Inhibition of insulin receptor phosphorylation is dependent of mTOR activity.

<p>(A) 3T3-L1 adipocytes were transfected with REDD1 siRNA. 48 h after transfection, adipocytes were pretreated with rapamycin (40 nM) for 1 hour before being stimulated with insulin for 5 minutes. Proteins were analyzed by immunoblots using indicated antibodies. (B) Quantification of phosphorylation of insulin receptor (n = 8), pThr308-PKB (n = 3) in insulin-induced conditions is shown. (C) Quantification of phosphorylation of pThr389-S6K (n = 3) is shown (*p<0.05, **p<0.01).</p

Insulin stimulates REDD1 expression in human and murine adipocytes.

<p>hMADS adipocytes (A) and 3T3-L1 adipocytes (B) were stimulated with insulin (100 nM) for the indicated period of times and analyzed by immunoblots with indicated antibodies. Quantification of three independent experiments done in duplicate is shown and REDD1 protein level is expressed compared to HSP90 or tubulin expression (*p<0.05; **p<0.01, ***p<0.001).</p

Downregulation of REDD1 expression inhibits insulin signaling pathway in 3T3-L1 adipocytes.

<p>3T3-L1 adipocytes were transfected with REDD1 siRNA 48 h after transfection, adipocytes were stimulated with insulin (10 nM) for 5 minutes. Proteins were analyzed by immunoblots using indicated antibodies. Quantification of phosphorylation of insulin receptor (n = 6) and PKB (n = 5) in response to insulin is shown (*p<0.05).</p

Insulin stimulates REDD1 expression through MEK dependent pathway in hMADS adipocytes.

<p>(A) hMADS adipocytes were stimulated with insulin (100 nM) for 20 minutes in absence or in presence of a pretreatment with U0126 (10 µM) or LY 294002 (50 µM) for 60 minutes. (B) 3T3-L1 adipocytes were stimulated with insulin (100 nM) for 10 and 20 minutes in absence or in presence of a pretreatment with U0126 (10 µM). Proteins were revealed by immunoblots using indicated antibodies. REDD1 protein level is expressed compared to HSP90 or tubulin expression and quantification of three independent experiments is shown (*p<0.05; **p<0.01).</p

Expression of constitutive active MEK inhibits REDD1 degradation.

<p>(A) HEK-293 cells were transfected with plasmids encoding for REDD1 in absence or in presence of a plasmid encoding for constitutive form of MEK (CA-MEK). 48 h after transfection, wells were treated with cycloheximide (CHX 10 µg/ml) for the indicated periods of time, and proteins were analyzed by immunoblots with indicated antibodies. REDD1 protein level is expressed compared to tubulin expression and quantification of three independent experiments is shown. (B) HEK-293 cells were transfected with plasmids encoding for REDD1 in absence or in presence of a plasmid encoding for a constitutive form of MEK (CA-MEK). 48 h after transfection, cells were treated with lactacystin (10 µM) for 5 hours. Proteins were analyzed by immunoblots with indicated antibodies. Quantification of three independent experiments is shown and REDD1 protein level is expressed compared to tubulin expression (**p<0.01).</p

Downregulation of REDD1 expression inhibits lipogenesis in 3T3-L1 adipocytes.

<p>3T3-L1 adipocytes were transfected with REDD1 siRNA. 48 h after transfection, 3T3-L1 adipocytes were incubated with [³H] sodium acetate (0.4 mmol/l, 0.5 µCi/ml) in absence or in presence of insulin 1 nM for 1 hour at 37°C. Radioactivity incorporated in cellular lipids was determined in a liquid scintillation counter. Results are expressed as fold of induction of pmol of acetate/mg of proteins (n = 3, *p<0.05). Proteins were analyzed by immunoblots using indicated antibodies.</p

Expression of constitutive active MEK increases REDD1 protein level in HEK-293 epithelial cells.

<p>HEK-293 cells were transfected with plasmids encoding for constitutive form of MEK (CA-MEK), REDD1 or both plasmids. (A) Proteins were analyzed by immunoblots using indicated antibodies. REDD1 protein level is expressed compared to HSP90 expression and quantification of seven independent experiments is shown (***p<0.001). (B) REDD1 mRNA level was determined by quantitative RT-PCR.</p