The expression of GPIHBP1, an endothelial cell binding site for lipoprotein lipase and chylomicrons, is induced by peroxisome proliferator-activated receptor-gamma

Abstract

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), a protein in the lymphocyte antigen 6 (Ly-6) family, plays a key role in the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1 binds lipoprotein lipase and chylomicrons and is expressed along the luminal surface of microvascular endothelial cells. Lipolysis is known to be regulated by metabolic factors and is controlled at multiple levels, including the number of LPL binding sites on capillaries. Here, we tested the possibility that GPIHBP1 expression could be regulated by dietary perturbations and by peroxisome proliferator-activated receptors (PPARs). Gpihbp1 transcript levels in the heart and in brown and white adipose tissue increased with fasting and returned toward baseline after refeeding. A PPARgamma agonist increased Gpihbp1 expression in adipose tissue, heart, and skeletal muscle, whereas PPARalpha and PPARdelta agonists had no effect. Gpihbp1 was expressed in endothelial cells of embryoid bodies generated from mouse embryonic stem cells, and Gpihbp1 expression in embryoid bodies was up-regulated by a PPARgamma agonist. Sequences upstream from exon 1 of Gpihbp1 contain a strong PPAR binding site, and that site exhibited activity in a luciferase reporter assay. Gpihbp1 transcript levels in brown and white adipose tissue were lower in endothelial cell PPARgamma knockout mice than in littermate control mice, suggesting that PPARgamma regulates Gpihbp1 expression in vivo. We conclude that GPIHBP1 is regulated by dietary factors and by PPARgamma.

Figure 1

Regulation of Gpihbp1 and Lpl Expression by Fasting and Refeeding RNA was isolated from fed mice, mice fasted for 16 h, and mice that had been fasted for 16 h and then refed a high-carbohydrate diet for 6 h (n = 10 male 10-wk-old C57BL/6 mice per group). Gpihbp1 and Lpl expression levels were measured by quantitative RT-PCR (mean ± se), and the data were normalized to β2-microglobulin expression. Bars show the amounts of Gpihbp1 and Lpl expression during fasting and refeeding, relative to those in fed mice, in heart (A), gonadal fat (B), brown adipose tissue (C), and liver (D).

Figure 2

Gpihbp1 and Lpl Expression in Mice on a High-Fat Diet RNA samples from various tissues were isolated from 11- to 12-month-old male mice fed chow (n = 7) or high-fat (n = 9) diets for 6 months. Gpihbp1 (A) and Lpl (B) expression on chow and high-fat diets were measured by quantitative RT-PCR and normalized to 36B4 expression. Bar graphs show expression levels on the high-diet relative to levels on the chow diet (mean ± se).

Figure 3

Identification of a PPRE Upstream of Exon 1 in Gpihbp1 A, Eight potential PPREs upstream of Gpihbp1 exon 1. Each potential PPRE, its distance from exon 1, and its orientation are listed. B, EMSA testing the ability of unlabeled potential PPREs from Gpihbp1 to compete with a well-characterized (10) ³²P-labeled ap2 PPRE for binding to PPARγ/RXRα heterodimers. C, EMSA testing the ability of the indicated ³²P-labeled Gpihbp1 PPRE to bind directly to PPARγ/RXRα heterodimers. D, Luciferase activity in HEK293T cells that had been transfected with a luciferase reporter construct containing zero, three, or six copies of the most proximal Gpihbp1 PPRE (PPRE 4) in the presence and absence of PPARγ/RXRα expression vectors (mean ± se). All cells were also transfected with a β-galactosidase expression vector, and luciferase activity was normalized to β-gal expression.

Figure 4

Changes in Gpihbp1 Expression in Response to PPAR Agonists Mice were treated with a PPARγ agonist (30 mg/kg rosiglitazone), a PPARα agonist (10 mg/kg GW7647), a PPARδ agonist (10 mg/kg GW742), or vehicle by oral gavage twice daily for 2 days (n = 10 per group). The expression of Gpihbp1 in different tissues was quantified by quantitative RT-PCR, and the data were normalized to 36B4 expression. Cd36, Ucp3, and ap2 expression levels were also measured. Bars show the amount of Gpihbp1 mRNA on different PPAR agonists (mean ± se), relative to those in vehicle-treated mice, for sc fat (A), brown adipose tissue (B), heart (C), muscle (quadriceps) (D), and liver (E).

Figure 5

Gpihbp1 Expression in Embryoid Bodies and Differentiated ES Cells A, Expression of Gpihbp1, as judged by quantitative RT-PCR, in mouse ES cells and embryoid bodies derived from ES cells. The data were normalized to 36B4 expression; shown are means ± se (n = 18). B, Epifluorescence microscopy showing the binding of a rat monoclonal antibody against mouse CD31 (red) and a rabbit antiserum against GPIHBP1 (green) to nonpermeabilized (non-perm.) embryoid bodies or embryoid bodies that had been permeabilized with Triton X-100. Images were taken with a ×40 objective. C and D, Gpihbp1 and Cd36 expression in mouse embryoid bodies (C) and differentiated mouse ES cells (D) that had been treated with 5 μm rosiglitazone or vehicle (dimethylsulfoxide) alone. Expression levels of Gpihbp1 and Cd36 were measured by quantitative RT-PCR (mean ± se; n = 4), and the data were normalized to the expression of 36B4. Bars show expression levels on rosiglitazone relative to those on vehicle alone.

Figure 6

Gpihbp1 Expression in Mice Lacking Endothelial Cell Expression of PPARγ Adipose tissue RNA was isolated from endothelial cell PPARγ knockout mice (Pparg^fl/fl Tie2cre) and control mice (Pparg^fl/fl) (n = 3 control and 6 knockout male 7-month-old mice; all mice were in a Ldlr^−/− background), and the expression of Gpihbp1 was measured by quantitative RT-PCR (mean ± se). Expression of Cd36, Lpl, and ap2 were also measured. Gpihbp1 expression was normalized to Cd31 expression to account for possible differences in endothelial cell content. All other genes were normalized to β2-microglobulin expression. Bars show the level of different transcripts in brown adipose tissue (A), white adipose tissue (B), heart (C), muscle (D), and liver (E) of Pparg^fl/fl Tie2cre mice, relative to those in Pparg^fl/fl mice (mean ± se).