Insulin induces REDD1 expression through hypoxia-inducible factor 1 activation in adipocytes

Abstract

REDD1 (regulated in development and DNA damage responses) is essential for the inhibition of mTORC1 (mammalian target of rapamycin complex) signaling pathway in response to hypoxia. REDD1 expression is regulated by many stresses such as hypoxia, oxidative stress, and energy depletion. However, the regulation of REDD1 expression in response to insulin remains unknown. In the present study, we demonstrate that in murine and in human adipocytes, insulin stimulates REDD1 expression. Insulin-induced REDD1 expression occurs through phosphoinositide 3-kinase/mTOR-dependent pathways. Moreover, using echinomycin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, and HIF-1alpha small interfering RNA, we demonstrate that insulin stimulates REDD1 expression only through the transcription factor HIF-1. In conclusion, our study shows that insulin stimulates REDD1 expression in adipocytes.

FIGURE 1.

Insulin induces REDD1 expression in murine and human adipocytes. 3T3-L1 adipocytes were incubated for 16 h in normoxia (21% O₂) or in hypoxia (1% O₂) in absence or presence of 100 nm insulin. A, REDD1 and 36B4 mRNA were measured by real time PCR (n = 3). B, cell lysates were analyzed by immunoblots with the indicated antibodies. C, human adipocytes were incubated for 16 h in normoxia (21% O₂) or in hypoxia (1% O₂) in absence or presence of 100 nm insulin. The cell lysates were analyzed by immunoblots with the indicated antibodies. D, 3T3-L1 adipocytes were stimulated for 16 h with 100 nm insulin, 100 nm insulin-like growth factor 1 (IGF1), or 50 ng/ml fibroblast growth factor (FGF), and cell lysates were analyzed by immunoblots with the indicated antibodies. Representative experiments of three independent experiments are shown. Error bars indicate ±S.E. ***, p < 0.005.

FIGURE 2.

REDD1 is induced by insulin in a time- and dose-dependent manner. A, 3T3-L1 adipocytes were stimulated with 100 nm insulin for 4, 8, or 16 h, and cell lysates were analyzed by immunoblots with the indicated antibodies. B, 3T3-L1 adipocytes were stimulated for 16 h with increasing insulin concentrations (from 0.1 to 100 nm), and cell lysates were analyzed by immunoblots with the indicated antibodies. Representative experiments and quantification of six independent experiments are shown. Error bars indicate ±S.E. *, p < 0.05.

FIGURE 3.

Insulin stimulates REDD1 expression through PI3K/mTOR-dependent pathways in human adipocytes. Human adipocytes were incubated in normoxia (21% O₂) or in hypoxia (1% O₂) for 16 h in the absence or presence of 100 nm insulin, with or without 10 μm U0126 (A), 50 μm LY294002 (B), 40 nm rapamycin (C), or 10 μm GFX (D). Cell lysates were analyzed by immunoblots with the indicated antibodies. Representative experiments and quantification of REDD1 expression obtained from five to eight independent experiments are shown. Error bars indicate ±S.E. * p < 0.05.

FIGURE 4.

Regulation of REDD1 expression in response to insulin in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated in normoxia (21% O₂) or in hypoxia (1% O₂) for 16 h in the absence or in presence of 100 nm insulin with or without 10 μm U0126, 50 μm LY294002, 40 nm rapamycin, or 10 μm GFX. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Quantifications of REDD1 expression obtained from three to five independent experiments are shown. Error bars indicate ±S.E. *, p < 0.05.

FIGURE 5.

Echinomycin inhibits REDD1 expression in response to insulin. A, 3T3-L1 adipocytes were stimulated with 100 nm insulin for 16 h, and HIF-1α and REDD1 protein expression was determined by immunoblotting. A representative experiment of three independent experiments is shown. B, 3T3-L1 adipocytes were stimulated in normoxia (21% O₂) or in hypoxia (1% O₂) in the absence or presence of 100 nm insulin and 20 μm echinomycin for 16 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies, and the quantification of REDD1 protein level obtained in three independent experiments is shown. Error bars indicate ±S.E. *, p < 0.05; **, p < 0.01.

FIGURE 6.

REDD1 expression induced by insulin is dependent on the transcription factor HIF-1. 3T3-L1 adipocytes were transfected with two distinct HIF-1α siRNA (#1 and #2) as described under “Experimental Procedures” and were incubated in normoxia (21% O₂) or in hypoxia (1% O₂) in the absence or presence of 100 nm insulin for 16 h. HIF-1α mRNA was measured by real time PCR (A), and cell lysates were analyzed by immunoblotting with the indicated antibodies. Representative experiments of at least three independent experiments are shown, as well as the quantification of several independent experiments.