Slc25a13-Knockout Mice Harbor Metabolic Deficits but Fail To Display Hallmarks of Adult-Onset Type II Citrullinemia

Abstract

Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn ؊/؊ mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn ؊/؊ mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.

Citrin/Mitochondrial Glycerol-3-phosphate Dehydrogenase Double Knock-out Mice Recapitulate Features of Human Citrin Deficiency

“…1). Comparisons between wild-type and Ctrn-KO mice also showed similar growth rates (42), and mGPD-KO mice showed only a small difference in growth rate compared with non-littermate wild-type mice (data not shown).…”

“…Animals-Mice homozygous for the targeted disruption of the Slc25a13 gene that encodes citrin (Ctrn Ϫ/Ϫ or Ctrn-KO) and for the mGPD gene that encodes mGPD (mGPD Ϫ/Ϫ or mGPD-KO) were generated as described previously (42,45). Both KO mutations were made congenic on the C57BL/6J genetic background by backcrossing for at least nine generations.…”

“…Single Ctrn-KO (Ctrn Ϫ/Ϫ , mGPD ϩ/ϩ ) and C57BL/6J wild-type mice were also generate as littermates by intercrossing mice heterozygous for the Ctrn-KO mutation. Genotyping was performed on DNA extracted from ear punch using procedures specific for each of the targeted disruptions made in the Ctrn-KO and mGPD-KO mice as described previously (42,45).…”

“…Of special note was the relative expression level of aralar, the brain-/muscletype AGC, that can functionally compensate for citrin (5). Aralar expression has not been detected previously in human liver by Northern or Western blot (3,4,6) and is most minimally detectable in mouse liver based on similar methods (7,42). The relative expression level of aralar in the liver of the double knock-out mice, detected by microarray analysis, was only slightly higher (1.40 Ϯ 0.19-fold) than that of wild-type mice.…”

“…We have previously created a citrin (Ctrn) knock-out mouse (42) by targeted disruption of the Slc25a13 gene. Although the resulting Ctrn Ϫ/Ϫ mice lacked Slc25a13 mRNA and citrin protein and demonstrated markedly reduced mitochondrial Asp transport and MA shuttle activity in vitro, there was no apparent phenotype in vivo.…”