Insulin-dependent decrease of 11beta-HSD2 activity and mRNA is reversible but HSD11B2 half-life is not affected.

<p>(<i>A</i>) 11beta-HSD2 activity was measured in HT-29 cells in presence (black bars) and absence (white bars) of 10⁻⁷ M insulin for 24 h. The insulin effect was reversible on washout with PBS 24 h or 48 h of follow-up (hatched bars). (<i>B</i>) HSD11B2 expression was assessed by qRT-PCR in HT-29 pretreated with the protein synthesis inhibitor CHX (10 µM) for 1 h and treated with insulin (10⁻⁷ M) for 24 h. Each data point is expressed as a percentage of the control value. (<i>C</i>) HT-29 cells were pretreated with (filled circles) or without (filled rhombus) insulin (10⁻⁷ M) for 12 h. The cells were then treated with 25 µM of the mRNA synthesis inhibitor DRB, without or with insulin (10⁻⁷ M) (defined as time zero). At the indicated time points thereafter, total RNA was isolated, and the steady state level of HSD11B2 mRNA assessed. Each data point is expressed as a percentage of the maximum determined at time zero.</p

C/EBP beta isoforms control HSD11B2 promoter activity.

<p>(<i>A</i>) HT-29 cells were transfected with the full length human HSD11B2 promoter cloned into pGL3-basic luciferase vector (p4.5 kb-HSD11B2, 400 ng) and a dose response of LAP expressing vector (pCMV-LAP, 6.25 to 400 ng). A schematic representation of the promoter of HSD11B2 is shown on the left side. The transcriptional initiation site is indicated by an arrow (+1). The empty pcDNA3 vector was used to equalize the amount of transfected DNA in every condition and the pCMV-hRL (100 ng) was used as transfection efficiency control. Cells were lysed for luciferase assays 24 h after transfection, and the reading were normalized by renilla activity. (<i>B</i>) HT-29 cells were transfected with the plasmids p4.5 kb-HSD11B2 (400 ng), pRL-CMV (100 ng), pCMV-LAP (50 ng) and an increasing quantity of pCMV-LIP (50 ng to 400 ng). (<i>C</i>) HT-29 cells were transfected with the wild type p4.5 kb-HSD11B2 and p0.2 kb-HSD11B2 constructs or with the C/EBP mutated constructs. (<i>D</i>) HT-29 cells were transfected with the wild type p4.5 kb-HSD11B2 or the C/EBP mutated construct together with increasing concentration of pCMV-LAP.</p

Lactate accumulation in the media upon insulin stimulation and insulin-dependent down-regulation of 11beta-HSD2 activity.

<p>(<i>A</i>) Dose-response effect of insulin on L-lactate production in cultured HT-29 cells after 24 h incubation. The concentration in lactate found in the media of HT-29 cells after 24 h of culture is reported above the bars (Mean +/− SEM). (<i>B</i>) 11beta-HSD2 activity in cultured HT-29 and HCT116 cells exposed to exogenous L-lactate for 3 h. (<i>C</i>) 24 h L-lactate production in cultured HT-29 cells exposed to DCA alone or in combination with insulin. (<i>D</i>) 11beta-HSD2 activity in cultured HT-29 cells exposed to DCA alone or in combination with insulin.</p

Insulin-dependent regulation of the 11beta-HSD2 protein level and role of the CCAAT/enhancer-binding protein (C/EBP) family.

<p>(<i>A</i>) Concentration-dependent effects of insulin on 11beta-HSD2, C/EBP alpha, and C/EBP beta protein levels. HT-29 cells were cultured for 24 h without and with increasing concentrations of insulin (10⁻⁹–10⁻⁵ M), then harvested for Western blotting to evaluate expression of 11beta-HSD2, C/EBP alpha, C/EBP beta. (<i>B</i>) Concentration-dependent effects of insulin on C/EBP alpha, C/EBP beta, and C/EBP delta mRNA expression. HT-29 cells were treated like in (A). The level of C/EBP alpha (open circles), C/EBP beta (open squares), and C/EBP delta (filled triangles) mRNA was measured using qRT-PCR with S18 as internal control. Expression levels in treated cells were normalized to untreated controls (100%). Representative data for at least three independent experiments. The relative intensity was determined by densitometric scanning. The ratio of relative densities of 11beta-HSD2 to beta-actin in cells cultured in the abscence of hormone was considered as 100% (control). The ratio of relative densities of nuclear extract proteins to HDAC in cells cultured without hormone was considered as 100% (control). * LIP was undetectable in the control samples, so the LAP/LIP ratio was not calculated. (<i>C, D</i>) Silencing of C/EBP alpha (<i>C</i>) and C/EBP beta (<i>D)</i> was performed using siRNA. The expression of C/EBP alpha, C/EBP beta (left panel) and HSD11B2 (right panel) mRNA was measured using qRT-PCR.</p

