Increased expression of enzymes of triglyceride synthesis is essential for the development of hepatic steatosis

Abstract

Molecular mechanisms underpinning nonalcoholic fatty liver disease (NAFLD) are not well understood. The earliest step of NAFLD is hepatic steatosis, which is one of the main characteristics of aging liver. Here, we present a molecular scenario of age-related liver steatosis. We show that C/EBPα-S193D knockin mice have age-associated epigenetic changes and develop hepatic steatosis at 2 months of age. The underlying mechanism of the hepatic steatosis in old wild-type (WT) mice and in young S193D mice includes increased amounts of tripartite p300-C/EBPα/β complexes that activate promoters of five genes that drive triglyceride synthesis. Knockdown of p300 in old WT mice inhibits hepatic steatosis. Indeed, transgenic mice expressing dominant-negative p300 have fewer C/EBPα/β-p300 complexes and do not develop age-dependent hepatic steatosis. Notably, the p300-C/EBPα/β pathway is activated in the livers of patients with NAFLD. Thus, our results show that p300 and C/EBP proteins are essential participants in hepatic steatosis.

Figure 1

Young S193D mice develop hepatic steatosis at the age of 2 months. A. Livers of 2-month-old (young) S193D mice and livers of 24-month-old WT mice were stained with Oil Red O. Scale bars: 10 μm. B. Amounts of TG in serum (upper) and liver (bottom) of young S193D and old WT mice are increased. Bar graphs show results of the analyses with eight mice of each genotype and each age group. * p<0.05, S193D versus WT, ^# p<0.05 old versus young. C. mRNA levels of enzymes of TG synthesis are increased in livers of S193D mice and in livers of old WT mice. Q-RT-PCR was performed with RNA isolated from livers of young WT and S193D mice (upper image) and from livers of WT old mice (bottom image). D. Protein levels of GPAT, DGAT1, and DGAT2 are increased in livers of S193D mice. Western blotting was performed with protein extracts isolated from the livers of young WT and S193D mice (upper) and old WT mice (bottom). Each membrane was re-probed with antibodies to β-actin. E. Immunostaining of livers of young WT, young S193D and old WT mice with antibodies to DGAT1 and to DGAT2. Note that livers of old WT mice and young S193D mice have enlarged hepatocytes (Jin et al., 2010). Scale bar: 10 μm. F. The number of DGAT1- and DGAT2-positive hepatocytes in livers of young WT, young S193D, and old WT mice. Bar graphs show the results of analyses of three animals of each group. * p<0.05, ** p<0.01, old WT versus young WT, ^# p<0.05 young S193D versus young WT.

Figure 2

p300-C/EBPα/β complexes occupy and activate the gene promoters driving TG-synthetic enzyme expression in livers of S193D mice. A. Promoters of GPAT, DGAT1, DGAT2, MGAT2, and AGPAT1 contain C/EBP sites. Locations and sequences of C/EBP having sites are shown. B. Electrophoretic mobility shift assay performed with the DGAT2 probe and nuclear extracts from S193D mouse livers. Antibodies to C/EBPαμ and C/EBPβ were included in the binding reactions. Positions of C/EBPα/β complexes and supershift (SS) are shown. C. C/EBP proteins recruit p300 to DGAT1 and DGAT2 promoters in livers of S193D mice and in livers of old WT mice. C/EBPα, C/EBPβ, p300, histone H3 acetylated at Lys9, or trimethylated on Lys9 were immunoprecipitated from chromatin solutions and examined in PCR reactions with primers amplifying C/EBP sites on the DGAT1 and DGAT2 promoters. In; 1/100 input. D. High-MW p300-C/EBPα/β complexes are abundant in livers of young S193D mice. Nuclear extracts from WT and S193D livers were separated by size-exclusion chromatography. The position of size-exclusion markers is shown at the top. Location of the p300 and C/EBP proteins in the fractions was determined by Western blotting with specific antibodies. C/EBPα-IP; C/EBPα was immunoprecipitated from each fraction and p300 and C/EBPβ were detected in these IPs. E. Amounts of C/EBPα and C/EBPβ proteins in cells transfected with individual DNAs coding for α, β, or both. In the case where both α and β were transfected, one-half the amount used in the single (α or β) transfections was used so that the total transfected equaled the amount used singly in α or β. Total lysates were isolated and examined by Western blotting with antibodies to C/EBPα and C/EBPβ. Bar graphs: Levels of C/EBP proteins were calculated as ratios to β-actin. F. p300-C/EBPα/β heterodimers activate the DGAT1 (upper) and DGAT2 (bottom) promoters more strongly than do individual C/EBPα and C/EBPβ proteins. DGAT1-luc and DGAT2-luc reporter constructs were cotransfected with constructs expressing C/EBPα, C/EBPβ, or both proteins. A similar experiment was performed in cells with inhibited p300. Data in Fig. 2E and F represent summary of 3–4 independent experiments.

