Expression of DNA-PKcs is enhanced during adipogenesis of human pre-adipocytes isolated from sub-cutaneous adipose tissue.

<p>Cells isolated from the stromal vascular fraction of collagenase digested subcutaneous tissue were grown for the indicated times in adipogenic medium as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003345#s2" target="_blank">Material and Methods</a> section. (A) Photographs of the cultured cells and (B) Western blots experiments using indicated antibodies. NS, non specific band that show equal loading between the different samples.</p

Adipocytes display an increased ability to repair DNA double strand breaks as compared to pre-adipocytes.

<p>(A) γH2AX foci formation detected by immunofluorescence using an antibody that specifically recognizes the phosphorylated form of H2AX, in pre-adipocytes and adipocytes cells, exposed for 1 h to 2 pM of CL1γ or 30 min after irradiation at 2 Gy. (B) Kinetics of repair of DNA double-strand breaks as determined by disappearance of γ-H2AX foci after irradiation at 2 Gy (left panel) or after treatment at 2 pM of CL1γ during 1 h (right panel), mean of 3 independent experiments±SEM. (C) Kinetics of DNA DSBs repair determined by pulse-field gel electrophoresis after treatment with CL1γ (40 or 500 pM, 1 h) (left panel) or after γ-irradiation at 80 Gy (right panel). Results were obtained from the quantification of two independent experiments for treatment at 40 pM and three independent experiments for 500 pM and 80 Gy (mean±SEM).</p

Adipocyte differentiation is associated with an early increase in DNA-PKcs expression.

<p>(A) Expression of DNA repair proteins (DNA-PKcs, Ku70, Ku80, XRCC4, Rad51 and ATM) or adipocyte differentiation marker (HSL) was analyzed by Western Blots in exponentially growing 3T3F442A fibroblast cells (Ex), in 3T3F442A fibroblast cells grown to confluence (Co) or after the indicated time of culture in adipogenic differentiation medium (B) Similar experiments were performed in exponentially growing pre-adipocytes (Ex), in pre-adipocytes grown to confluence (Co) or during the first two days of culture in adipogenic medium) (C) Expression of DNA-PKcs protein in a murine fibroblast cell line (Balb-C) grown to confluence in the presence or not of insulin in comparison to exponentially growing cells.</p

Markers of nuclear and microsomal proteins in the plasma membrane and caveolae fractions.

<p>Equal amounts of protein (10 μg) were subjected to SDS/PAGE, immunoblotting and densitometric scanning (Gel Doc 2000 imaging system, Bio-Rad). The results are expressed as the amount of the marker protein relative to the indicated subcellular fraction (set to 100%). The experiments shown are representative of 3 independent experiments. <i>Abbreviations</i>: <i>plasma membrane (PM) fraction; caveolae membrane/lipid raft (CM) fraction</i>.</p

Time-dependent response of membrane fluidity to sphingomyelin treatment.

<p>Cells were treated with SM-LA (15 μM) or vehicle for the indicated incubation times. The cells were labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), and the fluorescence anisotropy of the probe was determined. The measurements were performed at 37°C for 2 min immediately after the addition of the fluorescent probes. Each value is the mean±SEM of three independent experiments. <i>P</i><0.05, <i>P</i><0.005 and <i>P</i><0.001; SM-treated cells compared with control cells.</p

Hypothetical model depicting how sphingomyelin might regulate SREBPs in 3T3-F442A adipocytes.

<p>Illustration of how an SM-initiated signal transduction pathway leads to SREBP modulation. Two cell models of membrane SM enrichment were investigated in this report (1) by adding exogenous sphingomyelins in the culture medium of differentiated cells or (2) by inhibiting N-SMase with GW4869 or GSH. In turn, SM contents increase in membranes (caveolae). Membrane SM accumulation promotes a rigid state of the membrane and caveolin accumulation and regulates the Ras-Raf-ERK MAP kinase pathway, inhibiting SREBP-1. By inhibiting KSR, which is the putative target of ceramide, SM acts to amplify the signal provided to the Raf/ERK MAP kinase cascade. In turn, by affecting SREBP-1, SM induces SREBP-2. The increased levels of SREBP-2 are consistent with increased CHOL synthesis. In SM-enriched adipocytes, a higher amount of CHOL is required to maintain raft and caveolae assembly. Caveolin transports the neo-synthesized CHOL toward the cell surface, thus increasing the caveolin-2 levels in the plasma membrane. In SM-depleted cells, the opposite mechanism could be effective.</p

Real-time quantitative RT-PCR determination of SREBP-1 and SREBP-2 mRNA levels in SM-enriched adipocytes or unmodulated adipocytes.

<p>^a The control group was expressed as 1. <i>P</i><0.0001, <i>P</i><0.001, <i>P</i><0.01 and <i>P</i><0.05; SM-, GSH-, and PPMP-treated cells compared with untreated cells.</p><p>3T3-F442A adipocytes were treated with 15 μM natural SM (SM-LA, 24 h), 10 mM GSH (24 h), 20 μM PPMP (24 h) or vehicle. The mRNA levels of the studied genes were determined. The results are expressed as-fold variations over respective controls (R) after normalization to β-actin, as indicated in the Materials and methods. R values superior or equal to 2 were considered positive regulation of gene expression, whereas values lower than 0.5 indicated negative regulation. The results presented are the means of 3 independent experiments, which were performed twice each in duplicate.</p

SM-enriched adipocytes accumulate glucosylceramide, unlike SM-unmodulated cells.

<p>Cells were treated for 24 h with 30 μM SM-LA, 20 μM PPMP or vehicle. After lipid isolation, ceramide and glucosylceramide were quantified as described in the Materials and methods. The results are expressed in pmol of sphingosine per mg of protein. The results presented are the means of 3 independent experiments, which were performed in triplicate. The control group was expressed as 100, and the results are shown as percentages of the control cells. ^***<i>P</i><0.005, ^**<i>P</i><0.01 and ^*<i>P</i><0.05; SM-, GSH-, and PPMP-treated cells compared with control cells.</p

Relations between SM accumulation and SREBP-1, Ras, ERK, CREB and PPARγ proteins in human subcutaneous adipose tissue.

<p>R, coefficient of correlation; statistical significance was set at <i>P</i><0.05. Membrane SM levels refer to total membrane levels.</p

Subcellular distribution of sphingomyelin in 3T3-F442A adipocytes.

<p>*<i>P</i><0.05</p><p>**<i>P</i><0.01; SM-treated cells compared with control cells. <i>Abbreviations</i>: <i>post-nuclear supernatant (PNS)</i>, <i>plasma membrane (PM) fraction</i>, <i>non-caveolae membrane/non-lipid raft (NCM) fraction</i>, <i>caveolae membrane/lipid raft (CM) fraction</i>. <i>PNS represents the fraction of the cells without nuclei including cytosol</i>, <i>microsomes</i>, <i>membranes and others</i>.</p><p>Cells were treated with SM-LA (15 μM) for the indicated incubation times. The results, which are expressed as μg of SM per mg protein, are presented as percentages of control cells and are the mean±SEM of three independent experiments. The SM content of plasma membranes was 30±2 μg/mg protein in control adipocytes. The caveolae fraction represents approximately one-third (0.36) of the plasma membrane [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133181#pone.0133181.ref078" target="_blank">78</a>].</p