Phenotypical comparison of C57Bl6 and transgenic alb-SREBP-1c animals.

<p>Weight gain (A) and food intake (B) of male mice (n = 20 per genotype) were measured once a week starting at weaning and monitored for an observation period of 18 weeks. Food intake per body weight (C) and weight gain per food intake (D) were determined in each group of mice. Data are given as means including standard deviation (±S.D.). C57Bl6 vs. alb-SREBP-1c mice: **p<0.01.</p

Macroscopic and histological comparison of livers from C57Bl6 and alb-SREBP-1c mice.

<p>Panel (A) shows fatty liver macroscopically of a C57Bl6 (left) or alb-SREBP-1c (right) mouse. (B) Liver tissue of the Lobus caudatus, Lobus sinister- and Lobus dexter lateralis were used for (I) standard hematoxylin and eosin staining. (II) PAS staining was performed to determine glycogen content. (III) The tissues were also used for cryofixation, and Oil-red-O staining was used for lipid visualization. (IV) Fibers and the extra cellular matrix were visualized to determine tissue integrity. The overview magnification is 1∶10, and details are shown in 1∶100 magnification.</p

Macroscopic and histological comparison of livers from C57Bl6, alb-SREBP-1aΔP and alb-SREBP-1a mice.

<p>Male mice of each genotype (C57Bl6, alb-SREBP1aΔP, alb-SREBP-1a (n = 20 per genotype)) were housed as groups of 4 under standard conditions with unlimited access to water and regular chow (13.0 MJ/kg: 53% carbohydrates, 11% fat, 36% protein). (<b>A</b>) Livers of a C57Bl6 mouse (left), alb-SREBP-1aΔP (middle) or alb-SREBP-1a (right). All photographs were taken with the same magnification. (<b>B</b>) Liver tissue of the Lobus caudatus, Lobus sinister- and Lobus dexter lateralis were fixed in 4% paraformaldehyd/PBS and embedded in paraffin with automated standard histological procedures. (I) Standard hematoxylin and eosin staining was performed on 3 µm deparaffinized sections. (II) PAS staining was performed to determine glycogen contend. (III) The tissues were also used for cryofixation and Oil-red-O staining was used for lipid visualization. (IV) Fibers and extra cellular matrix were visualized using the “van Gierson kit” to determine tissue integrity. The overview magnification is 1∶10 and details are shown in 1∶80 magnification.</p

Verification of p38 specific phosphorylation sites in SREBP-1a-NT.

<p>(<b>A</b>) Coomassie brilliant blue stained SDS-PAGE of GST-SREBP-1a-NT and each of mutated SREBP-1a-NT phosphorylated by activated recombinant p38α (40 ng/µg protein). (<b>B</b>) Autoradiography of SDS-PAGE of GST-SREBP-1a-NT fusion protein, single mutated forms S63A, T426V or double mutant GST-SREBP-1a-NT S63A/T426V phosphorylated by activated recombinant p38α. Excised radioactive slices of p38α phosphorylated recombinant proteins were trypsin-digested and the resulting peptides GST-SREBP-1a-NT, S63A, T426V or double mutant S63A/T426V, as indicated in the figure, were subjected to anion exchange chromatography. Elution was performed with a KH₂PO₄ pH 4 buffer gradient. Reactions performed are described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032609#s4" target="_blank">Materials and Methods</a>”. (<b>C</b>) Autoradiography of SDS-PAGE of GST-SREBP-1a-NT fusion protein, single mutated forms S63A, T426V or double mutant GST-SREBP-1a-NT S63A/T426V phosphorylated by activated recombinant p38β or p38γ (40 ng/µg protein).</p

Weight gain and Food intake of C57Bl6, alb-SREBP-1aΔP and alb-SREBP-1a transgenic animals.

<p>Male C57Bl6, alb-SREBP-1aΔP and alb-SREBP-1a mice (n = 20 per genotype) were housed as groups of four under standard conditions with unlimited access to water and regular chow (13.0 MJ/kg: 53% carbohydrates, 11% fat, 36% protein). Weight gain (<b>A</b>) and Food intake (<b>B</b>) were measured once a week starting at weaning and monitored for an observation period of 18 weeks. Body weight (<b>C</b>), liver weight ((<b>D</b>) and WAT weight (<b>E</b>) were determined at sacrification. WAT contend per body weight (<b>F</b>), food uptake per body weight and (<b>G</b>) weight gain per food uptake (<b>H</b>) were determined in each group of mice. Data are given as means including standard deviation (±SD).</p

Fatty acid composition of fat.

<p>Fractional content of selected fatty acids. Data are mean ± SD (n = 20).</p><p>The specific composition of total fatty acid was determined by GC analyses in adipose tissues of C57Bl6 and alb-SREBP-1c transgenic animals (n = 20, each). Students t-test was performed to determine significance (observed alterations did not reach significance limit p<0.05).</p

Gene expression of lipid metabolic genes in livers of C57Bl6 and alb-SREBP-1c.

<p>The hepatic expression level of genes were determined by RT-PCR (n = 20 each). The relative RNA amount shown in arbitrary units was calculated and plotted ± S.D. (C57Bl6 vs. alb-SREBP-1a or alb-SREBP-1aΔP mice: **p<0.001; alb-SREBP-1aΔP vs. alb-SREBP-1a mice: ‡‡p<0.001.)</p

Macroscopic comparisons of C57Bl6 and transgenic alb-SREBP-1c animals.

<p>(A) First, sections of male mice at 24 weeks of age are shown. (B) Histology of visceral adipose tissue indicated hyperplasia but no signs of infiltration. All photographs were taken with the same magnification.</p

Effect of phosphorylation on DNA binding and acitvity of SREBP-1a <i>in vitro</i>.

<p>(<b>A</b>) His-SREBP-1a-NT fusion protein was incubated with or without JNK1 (40 ng/µg protein) as indicated. An EMSA of His-SREBP-1a-NT incubated with sre-1 fragment (upper panel) or E-box fragment (lower panel) is shown. To confirm equal loading, western-blot analyses with monoclonal anti-HisG-HRP antibody was performed. To control phosphorylation efficiency a JNK1 kinase assay with (γ³²P) ATP was performed and dried SDS-PAGE was exposed to X-ray film. For further details see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032609#s4" target="_blank">Materials and Methods</a>”. (<b>B</b>) His-SREBP-1a-NT fusion protein was incubated with or without p38α (40 ng/µg protein) as indicated. An EMSA of His-SREBP-1a-NT incubated with sre-1 fragment (upper panel)or E-box fragment (lower panel) is shown. To confirm equal loading, western-blot analysis with monoclonal anti-HisG-HRP antibody were performed. To control phosphorylation efficiency a p38α kinase assay with (γ³²P) ATP was performed and dried SDS-PAGE was exposed to X-ray film. For further details see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032609#s4" target="_blank">Materials and Methods</a>”.</p

Comparison of body composition of C57Bl6 and transgenic alb-SREBP-1c animals.

<p>Body weight (A), lean body mass (B), subcutaneous adipose tissue (C), visceral adipose tissue (E) and liver weight were determined at scarification at 24 weeks of age of male mice (n = 20 per genotype) and are given directly (A, B, C, E, G) and in relation to body weight (BW) (D, F, H). C57Bl6 vs. alb-SREBP-1c mice: *p<0.05; **p<0.01.</p