Phosphoenolpyruvate carboxykinase is necessary for the integration of hepatic energy metabolism

Abstract

We used an allelogenic Cre/loxP gene targeting strategy in mice to determine the role of cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in hepatic energy metabolism. Mice that lack this enzyme die within 3 days of birth, while mice with at least a 90% global reduction of PEPCK, or a liver-specific knockout of PEPCK, are viable. Surprisingly, in both cases these animals remain euglycemic after a 24-h fast. However, mice without hepatic PEPCK develop hepatic steatosis after fasting despite up-regulation of a variety of genes encoding free fatty acid-oxidizing enzymes. Also, marked alterations in the expression of hepatic genes involved in energy metabolism occur in the absence of any changes in plasma hormone concentrations. Given that a ninefold elevation of the hepatic malate concentration occurs in the liver-specific PEPCK knockout mice, we suggest that one or more intermediary metabolites may directly regulate expression of the affected genes. Thus, hepatic PEPCK may function more as an integrator of hepatic energy metabolism than as a determinant of gluconeogenesis.

FIG. 1

pck alleles generated by gene targeting and Cre-mediated recombination. (A) Top, partial map of the pck^w allele. Exons are indicated as solid rectangles. The location of the DNA fragment used as the Southern hybridization probe is shown. Middle, map of the PEPCK gene targeting vector. The vector contains a pgk-neo cassette, a pgk-tk cassette, and three loxP sites (triangles). Two of the loxP sites flank neo, and the third is located between exons 4 and 5 in the PEPCK gene. The pck^lox+neo allele was generated by homologous recombination (HR) in ES cells. Bottom, the pck^lox and pck^del alleles, derived from pck^lox+neo by Cre-mediated recombination. Exons 4 and 5 and neo were excised by Cre microinjection of single-cell pck^lox+neo/w embryos. (B and C) PCR genotype analysis. Tail DNA was amplified by both primer pair W (5′-TCTGTCAGTTCAATACCAATCT-3′), 5′-AATGTTCTCTGCAAGTCCTGGTG-3′) and primer pair D (5′-ATCAGCTTTAGTCGTCTCTGGT-3′, 5′-AATGTTCTCTGCAAGTCCTGGTG-3′) or primer pair F (5′-TCTGTCAGTTCAATACCAATCT-3′, 5′-AGCCTCTGTTCCACATACACTTCA-3′. The amplified alleles and their sizes are shown on the right and left, respectively. (D) Western blot analysis of liver homogenates from pck^w/w (lane 1), pck^del/w (lane 2), and pck^del/del (lane 3) mice.

FIG. 1

pck alleles generated by gene targeting and Cre-mediated recombination. (A) Top, partial map of the pck^w allele. Exons are indicated as solid rectangles. The location of the DNA fragment used as the Southern hybridization probe is shown. Middle, map of the PEPCK gene targeting vector. The vector contains a pgk-neo cassette, a pgk-tk cassette, and three loxP sites (triangles). Two of the loxP sites flank neo, and the third is located between exons 4 and 5 in the PEPCK gene. The pck^lox+neo allele was generated by homologous recombination (HR) in ES cells. Bottom, the pck^lox and pck^del alleles, derived from pck^lox+neo by Cre-mediated recombination. Exons 4 and 5 and neo were excised by Cre microinjection of single-cell pck^lox+neo/w embryos. (B and C) PCR genotype analysis. Tail DNA was amplified by both primer pair W (5′-TCTGTCAGTTCAATACCAATCT-3′), 5′-AATGTTCTCTGCAAGTCCTGGTG-3′) and primer pair D (5′-ATCAGCTTTAGTCGTCTCTGGT-3′, 5′-AATGTTCTCTGCAAGTCCTGGTG-3′) or primer pair F (5′-TCTGTCAGTTCAATACCAATCT-3′, 5′-AGCCTCTGTTCCACATACACTTCA-3′. The amplified alleles and their sizes are shown on the right and left, respectively. (D) Western blot analysis of liver homogenates from pck^w/w (lane 1), pck^del/w (lane 2), and pck^del/del (lane 3) mice.

