Abstract
Glucocorticoids are important regulators of lipid homeostasis, and chronically elevated glucocorticoid levels induce hypertriglyceridemia, hepatic steatosis, and visceral obesity. The occupied glucocorticoid receptor (GR) is a transcription factor. However, those genes regulating lipid metabolism under GR control are not fully known. Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor), a protein inhibitor of lipoprotein lipase, is synthesized and secreted during fasting, when circulating glucocorticoid levels are physiologically increased. We therefore tested whether the ANGPTL4 gene (Angptl4) is transcriptionally controlled by GR. We show that treatment with the synthetic glucocorticoid dexamethasone increased Angptl4 mRNA levels in primary hepatocytes and adipocytes (2-3-fold) and in the livers and white adipose tissue of mice (approximately 4-fold). We tested the mechanism of this increase in H4IIE hepatoma cells and found that dexamethasone treatment increased the transcriptional rate of Angptl4. Using bioinformatics and chromatin immunoprecipitation, we identified a GR binding site within the rat Angptl4 sequence. A reporter plasmid containing this site was markedly activated by dexamethasone, indicative of a functional glucocorticoid response element. Dexamethasone treatment also increased histone H4 acetylation and DNase I accessibility in genomic regions near this site, further supporting that it is a glucocorticoid response element. Glucocorticoids promote the flux of triglycerides from white adipose tissue to liver. We found that mice lacking ANGPTL4 (Angptl4(-/-)) had reductions in dexamethasone-induced hypertriglyceridemia and hepatic steatosis, suggesting that ANGPTL4 is required for this flux. Overall, we establish that ANGPTL4 is a direct GR target that participates in glucocorticoid-regulated triglyceride metabolism.
Highlights
Fasting increases circulating levels of angiopoietin-like 4 (ANGPTL4, a fasting-induced adipose factor)
Glucocorticoids Increase ANGPTL4 gene (Angptl4) Expression in Vitro and in Vivo—To determine whether Angptl4 expression is regulated by glucocorticoids in primary rat hepatocytes and human adipocytes, we treated these cells with the synthetic glucocorticoid DEX for 5 h
We first confirmed that DEX treatment increased the expression of Pepck and Per1 in hepatocytes and adipocytes, respectively, and did not affect mRNA levels of Angptl3 and Abcd4, two genes not known to be regulated by glucocorticoids (Fig. 1A)
Summary
H4IIE rat hepatoma cells (a gift from Dr Daryl Granner, Vanderbilt University) were cultured in Dulbecco’s modified Eagle’s medium with 5% fetal bovine serum (Invitrogen). 50 l of beads (in binding/wash buffer) were added to RNA, incubated at 42 °C for 20 min, and shaken for 2 h at room temperature. To synthesize random-primed cDNA, 0.5 g of total RNA (10 l), 4 l of 2.5 mM dNTP, and 2 l of random primers (New England Biolabs) were mixed and incubated at 70 °C for 10 min. The resultant cDNA was diluted to 200 l, and 2.5 l was used to perform qPCR in a 25-l reaction containing Taq DNA polymerase (1.25 units; Promega), 1ϫ reaction buffer, 1.5 mM MgCl2, 0.5 mM dNTP (Invitrogen), 0.2ϫ SYBR Green I dye (Molecular Probes), and 250 nM of each primer. Cells were harvested and incubated in lysis buffer (10 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 10 mM NaCl, 150 mM sucrose, and 0.5% Nonidet P-40) at 4 °C for 5 min. One was incubated in 100 l of 2ϫ in vitro transcription buffer
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