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

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

Surrogate parameters for insulin resistance and insulin sensitivity.

<p>Data are mean ± SD (n = 20). T-test C57Bl6 vs. alb-SREBP-1c: **p<0.01.</p><p>Surrogate indices were calculated from fasting blood glucose and plasma insulin concentrations as follows: QUICKI = 1/[log(I0)+log(G0)], where I0 is fasting insulin (µU/ml) and G0 is fasting glucose (mg/dl); and HOMA-IR = (G0 * I0)/22.5, with glucose expressed as mmol/l and insulin expressed as µU/ml.). Data were calculated from all mice investigated. Students t-test was performed to determine significance (C57Bl6 vs. alb-SREBP-1c mice: **p<0.01).</p

Gene expression of metabolic genes in liver.

<p>Data are given as mean ± SD (n = 20, each genotype) in arbitrary units normalized to 18 S RNA contend. T-test C57Bl6 vs. alb-SREBP-1c: **p<0.01.</p><p>The hepatic expression level of genes was determined by RT-PCR (n = 20 each). The relative RNA amount shown in arbitrary units was calculated and plotted ± S.D. Students t-test was performed to determine significance (C57Bl6 vs. alb-SREBP-1c mice: **p<0.01).</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

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

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

Physiological parameters and serum lipid composition.

<p>Data are mean ± SD (n = 20). T-test C57Bl6 vs. alb-SREBP-1c: **p<0.01.</p><p>Clinical parameters were measured in C57Bl6 and alb-SREBP-1c mice (n = 20, for each). Triglycerides cholesterol, total protein and liver enzymes (ALT, AST) were determined on a Hitachie 912 laboratory automat. Students t-test was performed to determine significance (C57Bl6 vs. alb-SREBP-1c mice: **p<0.01).</p

Fatty acid composition of liver.

<p>Fractional content of selected fatty acids. Data are mean ± SD (n = 20). T-test C57Bl6 vs. alb-SREBP-1c: **p<0.01.</p><p>The specific composition of total fatty acid was determined by GC analyses in liver tissues of C57Bl6 and alb-SREBP-1c transgenic animals (n = 20 for each). Students t-test was performed to determine significance (C57Bl6 vs. alb-SREBP-1c mice: **p<0.01).</p

Cytokine profile in serum of C57Bl6 and transgenic alb-SREBP-1c animals.

<p>The cytokine content in serum was analyzed using the Proteome Profiler™; R&D Systems, (Abingdon, UK). Spot intensities were normalized to background and positive controls set to 100% intensity. Presented numbers on membranes mark targets as follows: (1) C5a; (2) CSF-3; (3) sICAM; (4) INF-γ; (5) IL-12-p70; (6) CXCL-1; (7) CSF-1; (8) MCP-1; (9) TIMP-1; (10) TREM-1. Abundance of: CXCL13, CSF-2, CCL-1, CCL-11, IL1-α, IL1-ß, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13, IL-16, IL-17, IL-23, IL-27, CXCL-10, CXCL-11, MCP-5, CXCL-9, CCR-1a, CCL-4, CCL-2, CCL5, CXCL-12, CCL-17 or TNF-α was not detected in serum. Data are given as means ± S.D. (n = 6, each) of normalized intensity. Significance was calculated by 2-way ANOVA. C57Bl6 vs. alb-SREBP-1c mice: *p<0.01.</p