MnSOD knock-down in predifferentiated and differentiated 3T3-L1 cells.

<p>(A) MnSOD, PARP, HO-1 and GAPDH in mature 3T3-L1 cells treated with scrambled (Scr) or MnSOD siRNA. Staurosporine treated cells (ST) are shown as control. (B) Full-length PARP in scrambled (Scr) and MnSOD siRNA treated cells partly shown in A, data of 3 experiments have been calculated. (C) Cleaved PARP in scrambled (Scr) and MnSOD siRNA treated cells partly shown in A, data of 3 experiments have been calculated. (D) Antioxidative capacity of the cells described in A. Data of 6 experiments were calculated. (E) HO-1 in scrambled (Scr) and MnSOD siRNA treated cells partly shown in A, data of 3 experiments have been calculated. (F) MnSOD, PARP, HO-1 and GAPDH in 3T3-L1 cells differentiated from cells treated with siRNAs 6 d after initiation of differentiation. (G) Triglyceride levels (ng/µg cellular protein) in the cells described in F, data of 6 experiments were calculated. (H) Lactate dehydrogenase in the supernatants of cells described in F, data of 6 experiments have been calculated (arbitrary units = au). (I) Full-length PARP in scrambled (Scr) and MnSOD siRNA treated cells partly shown in F, data of 6 experiments have been calculated. (J) Cleaved PARP in scrambled (Scr) and MnSOD siRNA treated cells partly shown in F, data of 6 experiments have been calculated. (K) HO-1 in scrambled (Scr) and MnSOD siRNA treated cells partly shown in F, data of 6 experiments have been calculated.</p

MnSOD in adipose tissues of male rodents fed high fat diets.

<p>MnSOD mRNA in subcutaneous (A), epididymal (B), perirenal (C) and brown adipose tissue (BAT, D) of mice fed a standard (SD) or a high fat diet (HFD). MnSOD protein in subcutaneous (E, F), epididymal (G, H), and perirenal (I, J) fat of mice fed a SD or HFD. Coomassie (Coom) stained gel is shown as loading control. (K) MnSOD and CD163 in subcutaneous and epididymal adipose tissue of mice fed a SD and in stromavascular cell fraction (SVF) isolated from these fat depots. (L) MnSOD protein/adipocyte area in subcutaneous (6 mice per group) adipose tissues of mice fed a SD or HFD. (M) MnSOD protein/adipocyte area in epididymal (4 mice per group) adipose tissues of mice fed a SD or HFD. (N) MnSOD protein in perirenal fat of rats fed a SD or a HFD. Coomassie (Coom) stained gel is shown as loading control. (O) Quantification of the data partly shown in N.</p

MnSOD in different adipose tissue depots.

<p>(A) MnSOD protein in subcutaneous (sc) and visceral (vis) fat of two mice. (B) MnSOD protein in paired samples of subcutaneous (sc) and visceral (vis) human adipocytes of two extremely obese patients. (C) Immunohistochemistry of MnSOD in subcutaneous fat of mice kept on a standard chow (SD) or high fat diet (HFD).</p

Free Fatty Acids, Lipopolysaccharide and IL-1α Induce Adipocyte Manganese Superoxide Dismutase Which Is Increased in Visceral Adipose Tissues of Obese Rodents

<div><p>Excess fat storage in adipocytes is associated with increased generation of reactive oxygen species (ROS) and impaired activity of antioxidant mechanisms. Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme involved in detoxification of ROS, and objective of the current study is to analyze expression and regulation of MnSOD in obesity. MnSOD is increased in visceral but not subcutaneous fat depots of rodents kept on high fat diets (HFD) and ob/ob mice. MnSOD is elevated in visceral adipocytes of fat fed mice and exposure of differentiating 3T3-L1 cells to lipopolysaccharide, IL-1α, saturated, monounsaturated and polyunsaturated free fatty acids (FFA) upregulates its level. FFA do not alter cytochrome oxidase 4 arguing against overall induction of mitochondrial enzymes. Upregulation of MnSOD in fat loaded cells is not mediated by IL-6, TNF or sterol regulatory element binding protein 2 which are induced in these cells. MnSOD is similarly abundant in perirenal fat of Zucker diabetic rats and non-diabetic animals with similar body weight and glucose has no effect on MnSOD in 3T3-L1 cells. To evaluate whether MnSOD affects adipocyte fat storage, MnSOD was knocked-down in adipocytes for the last three days of differentiation and in mature adipocytes. Knock-down of MnSOD does neither alter lipid storage nor viability of these cells. Heme oxygenase-1 which is induced upon oxidative stress is not altered while antioxidative capacity of the cells is modestly reduced. Current data show that inflammation and excess triglyceride storage raise adipocyte MnSOD which is induced in epididymal adipocytes in obesity.</p></div

MnSOD in 3T3-L1 cells differentiated in the presence of free fatty acids.

