Preventing Phosphorylation of Sterol Regulatory Element-Binding Protein 1a by MAP-Kinases Protects Mice from Fatty Liver and Visceral Obesity

The transcription factor sterol regulatory element binding protein (SREBP)-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP–1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP–1a mice the phosphorylation–deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role, in vivo

Liver-Specific Expression of Transcriptionally Active SREBP-1c Is Associated with Fatty Liver and Increased Visceral Fat Mass

The pathogenesis of fatty liver is not understood in detail, but lipid overflow as well as de novo lipogenesis (DNL) seem to be the key points of hepatocyte accumulation of lipids. One key transcription factor in DNL is sterol regulatory element-binding protein (SREBP)-1c. We generated mice with liver-specific over-expression of mature human SREBP-1c under control of the albumin promoter and a liver-specific enhancer (alb-SREBP-1c) to analyze systemic perturbations caused by this distinct alteration. SREBP-1c targets specific genes and causes key enzymes in DNL and lipid metabolism to be up-regulated. The alb-SREBP-1c mice developed hepatic lipid accumulation featuring a fatty liver by the age of 24 weeks under normocaloric nutrition. On a molecular level, clinical parameters and lipid-profiles varied according to the fatty liver phenotype. The desaturation index was increased compared to wild type mice. In liver, fatty acids (FA) were increased by 50% (p<0.01) and lipid composition was shifted to mono unsaturated FA, whereas lipid profile in adipose tissue or serum was not altered. Serum analyses revealed a ∼2-fold (p<0.01) increase in triglycerides and free fatty acids, and a ∼3-fold (p<0.01) increase in insulin levels, indicating insulin resistance; however, no significant cytokine profile alterations have been determined. Interestingly and unexpectedly, mice also developed adipositas with considerably increased visceral adipose tissue, although calorie intake was not different compared to control mice. In conclusion, the alb-SREBP-1c mouse model allowed the elucidation of the systemic impact of SREBP-1c as a central regulator of lipid metabolism in vivo and also demonstrated that the liver is a more active player in metabolic diseases such as visceral obesity and insulin resistance

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

Identification of S117 as major JNK related phosphorylation site in SREBP-1a.

<p>(<b>A</b>) 10 µg GST-SREBP-1a-NT and GST-SREBP-1a-NT S117A were phosphorylated by activated recombinant JNK1 or JNK2 (40 ng/µg protein) or recombinant p38 isoforms p38α, p38β, p38γ (40 ng/µg protein) <i>in vitro</i> and separated by 10% SDS-PAGE. A coomassie blue stained gel (left), autoradiography of SDS-PAGE of JNK1, JNK2, p38α, p38β or p38γ phosphorylation is shown. The arrow marks band of SREBP-1a. The excised radioactive slices of GST-SREBP-1a-NT or GST-SREBP-1a-NT S117A phosphorylated by JNK1 (<b>B</b>) or phosphorylated by p38α (<b>C</b>) were digested by trypsin and the resulting peptides were subjected to anion exchange chromatography. The graph shows the elution fraction plotted against the incorporated radioactivity. 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>”.</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

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

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

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