Figure 3

p300 and p300-mediated elevation of DGAT1 and DGAT2 are responsible for the development of hepatic steatosis in old WT mice and in young S193D mice. A. Inhibition of p300 by siRNA in livers of old WT mice. Western blotting shows p300 in liver and in adipose tissues of mice treated with p300-specific siRNA and with control non-targeting RNA (NTG). β-actin is a control for protein loading. B. Oil Red O staining of livers of old WT mice treated with NTG and with siRNA to p300. C. Amounts of TG in livers of old WT mice treated with siRNA to p300. Bar graph is an average of 3 independent experiments. D. Knock-down of DGAT1 and DGAT2 inhibits hepatic steatosis in S193D mice. Western blotting was performed using antibodies to DGAT1 and DGAT2 with protein extracts isolated from liver and adipose tissues. β-Actin shows protein loading. NTG, treatments of S193D mice with control non-targeting RNA. E. A typical picture of Oil Red O staining of livers at 7 days after delivery of DGAT1 and DGAT2 siRNAs. NTG; treatments of WT and S193D mice treated with non-specific RNA. F. Bar graphs show amounts of TG in the livers of mice treated with DGAT1 and DGAT2 siRNAs as an average of three independent experiments.

Figure 4

Development of hepatic steatosis by HFD is accelerated in S193D mice. A. A typical picture of H&E (left) and Oil Red O (right) staining of WT and S193D livers under HFD conditions. Upper image shows staining of the livers under normal diet (N diet). Scale bar: 40 μm. B and C. Amounts of TGs in serum and in livers of WT and S193D mice at different time points of HFD. Data from three animals of each genotype are presented as bar graphs. D. Amounts of DGAT1 and DGAT2 in livers after HFD. Western blotting was performed with protein extracts isolated at 3 and 12 weeks after initiation of HFD. Bar graphs show ratios of DGAT1 and DGAT2 to β-actin. E. Examination of DGAT2 expression in livers of WT and S193D mice by immunostaining. Bar graphs show the number of DGAT2-positive hepatocytes in livers of WT and S193D mice. Scale bar: 10 μm.

Figure 5

Transgenic mice expressing dominant-negative p300 (dnp300) do not develop hepatic steatosis with age and under HFD conditions. A. Generation of transgenic animals expressing dnp300. Scheme showing the localization of the CH3 domain used in the generation of the transgenic animals. B. PCR genotyping gel indicating three animals display genomic insertion of the transgene. Dn and wt; PCR from control plasmids containing dominant-negative and WT cDNAs. Western blotting (bottom image) shows expression of c-myc–dnp300 in livers of transgenic animals. CRM; cross reactive molecule. C. The total fat is reduced in transgenic mice. A whole-body scan was performed on WT and dnp300 mice. Bar graphs represent an average of 5 animals of each genotype. D. Numbers of fat droplets are reduced in livers from dnp300 mice (20 months of age) as compared with the numbers WT animals (20 months of age). Hepatocytes from WT or dnp300 mice were stained with Oil Red O. The number of hepatocytes with fat droplets was calculated in livers of WT and dnp300 mice. The bar graph picture shows a summary of three repeats with five animals of each genotype. E. dnp300 disrupts C/EBPα/β-p300 complexes in livers of old mice. Protein extracts from livers of 20-month-old WT and dnp300 mice were separated on the SEC400 column. Fractions were analyzed as described in the legend to Fig. 2D. F. dnp300 mice do not develop hepatic steatosis within 12 weeks of HFD protocol.

Figure 6

Dnp300 inhibits development of steatosis in S193D mice. A. Genotyping of the double S193D-dnp300 mice. The picture shows PCR-based genotyping of dnp300 and digestions of the PCR products with BamHI enzyme (S193D genotyping) is shown. B. A typical picture of livers of WT and S193D-dnp300 mice at 12 weeks after initiation of HFD. C. H&E and Oil Red O staining of the livers. Scale bar of H&E staining: 40 μm. Scale bar of Oil Red O staining: 10 μm. D. Expression of DGAT2 in livers from WT and S193D-dnp300 mice. Bar graphs show number of DGAT2-positive hepatocytes as an average of three repeats with three mice of each genotype. E and F. Examination of the p300-C/EBPα/β complexes on the DGAT1 and DGAT2 promoters using the ChIP assay. The ChIP was performed as described in the legend to Fig. 2C.

Figure 7

C/EBPα-p300-DGAT1/2 pathway is elevated in livers of patients with non-alcoholic fatty liver disease. A. Generation of antibodies to human p-Ser190 isoform of C/EBPα. Sequences of mouse and human C/EBPα surrounding Ser193 and Ser190 are shown. Asterisks show differences in the sequences. Bottom: Western blotting with antibodies to p-Ser190 human C/EBPα. F+PP2A; proteins from patients with fatty liver were treated with phosphatase PP2A. B. Immunostaining of normal livers and livers from patients with fatty liver diseases using antibodies to p-Ser190 isoform of C/EBPα. The slides were stained with DAPI. C. Percentage of p-Ser190-C/EBPα positive hepatocytes in livers of normal patients and in livers of patients with fatty liver disease. D. Amounts of p-Ser190–C/EBPα-p300 complexes are increased in livers of NAFLD patients. The same liver sections were stained with antibodies to p-Ser190-C/EBPα, to p300, and with DAPI. Merge shows colocalization of C/EBPα and p300 in nucleus. Bar graphs show total number of hepatocytes with colocalization of C/EBPα and p300 and number of hepatocytes with multiple foci of colocalizations. E. Amounts of DGAT2 are increased in livers of patients with fatty liver diseases. Liver sections were stained with antibodies to DGAT2 and with DAPI. Bar graphs show percentage of DGAT2-positive hepatocytes. F. A model showing hypothetical mechanisms for the age-associated hepatic steatosis.