Liver steatosis and increased ChREBP expression in mice carrying a liver specific SIRT1 null mutation under a normal feeding condition

Abstract

SIRT1, a homolog of yeast Sir2, is a type III NAD(+) dependent histone and protein deacetylase. Previous studies of mice carrying liver specific deletion of exon 4 of the Sirt1 gene revealed opposite responses of mutant mice to a high-fat diet in terms of fatty liver formation, which obscures the function of SRIT1 in liver development and lipid metabolism. To investigate this, we deleted exons 5 and 6 of Sirt1 in the liver by using a Cre-loxP approach. Western blot using an antibody to N-terminal SIRT1 does not detect a truncated protein in the liver of the mutant mice (Sirt1(flox5-6/flox5-6);Alb-Cre), suggesting a null mutation for SIRT1 is generated in the liver. Unlike the previously reported phenotypes, the Sirt1(flox5-6/flox5-6);Alb-Cre mice develop fatty liver under a normal feeding condition. The disease starts at two months of age and incidence increases as the animals become older, affecting 78% of them when they are over one year of age. We showed that the steatosis is accompanied by altered expression of a number of genes, including increased expression of ChREBP, which acts as one of the central determinants of lipid synthesis in the liver. This data uncovers an important role of SIRT1 in regulating lipid metabolism in the liver, and the SIRT1 mutant mice may serve as an animal model for studying human fatty liver disease and facilitate the development of effective therapeutic approach for the disease.

Keywords: ChREBP; SIRT1; SREBP-1c; fatty liver; mice.

Figure 1

Generation of liver specific SIRT1 knockout mice. (A) To generate a Sirt1 conditional mutant allele, an existing mutant allele that carries a ploxPneo gene in the intron 4 and a third loxP in the intron 6 of the Sirt1 locus was used (upper panel). After breeding mice carrying the 3-loxP allele of Sirt1 gene with EIIa-Cre transgenic mice, Cre-mediated recombination between loxP1 and loxP2 was identified using primers 1 and 2 (P1 and P2) to screen for offspring that has the neo gene deleted while still keeping the loxP3 (lower panel). (B) PCR analysis to identify offspring that carry Cre-mediated recombination between loxP1 and loxP2 (using primers 1 and 2: ~700 bp for Sirt1^flox5-6 allele and ~640 bp for wild type allele) while keeping loxP3 (using primers 3 and 4: ~420 bp for mutant allele and ~360 bp for wild type allele). The first three lanes are liver DNA from Sirt1 ^{flox5-6/flox5-6} mice and these primers only detect the 700 pb and 420 bp bands, while the last two lanes are samples from wild type mice and only detect the 640 bp and 360 bp bands. (C) Sirt1 ^{flox5-6/flox5-6} mice were crossed with mice carrying an album promoter driving Cre transgene (Alb-Cre) to generate liver specific SIRT1 knockout mice (Sirt1^{flox5-6/flox5-6};Alb-Cre, or Sirt1^LKO). (D) Sirt1^LKO mice were validated by PCR analysis of genomic DNA isolated from liver tissue with primers P1/P4 to demonstrate the deletion of exon 5 and exon 6. (E) Western blot with an antibody against SIRT1 N-terminus to demonstrate that there is no truncated SIRT1 in our SIRT1 liver specific knockout mice. The samples were from 2 months old male mice. More than 20 pairs of animals were analyzed. Data from 2 pairs are shown here.

Figure 2

SIRT1 liver specific knockout causes liver steatosis. (A-C) H&E staining and Oil Red O staining of 2 months (A), 6 months (B) and 14 months (C) old male mice liver respectively. (D) Oil Red O staining of 14 months old male liver with higher magnification. The inserted table is the summary of fatty liver cases at different age among control (Sirt1^flox5-6) and liver specific SIRT1 knockout (Sirt1^LKO) males. (E-G) Liver triglyceride (TG) content (E), plasma free fatty acid (FFA) amount (F) and plasma triglyceride (TG) content (G) of 9 months old male mice (n=11). *p<0.05.

Figure 3

SIRT1 liver specific knockout leads to increased expression of lipid metabolism genes. (A) The expression of genes involved in glycolysis, β-oxidation, esterificaiton and fat uptake is not changed in the livers of Sirt1^LKO male mice at 2 months of age (n≥6). (B) Sirt1^LKOmice display elevated expression of FAS, ACC1 and ELOVL6 at 2 months (n≥6). *p<0.05. (C) At 6 months of age, the expression of FAS, ACC1, ELOVL6 and SCD1 remains increased in Sirt1^LKOmice (n≥6). *p<0.05. (D-E) The expression of SREBP-1c is down regulated in Sirt1^LKOmice at 2 months of age at both mRNA level (C) and protein level (D). (F) In vitro, deletion of SIRT1 (compare last two bars) or knocking down SIRT1 by siRNA (the first two bars) reduces mRNA level of SREBP-1c, while over expressing SIRT1 (middle two bars) increases SREBP-1c mRNA level. * p<0.01

Figure 4

SIRT1 liver-specific knockout leads to increased expression of ChREBP. (A) The expression of CREB, CEBPβ and CEBPδ is up-regulated in the liver of Sirt1^LKO male mice at 2 months of age (n≥6). *p<0.05 (B) Sirt1^LKOmice display consistent elevated mRNA level of ChREBP at both 2 months and 6 months of age (n≥6). *p<0.05 . (C) At 2months of age, Sirt1^LKOmice contain higher ChREBP protein level. (D) In vitro, deletion of SIRT1 (last two bars) or knocking down SIRT1 by siRNA (first two bars) increased the mRNA level of ChREBP, while over expressing SIRT1 (middle two bars) reduces ChREBP mRNA level. * p<0.01. (E-G) Chromatin Immunoprecipitation (ChIP) assay demonstrates deletion of SIRT1 leads to increased acetylation of histone H3K9 and histone H4K16 on 3 fragments (365-539, 984-1173 and 1614-1752) upstream of starting codon ATG of ChREBP promoter.