Schematic representation of the insulin pathway and its regulation by sustained insulin stimulation in HT-29.

<p>mRNAs were quantified 24 h after insulin (10⁻⁷ M) treatment using RT² Profiler PCR Arrays PAHS-30C. Up-regulated transcripts are shown in red and down-regulated transcripts are shown in green.</p

Sustained insulin treatment diminished the 11beta-HSD2 expression and activity in HT-29 cells.

<p>(<i>A</i>) 11beta-HSD2 activity was measured by ³H-cortisol/cortisone conversion assay in colonic cell lines 24 h after incubation with insulin (10⁻¹¹–10⁻⁷ M). The activity measured for HCT116 in absence of insulin was set as 100%. (<i>B</i>) Dose-response effect of insulin (10⁻⁹–10⁻⁵ M) on HSD11B2 mRNA (gray bars) and activity (curve) in HT-29 cells treated for 24 h. (<i>C</i>) Time-dependent effect of insulin (10⁻⁷ M) on HSD11B2 mRNA (gray bars) and activity (curve) in HT-29 cells. (<i>D</i>) Time-dependent effect of insulin (10⁻⁷ M) on 11beta-HSD2 protein level.</p

Binding of C/EBP alpha/beta on human HSD11B2 promoter.

<p><i>(A) Nuclear proteins isolated from HT-29 cells bind to identified C/EBP alpha/beta sites.</i>4 µg of nuclear extracts isolated from insulin treated (for the indicated period of time, 10⁻⁷ M) or untreated HT-29 cells were incubated with radiolabeled probe encompassing the consensus C/EBP alpha/beta site in the presence or absence of non-radiolabeled (100×) competitor probe (cons C/EBP alpha/beta or mut C/EBP alpha/beta) (lanes1–7). Arrows indicate C/EBP alpha/beta / DNA shifts (C1, C2, C3) separated from free probe by gel electrophoresis. The complex C3 is formed in presence of radiolabeled −198 C/EBP alpha/beta probe (lanes 14–17) while complex C2 is formed in presence of radiolabeled −4362 C/EBP alpha/beta probe (lane 22–25). <i>(B) Nuclear proteins isolated from HT-29 cells bind to the consensus SP1 site.</i> Nuclear extracts isolated from insulin treated (for the indicated period of time, 10⁻⁷ M) or untreated HT-29 cells were incubated with radiolabeled probe encompassing the consensus SP1 site with and without non-radiolabeled (100X) competitor probe (cons SP1, lane 5or mut SP1, lane 6). The arrow indicates SP1/DNA shifts separated from free probe by gel electrophoresis. The complex intensity increased modestly with insulin treatment. The specific shift was abolished by the cold cons SP1 probe (lane 5) while not affected when mut SP1 probe was used as competitor (lane 6). <i>(C) Chromatin immunoprecipitation (ChIP) analysis of C/EBP alpha and C/EBP beta during insulin stimulation in HT-29 cells.</i> ChIPs were performed from untreated (W/O) and insulin induced (1–24 h) HT-29 cells using antibodies specific for C/EBP alpha (middle panel) and C/EBP beta (bottom panel), a no-antibody control (NO). The precipitated chromatin was analyzed using primers specific for the human HSD11B2 promoter. The DNA fragments were amplified with PCR primers to detect a 210 bp fragment containing the potential −177 and −198 C/EBP sites within the HSD11B2 promoter. Input chromatin is represented in upper panel.</p