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. 2013 Apr 30;110(18):7423-8.
doi: 10.1073/pnas.1305805110. Epub 2013 Apr 15.

Targeted mutagenesis of mitochondrial carbonic anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose metabolism

Gul N Shah  1 , Timothy S Rubbelke, Joshua Hendin, Hien Nguyen, Abdul Waheed, James D Shoemaker, William S Sly
Affiliations

Targeted mutagenesis of mitochondrial carbonic anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose metabolism

Gul N Shah et al. Proc Natl Acad Sci U S A. .

Abstract

Prior studies with carbonic anhydrase (CA) inhibitors implicated mitochondrial CA in ureagenesis and gluconeogenesis. Subsequent studies identified two mitochondrial CAs. To distinguish the contribution of each enzyme, we studied the effects of targeted disruption of the murine CA genes, called Car5A and Car5B. The Car5A mutation had several deleterious consequences. Car5A null mice were smaller than wild-type littermates and bred poorly. However, on sodium-potassium citrate-supplemented water, they produced offspring in expected numbers. Their blood ammonia concentrations were markedly elevated, but their fasting blood sugars were normal. By contrast, Car5B null mice showed normal growth and normal blood ammonia levels. They too had normal fasting blood sugars. Car5A/B double-knockout (DKO) mice showed additional abnormalities. Impaired growth was more severe than for Car5A null mice. Hyperammonemia was even greater as well. Although fertile, DKO animals were produced in less-than-predicted numbers even when supplemented with sodium-potassium citrate in their drinking water. Survival after weaning was also reduced, especially for males. In addition, fasting blood glucose levels for DKO mice were significantly lower than for controls (153 ± 33 vs. 230 ± 24 mg/dL). The enhanced hyperammonemia and lower fasting blood sugar, which are both seen in the DKO mice, indicate that both Car5A and Car5B contribute to both ammonia detoxification (ureagenesis) and regulation of fasting blood sugar (gluconeogenesis). Car5A, which is expressed mainly in liver, clearly has the predominant role in ammonia detoxification. The contribution of Car5B to ureagenesis and gluconeogenesis was evident only on a Car5A null background.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Generation, genotyping and characterization of Car5A KO mice. (A) Structure of the Car5A gene (Top), the targeting construct (Middle), and the predicted structure of the disrupted Car5A gene after homologous recombination and excision of the neomycin-resistance (Neo) cassette (Bottom). The open box indicates the position of the external probe. Only the relevant restriction sites are shown. The numbered solid boxes represent exons. Neo and TK (thymidine kinase) refer to positive and negative selection markers, respectively. (B) Genotyping of Car5A KO colony. Genotyping by PCR was done on mouse-tail genomic DNA. PCR products present in WT (0.6 kb) and mutant (0.55 kb) mice were present in the heterozygotes. (C) Car5A transcripts in liver and kidney from WT and KO mice. Total RNA (≤1 µg) was reverse-transcribed and amplified by PCR. A 0.35-kb band was amplified from both tissues from the WT animals but not from the KO mice. (D) Western blot analysis. The membrane proteins (50 μg) from liver and kidney of CA VA WT and KO mice were electrophoresed on a 12% (wt/vol) SDS-PAGE gel. After blotting, the membranes were probed with rabbit anti-mouse CA VA antibodies. A 29-kDa band present in both liver and kidney from the WT animals was absent in the same tissues from KO animals.
Fig. 2.
Fig. 2.
Generation, genotyping, and characterization of Car5B KO mice. (A) Structure of the Car5B gene (Top), the targeting construct (Middle), and the predicted structure of the disrupted Car5A gene after homologous recombination and excision of the neomycin-resistance (Neo) cassette (Bottom). The open box indicates the position of the external probe. Only the relevant restriction sites are shown. The numbered solid boxes represent exons. Neo and TK (thymidine kinase) refer to positive and negative selection markers, respectively. (B) Genotyping of the Car5B KO colony. Genotyping by PCR was done on mouse-tail genomic DNA. PCR products present in WT (0.52 kb) and mutant (0.44 kb) mice were present in the heterozygotes. (C) Car5B transcripts in liver and kidney amplified from WT mice but not from KO mice. (D) Western blot analysis. The membrane proteins (50 μg) from liver and kidney of CA VB WT and KO mice were electrophoresed on a 12% (wt/vol) SDS-PAGE gel. After blotting, the membranes were probed with rabbit anti-mouse CA VB antibodies. A 31-kDa band present in both liver and kidney from the WT animals was absent in the same tissues from KO animals.
Fig. 3.
Fig. 3.
Comparison of weights of Car5A/B DKO female and male mice with WT controls of the same sex. All mice were fed normal rat chow ad libitum and given citrate-supplemented water.
Fig. 4.
Fig. 4.
Mortality. Percentage survival was plotted from birth to age 56 d. Initial numbers in each group are shown.
Fig. 5.
Fig. 5.
Ammonia concentration in the blood of WT, Car5A, and Car5B null mice and the DKO animals. Plasma ammonia levels were measured in the fed state for each of the genotypes shown. The statistically significant elevations in the Car5A mice are described in Results, as is the even greater elevation seen in the DKO mice.
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