FIG. 1

pck alleles generated by gene targeting and Cre-mediated recombination. (A) Top, partial map of the pck^w allele. Exons are indicated as solid rectangles. The location of the DNA fragment used as the Southern hybridization probe is shown. Middle, map of the PEPCK gene targeting vector. The vector contains a pgk-neo cassette, a pgk-tk cassette, and three loxP sites (triangles). Two of the loxP sites flank neo, and the third is located between exons 4 and 5 in the PEPCK gene. The pck^lox+neo allele was generated by homologous recombination (HR) in ES cells. Bottom, the pck^lox and pck^del alleles, derived from pck^lox+neo by Cre-mediated recombination. Exons 4 and 5 and neo were excised by Cre microinjection of single-cell pck^lox+neo/w embryos. (B and C) PCR genotype analysis. Tail DNA was amplified by both primer pair W (5′-TCTGTCAGTTCAATACCAATCT-3′), 5′-AATGTTCTCTGCAAGTCCTGGTG-3′) and primer pair D (5′-ATCAGCTTTAGTCGTCTCTGGT-3′, 5′-AATGTTCTCTGCAAGTCCTGGTG-3′) or primer pair F (5′-TCTGTCAGTTCAATACCAATCT-3′, 5′-AGCCTCTGTTCCACATACACTTCA-3′. The amplified alleles and their sizes are shown on the right and left, respectively. (D) Western blot analysis of liver homogenates from pck^w/w (lane 1), pck^del/w (lane 2), and pck^del/del (lane 3) mice.

FIG. 2

Analysis of PEPCK expression in mice that are homozygous for the pck^lox+neo and pck^lox alleles and that are compound heterozygotes of pck^lox+neo and pck^del alleles. (A) Hepatic and renal PEPCK activity in pck^{lox+neo/lox+neo} and pck^lox/lox mice fasted for 24 h. ∗∗∗, P < 0.001, n = 4. (B) Western blot analysis of liver and kidney tissues from 24-h-fasted mice. Lanes 1, 2, and 3 represent pck^w/w, pck^{lox+neo/lox+neo}, and pck^lox/lox mice, respectively. (C) Plasma glucose concentration in pck^{lox+neo/lox+neo} and pck^lox+neo/del mice at fed and 24-fasted states (n = 7 to 9). (D) Northern blot analysis of liver, kidney, and brown and white adipose tissues (BAT and WAT) from 24-h-fasted mice. Lanes 1 and 2 represent pck^w/w and pck^lox+neo/del mice, respectively. cyclo., cyclophilin.

FIG. 3

Liver-specific recombination in pck^lox/lox+Alb-cre (lanes 1) and pck^lox/lox (lanes 2) mice. (A) Northern blot analysis of overnight-fasted 5- to 6-week-old mice; BAT, brown adipose tissue; cyclo., cyclophilin. (B) Western blot analysis of liver tissue from overnight-fasted mice.

FIG. 4

Changes of blood glucose concentrations during exercise in pck^lox/lox+Alb-cre mice. ∗∗∗, P < 0.001 versus pck^lox/lox at each time point; n = 5.

FIG. 5

Hepatic steatosis in 24-h-fasted pck^lox/lox+Alb-cre mice. (A and B) Necropsy indicates increased liver size with pale color in ∼9-week-old pck^lox/lox+Alb-cre mice compared to pck^lox/lox mice. (C and D) Oil red O histochemistry for neutral lipids in liver sections. Larger lipid droplets (red) are shown in livers of pck^lox/lox+Alb-cre mice than in those of pck^lox/lox mice. (E and F) Hematoxylin-eosin histological staining for liver sections. Open circles in liver sections of pck^lox/lox+Alb-cre mice suggest lipid vacuoles. Scale bars in panels C to F represent 50 μm.

FIG. 6

Altered gene expression for energy metabolism enzymes in 24-h-fasted pck^lox/lox+Alb-cre mice. Northern blots were first probed with the specific cDNA; then the membranes were stripped and reprobed with cyclophilin cDNA. The relative abundance for each mRNA was normalized to the cyclophilin mRNA level. (A) Lipid-metabolizing enzymes. MCD, malonyl-CoA decarboxylase; VLCAD, very long-chain fatty acyl-CoA dehydrogenase; LCAD, long-chain fatty acyl-CoA dehydrogenase; MCAD, medium-chain fatty acyl-CoA dehydrogenase; COT, carnitine octanoyltransferase; CAT, carnitine acetyltransferase; ACO, acyl-CoA oxidase; PBE, enoyl-CoA hydratase–l-3-hydroxyacyl-CoA dehydrogenase bifunctional protein. (B) Gluconeogenic, TCA cycle, and other enzymes. cAAT and mAAT, cytosolic and mitochondrial aspartate aminotransferase, respectively; cMDH and mMDH, cytosolic and mitochondrial malate dehydrogenase, respectively; CS, citrate synthase; IDH, isocitrate dehydrogenase; SCS, succinyl-CoA synthetase; LDH, lactate dehydrogenase; ALT, alanine aminotransferase; PC, pyruvate carboxylase; G6Pase, glucose-6-phosphatase; FBPase, fructose-1,6-bisphosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ∗, P < 0.05, ∗∗, P < 0.01, and ∗∗∗, P < 0.001; n = 4.