C/EBPbeta, when expressed from the C/ebpalpha gene locus, can functionally replace C/EBPalpha in liver but not in adipose tissue

Abstract

Knockout of C/EBPalpha causes a severe loss of liver function and, subsequently, neonatal lethality in mice. By using a gene replacement approach, we generated a new C/EBPalpha-null mouse strain in which C/EBPbeta, in addition to its own expression, substituted for C/EBPalpha expression in tissues. The homozygous mutant mice C/ebpalpha(beta/beta) are viable and fertile and show none of the overt liver abnormalities found in the previous C/EBPalpha-null mouse line. Levels of hepatic PEPCK mRNA are not different between C/ebpalpha(beta/beta) and wild-type mice. However, despite their normal growth rate, C/ebpalpha(beta/beta) mice have markedly reduced fat storage in their white adipose tissue (WAT). Expression of two adipocyte-specific factors, adipsin and leptin, is significantly reduced in the WAT of C/ebpalpha(beta/beta) mice. In addition, expression of the non-adipocyte-specific genes for transferrin and cysteine dioxygenase is reduced in WAT but not in liver. Our study demonstrates that when expressed from the C/ebpalpha gene locus, C/EBPbeta can act for C/EBPalpha to maintain liver functions during development. Moreover, our studies with the C/ebpalpha(beta/beta) mice provide new insights into the nonredundant functions of C/EBPalpha and C/EBPbeta on gene regulation in WAT.

FIG. 1

Targeted modification of the C/ebpα gene locus. (A) C/ebpα gene (top), targeted allele (middle), and the expected Cre-loxP-mediated removal of the PGK.neo transgene (bottom). (B and C) Southern blot analysis of representative F₂ (before crossing with the EIIa-cre mice) mouse tail biopsies. Tail DNA was digested with HindIII (B) and with HindIII and BamHI (C) and probed with the probes shown in panel A. (D) Southern blot analysis of representative F₂ mouse tail biopsies after crossing with the EIIa-cre mice. DNA was digested with HindIII and probed with the probes shown in panel A. +, wild-type allele; β, targeted C/ebpα allele.

FIG. 2

Normal growth and liver functions of C/ebpα^β/β mice. (A) Growth curves for C/ebpα^β/β mice on a standard diet. The value for each point is the average body weight of five mice of the same genotype. The standard deviation for each group is within 10% of the respective mean value and is not shown. (B) Histological sections and H&E staining of livers of 9-week-old C/ebpα^β/β mice. Magnification, ×360 (original magnification, ×400). (C) Northern blotting analysis of representative liver biopsies of C/ebpα^β/β mice. Liver total RNAs (20 μg) from C/ebpα^β/β mice of different ages were denatured and electrophoresed on a formaldehyde-containing 1% agarose gel, blotted to a nylon membrane, and probed with the indicated cDNA probes. Each lane contains RNA from an individual animal. E, embryonic stage; NB, newborn; W, week. (D) Western analysis of C/EBPβ protein levels of representative liver and WAT biopsies of C/ebpα^β/β mice. Total cellular protein (50 μg) from livers from mice of different ages and from WAT of 7-week-old mice were separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, blotted to a nitrocellulose membrane, and probed with an antibody against mouse C/EBPβ. Each lane contains protein from an individual animal. E, embryonic stage; W, week. (E) Northern blotting analysis of representative liver biopsies of C/ebpα^β/β mice after the LPS treatment. Liver total RNA (20 μg) from 4-week-old C/ebpα^β/β mice injected with LPS were denatured and electrophoresed on a formaldehyde-containing 1% agarose gel, blotted to a nylon membrane, and probed with the indicated oligonucleotide probes. Each lane contains RNA from an individual animal. +, wild-type allele; β, targeted C/ebpα allele.

FIG. 3

Inhibited adipocyte hypertrophy and gene expression in WAT of C/ebpα^β/β mice. (A) Appearance of epididymal fat pads (panels a and b), histological sections, and H&E staining of epididymal fat pads (panels c and d) (magnification, ×86; original magnification, ×100) and histological sections and oil red O staining of epididymal fats (panels e and f) of 4-week-old C/ebpα^β/β mice. (B) Appearance (panels a and b) and histological sections and H&E staining (panels c and d) (magnification, ×344; original magnification, ×400) of brown fat pads of 4-week-old C/ebpα^β/β mice. (C through E) Northern blotting analysis of representative epididymal fat (C and E) and brown fat (D) biopsies of 4-week-old C/ebpα^β/β mice. Total RNA (8 μg) from 4-week-old C/ebpα^β/β mice was denatured and electrophoresed on a formaldehyde-containing 1% agarose gel, blotted to a nylon membrane, and probed with the indicated cDNA probes. Each lane contains RNA from an individual animal. F, epididymal fat pads; T, testes; +, wild-type allele; β, targeted C/ebpα allele.

FIG. 3

Inhibited adipocyte hypertrophy and gene expression in WAT of C/ebpα^β/β mice. (A) Appearance of epididymal fat pads (panels a and b), histological sections, and H&E staining of epididymal fat pads (panels c and d) (magnification, ×86; original magnification, ×100) and histological sections and oil red O staining of epididymal fats (panels e and f) of 4-week-old C/ebpα^β/β mice. (B) Appearance (panels a and b) and histological sections and H&E staining (panels c and d) (magnification, ×344; original magnification, ×400) of brown fat pads of 4-week-old C/ebpα^β/β mice. (C through E) Northern blotting analysis of representative epididymal fat (C and E) and brown fat (D) biopsies of 4-week-old C/ebpα^β/β mice. Total RNA (8 μg) from 4-week-old C/ebpα^β/β mice was denatured and electrophoresed on a formaldehyde-containing 1% agarose gel, blotted to a nylon membrane, and probed with the indicated cDNA probes. Each lane contains RNA from an individual animal. F, epididymal fat pads; T, testes; +, wild-type allele; β, targeted C/ebpα allele.

FIG. 4

Reduced serum adipsin and leptin levels and increased food intake in C/ebpα^β/β mice. (A) Sera were collected from groups of mice of each genotype at the age of 4 weeks for analysis of hemolytic activity and insulin and leptin levels. Values presented are the means ± standard errors for at least five serum samples. The P value indicates the level of significance for differences between C/ebpα^β/β and wild-type mice. (B) Weekly food intake during a 6-week diet experiment. Values presented are the means ± standard errors for six mice. +, wild-type allele; β, targeted C/ebpα allele.

FIG. 5

Decreased expressions of transferrin and CDO genes in WAT, but not in liver, of C/ebpα^β/β mice. Northern blotting analysis of representative tissue biopsies of C/ebpα^β/β mice. Total RNA isolated from tissues of C/ebpα^β/β mice were denatured and electrophoresed on a formaldehyde-containing 1% agarose gel, blotted to a nylon membrane, and probed with the indicated cDNA probes. Each lane contains RNA from an individual animal. +, wild-type allele; β, targeted C/ebpα allele.