A number of functions have been attributed to TSP1, including antiangiogenic activity (17
) and, more recently, as a proatherogenic protein that may provide an important link between diabetes and vascular complications (36
). Studies (5
) have reported the expression of TSP1 in adipose tissue, identifying TSP1 as a gene differentially expressed in different adipose depots of morbidly obese humans (5
) and overexpressed in obese Zucker rats (6
). However, the expression of TSP1 in subjects with varying degrees of obesity or the potential association of TSP1 to insulin resistance or metabolic syndrome has not yet been explored. This is the first study to examine the association of TSP1 to obesity, insulin resistance, inflammation, and metabolic syndrome and to establish TSP1 as an adipokine.
Whole adipose tissue is a complex tissue that, in addition to adipocytes, contains the SVF comprised of endo-thelial cells, monocytes, macrophages, pericytes, fibroblasts, and pluripotent stem cells, including preadipo-cytes, many of which are known sources of TSP1 (12
). Therefore, we first determined whether TSP1 is expressed by adipocytes. Separating whole adipose tissue into adi-pocyte and SVF revealed that the expression of the TSP1 gene was significantly higher in the adipocyte fraction compared with the SVF. Our observation, made from whole adipose tissue and its fractions, was further confirmed by examining the accumulation of TSP1 mRNA in adipocytes differentiated in vitro from preadipocytes of ADHASC and SGBS cells compared with THP-1–derived macrophages. As was observed in whole adipose tissue and its fractions, the mRNA expression in differentiated adipocytes was very high compared with the expression by macrophages. A similar profile was seen on examining TSP1 protein expression in cell lysates, suggesting that the TSP1 secreted into the medium was primarily derived from adipocytes, identifying TSP1 as an adipokine. The TSP1 gene was expressed in preadipocytes in vitro at comparable levels to differentiated adipocytes, suggesting that some of the observed expression of TSP1 in the SVF derives from preadipocytes. Previous studies have described complex regulation of TSP1 during adipocyte differentiation, with decreased TSP1 expression at day 2 and increased expression at day 6 (16
). The demonstrated antiproliferative properties of TSP1 (38
) may play a role in this regulation.
To better understand the role of TSP1 in humans with varying degrees of obesity and insulin resistance, we measured TSP1 mRNA in humans covering a wide range of BMI and Si
. TSP1 showed positive association with BMI and negative association with Si
, suggesting that it plays a role directly or indirectly in insulin resistance. Obesity-induced insulin resistance may be due to numerous factors such as increased adipose-derived circulating free fatty acids, increased secretion of inflammatory adipokines (39
), or decreased secretion of adiponectin (41
). Many adipose tissue inflammatory factors are secreted predominantly from adipose-derived macrophages as opposed to adipocytes (1
). Although our data demonstrate that TSP1 was primarily expressed in adipocytes, we found a significant positive association between TSP1 gene expression and the inflammatory markers MCP-1 and CD68, both of which are derived predominantly from macrophages (29
). MCP-1 is a chemoattractant whose increased production in obesity aids in the infiltration of macrophages into adipose tissue contributing to the inflammatory state. TSP1 also has chemotactic properties with smooth muscle cells and macrophages and is responsible for macrophage migration into the site of injury aiding in wound healing (17
). Moreover, TSP1 gene and protein expression were augmented in both adipocytes and macrophages following coculture, as was TSP1 secretion, demonstrating a direct interaction between these cells that may exacerbate the inflammatory state during obesity. Taken together, these results suggest that TSP1 is an important link between macrophage-driven inflammation and altered adipocyte function in metabolic syndrome. However, our results did not confirm a previous finding that the TSP1 gene is preferentially expressed in VAT compared with SAT (5
). Although expression in VAT was slightly higher than SAT, the difference was not significant. While the initial report was based on mRNA from VAT and SAT of two obese individuals, our study included 14 obese subjects.
Elevated plasma PAI-1 is recognized as a core feature of metabolic syndrome, contributing to the prothrombotic state (23
). PAI-1 is produced by endothelial cells and adipose tissue (21
) and, as demonstrated for CD68 and MCP-1, is preferentially expressed in the SVF of adipose tissue. PAI-1 gene expression is controlled by TGF-β through SMAD phosphorylation and subsequent binding to the PAI-1 promoter (45
). PAI-1 is a serine protease inhibitor that influences fibrinolysis by inhibiting both tissuetype plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA) (46
). We found that TSP1 gene expression correlated positively both with PAI-1 mRNA in adipose tissue and with plasma PAI-1. It is possible that this correlation is functional in adipose through TSP1-dependent TGF-β activation leading to upregulation of PAI-1 gene expression, which may contribute to the elevated circulating PAI-1 in insulin resistance and metabolic syndrome.
TZDs are insulin-sensitizing drugs that act as antidiabetes and anti-inflammatory agents. We examined the effect of the TZD pioglitazone on TSP1 gene expression in IGT subjects compared with another glucose-lowering drug, metformin. Pioglitazone-treated IGT subjects showed a significant decrease in TSP1 gene expression, whereas there was no significant change in TSP1 gene expression in subjects treated with metformin. SGBS adipocytes that were treated with pioglitazone also showed significant decrease in TSP1, as well as PAI-1 gene expression. These results are consistent with data showing that TSP1 is downregulated in response to TZDs during adipogenic differentiation in 3T3L1 cells (16
) and that PAI-1 expression is attenuated in adipose tissue in response to TZDs (34
). Recent work suggests that nuclear corepressors may repress peroxisome proliferator–activated receptor γ–mediated transcription on specific promoters in the adipocyte, providing a potential mechanism for downregulation (47
). It has also been reported that in macrophages, TZDs downregulate transcriptional activation through sumoylation of the peroxisome proliferator–activated receptor γ ligand-binding domain (48
), which may be relevant to inhibition of SVF gene expression. Thus, TZDs have the potential to downregulate PAI-1 gene expression directly in both macrophages and adipocytes. In addition, TZDs induced macrophage apoptosis and may thus reduce PAI-1 expression (31
). Finally, TZDs downregulated TSP1 expression in adipocytes that may interfere with TGF-β–dependent expression of PAI-1 in both macrophages and adipocytes.
In summary, TSP1 is an adipokine positively associated with BMI, PAI-1 levels, and markers of inflammation and negatively associated with Si. TSP1 has diverse biological functions, and, therefore, downregulation of TSP1 may mediate many of the wide range of beneficial effects of pioglitazone in metabolic syndrome and reduction of inflammation.