Thrombospondin-1 is an adipokine associated with obesity, adipose inflammation, and insulin resistance

Abstract

Objective: We examined the relationship between the expression of thrombospondin (TSP)1, an antiangiogenic factor and regulator of transforming growth factor-beta activity, obesity, adipose inflammation, and insulin resistance.

Research design and methods: TSP1 gene expression was quantified in subcutaneous adipose tissue (SAT) of 86 nondiabetic subjects covering a wide range of BMI and insulin sensitivity, from visceral adipose (VAT) and SAT from 14 surgical patients and from 38 subjects with impaired glucose tolerance randomized to receive either pioglitazone or metformin for 10 weeks. An adipocyte culture system was also used to assess the effects of pioglitazone and coculture with macrophages on TSP1 gene expression.

Results: TSP1 mRNA was significantly associated with obesity (BMI) and insulin resistance (low insulin sensitivity index). Relatively strong positive associations were seen with markers of inflammation, including CD68, macrophage chemoattractant protein-1, and plasminogen activator inhibitor (PAI)-1 mRNA (r >/= 0.46, P = 0.001 for each), that remained significant after controlling for BMI and S(i). However, TSP1 mRNA was preferentially expressed in adipocyte fraction, whereas inflammatory markers predominated in stromal vascular fraction. Coculture of adipocytes and macrophages augmented TSP1 gene expression and secretion from both cell types. Pioglitazone (not metformin) treatment resulted in a 54% decrease (P < 0.04) in adipose TSP gene expression, as did in vitro pioglitazone treatment of adipocytes.

Conclusions: TSP1 is a true adipokine that is highly expressed in obese, insulin-resistant subjects; is highly correlated with adipose inflammation; and is decreased by pioglitazone. TSP1 is an important link between adipocytes and macrophage-driven adipose tissue inflammation and may mediate the elevation of PAI-1 that promotes a prothrombotic state.

FIG. 1

Correlations between SAT TSP1 mRNA and BMI (n = 82) (A) andS_i(n=73) (B). TSP1 mRNA was quantified by real-time RT-PCR analysis and was expressed relative to endogenous 18S RNA. S_i was determined by the frequently sampled intravenous glucose tolerance test method. TSP1 mRNA was plotted against the natural log of S_i. TSP mRNA is positively correlated with BMI and negatively correlated with S_i. Elimination of the four subjects in Fig. 1A with BMI >40 kg/m² yields r = 0.39, P = 0.0004, n = 78.

FIG. 2

Correlation between SAT TSP1 mRNA and PAI-1 mRNA (n = 80) (A) and plasma PAI-1 (n = 41) (B). TSP1 and PAI-1 mRNA levels were quantified by real-time RT-PCR analysis and were expressed relative to endogenous 18S RNA. Plasma TSP1 was measured by enzyme-linked immunosorbent assay as described in research design and methods. TSP1 mRNA was plotted against the natural log of PAI-1 mRNA and plasma PAI-1 levels. TSP1 mRNA is positively correlated to PAI-1 mRNA and plasma levels.

FIG. 3

Correlation between SAT TSP1 mRNA and mRNAs encoding adipose markers of inflammation CD68 (n = 81) (A) and MCP-1 (n = 81) (B). mRNA levels were quantified by real-time RT-PCR analysis and were expressed relative to endogenous 18S RNA. The TSP1 mRNA was plotted against the natural log of CD68 mRNA and MCP-1 mRNA. TSP1 mRNA was positively correlated with both CD68 and MCP-1 mRNAs.

FIG. 4

TSP1 protein expression and secretion in differentiated ADHASC and macrophages is augmented in coculture. A: Representative blot of TSP1 protein expression in cell lysates and loading control for cell lysates of differentiated ADHASC alone (Ad), cocultured differentiated ADHASC (Ad from AdMc coculture), cocultured THP-1 macrophages (Mc from AdMc coculture), and THP-1 macrophages alone (Mc). B: Representative blot of secreted TSP1 in medium from cultures of differentiated ADHASC alone (Ad), differentiated ADHASC cocultured with macrophages (AdMc), and THP-1 macrophages alone. Cells were cultured for 48 h. Equal volumes of medium was resolved under reducing conditions and subjected to Western blot to detect TSP1. Densitometric analysis of Western blots of medium for TSP1. The densitometric values are a means ± SE of three independent experiments. *P < 0.01 vs. AdMc; †P < 0.02 vs. AdMc; ‡P < 0.05 (Ad vs. Mc).

FIG. 5

Effect of pioglitazone on TSP1 and PAI-1 expression. A:IGT subjects were treated with pioglitazone (n= 17) or metformin (n= 21) for 10 weeks, and TSP1 mRNA expression was quantified in adipose tissue by real-time RT-PCR. B:Effect of pioglitazone (n= 12) and metformin (n= 15) treatment of IGT subjects on plasma PAI-1. C:Effect of 48 h pioglitazone treatment of SGBS adipocytes in vitro on TSP1 and PAI-1 mRNA expression. The treatment of SGBS adipocytes was performed in duplicate and repeated twice. mRNA levels were quantified by real-time RT-PCR analysis and were expressed relative to endogenous 18S RNA.