.