<p>(A) Photomicrograph of 3T3-L1 cells differentiated with the standard protocol and in the presence of 200 µM palmitate (PA), oleate (OA) or linoleate (LA). (B) MnSOD in 3T3-L1 cells differentiated with the standard protocol (C) and in the presence of 200 µM PA, OA or LA. MnSOD in 3T3-L1 cells differentiated in the presence of increasing concentrations of PA (C), OA (D) or LA (E). (F) Quantification of the data of 3 PA incubated, 4 OA incubated and 3 LA incubated cells partly shown in B to E. (G) MnSOD, FABP4 and Cox-4 protein in preadipocytes (Pre) and cells differentiated for 1 d, 2 d, 3 d, 6 d and 9 d in the presence (+) or absence (−) of 400 µM oleic acid. (H) MnSOD, FABP4, Cox-4 and GAPDH in 3T3-L1 cells differentiated in the presence of 200 µM PA, OA or LA. (I) Full length (PARP, 116 kDa) and cleaved PARP (PARP, 89 kDa) in 3T3-L1 cells differentiated in the presence of 200 µM PA, OA or LA. ST indicates lysate of staurosporine treated 3T3-L1 cells as positive control which was analyzed on the identical gel in a non-adjacent lane. (J) Number of mature adipocytes when cells were differentiated in the presence of 200 µM PA, OA or LA. Data of 4 experiments are shown.</p

MnSOD in adipose tissue of ob/ob mice.

<p>(A) MnSOD and Cox-4 protein in subcutaneous fat of ob/ob mice and wild type (WT) animals. Coomassie (Coom) stained gel is shown as loading control. (B) MnSOD and Cox-4 protein in epididymal fat of ob/ob mice and wild type animals. Coomassie (Coom.) stained gel is shown as loading control. (C) Quantification of the immunoblots partly shown in A and B.</p

MnSOD in 3T3-L1 cells differentiated in the presence of glucose, LPS and IL-1α.

<p>(A) MnSOD and stearoyl CoA desaturase 1 (SCD1) protein in preadipocytes (Pre), 1 d, 2 d, 3 d, 6 d and 9 d differentiated cells. (B) MnSOD and SCD1 in primary human preadipocytes (Pre) and the respective mature cells (Mat) of two different donors. (C) MnSOD in 3T3-L1 cells differentiated in the presence of 25.0 mM (HG) and 5.5 mM (LG) glucose. (D) Quantification of the results of 4 experiments partly shown in C. (E) MnSOD in 3T3-L1 cells differentiated in the presence of LPS (10 ng/ml). (F) Quantification of the results of 3 experiments partly shown in E. (G) MnSOD in 3T3-L1 cells differentiated in the presence of increasing concentrations of IL-1α.</p

IL-6, TNF and SREBP2 knock-down in 3T3-L1 cells.

<p>(A) IL-6 in the supernatants of 3T3-L1 cells differentiated with the standard protocol and in the presence of 200 µM palmitate (PA), oleate (OA) or linoleate (LA) * is p-value <0.001. (B) TNF in the supernatants of 3T3-L1 cells differentiated with the standard protocol (C) and in the presence of 200 µM PA, OA or LA. * is p-value <0.001. (C) MnSOD in 3T3-L1 cells differentiated in the presence of TNF and IL-6. (D) Quantification of the data of 3 TNF/IL-6 incubated cells partly shown in C. (E) MnSOD and SREBP2 in adipocytes differentiated from preadipocytes treated with scrambled (Scr) or SREBP2 siRNA. Cells were differentiated with the standard protocol or in the presence of 200 µM OA. (F) Quantification of MnSOD in the cells described in E. Ratio of MnSOD protein in SREBP si RNA treated cells to scrambled siRNA transfected cells in % is shown. Data of 4 experiments have been calculated.</p

Seven SNPs show sex difference.

a<p>Trait and sex for which the SNP was selected;</p>b<p>Gene labels state the nearest gene or the gene as published previously; details on all genes near the association signal can be found in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003500#pgen.1003500.s002" target="_blank">Figure S2</a>;</p>c<p>One-sided P-Values.</p>d<p>larger sample size due to one additional study that did not have hip circumference, and therefore could not contribute to WHRadjBMI.</p>e<p>smaller sample size as this SNP was not on Metabochip.</p><p>Shown are the seven SNPs with significant (at 5% false discovery rate) sex difference in the follow-up data. These seven SNPs exhibit genome-wide significant association in women (joint discovery and follow-up <i>P_women</i><5×10−8) and only two of these show nominally significant association in men (joint <i>P_men</i><0.05). The three loci MAP3K1, HSD17B4, and PPARG are shown here for the first time for their anthropometric trait association as well as for sex-difference.</p

Genome-wide scan for sex-specific genome-wide association highlights numerous loci.

<p>(a) Manhattan plot showing the men-specific (upward, up to 60,586 men) and women-specific (downward, up to 73,137 women) association P-values from the discovery with the 619 selected loci colored by the phenotype for which the locus was selected; (b) QQ-plot showing the sex-specific association P-values as observed against those expected under the null overall phenotypes (black) and for each phenotypes separately (colored).</p