Genetic background and sex determine citrulline and de novo arginine production in mice
Authors: Marini Juan C , Agarwal Umang , Didelija Inka C 
Published: November 2023

We have previously shown sex and genetic background differences in citrulline production and in de novo arginine synthesis in mice. These differences were the consequence of a higher expression of intestinal ornithine transcarbamylase (OTC), a mitochondrial enzyme involved in the synthesis of citrulline, in mice of Swiss background when compared with BL6 animals. The OTC gene is in the X chromosome and thus females have the potential to overexpress this gene due to incomplete X inactivation. Also many gene polymorphisms have been described among different genetic backgrounds, which may also result in a differential expression and citrulline production in mice. Likewise, genetic polymorphisms may result in a differential utilization of arginine among mouse strains. To test the effect of sex and genetic background on the arginine metabolome and fluxome C57BL6/J (B) and SJL/J (S) mice were mated to produce a F1 BB, BS, SB and SS offspring. At 6–8 weeks of age and after a 3 h fast, male and female mice were infused for 4 h with tracers to determine citrulline, ornithine and arginine fluxes, as well as the de novo arginine synthesis rate and arginine disposal by arginase. A phenylalanine tracer was also infused to determine protein breakdown and arginine contribution from this source to the overall arginine flux. The flux of citrulline was higher (P < 0.01) in females and in mice with a SJL progenitor. This resulted in a greater (P < 0.01) de novo arginine synthesis in females and in mice with a SJL mother. No difference (P > 0.15) between sexes or genotypes was observed for the fraction of citrulline utilized for plasma arginine synthesis (75±2%). Arginine and phenylalanine fluxes were higher (P<0.03) in mice with both SJL parents. Furthermore, arginine flux was higher (P<0.02) in males than in females. This was accompanied by a higher ornithine flux and a higher rate of conversion of plasma arginine into ornithine in females, indicating a higher arginase activity. The ratio of arginine from protein turnover to phenylalanine flux was lower in females (1.16 vs 1.50, P<0.001) demonstrating that some of the arginine disposal takes place immediately after protein breakdown. Plasma arginine and citrulline concentrations were not different (P > 0.08) among the different genotypes or the two sexes. Likewise there were no sex differences for ornithine concentration; however, plasma ornithine was higher (P < 0.03) in mice with at least one SJL progenitor. These results show that females produce more citrulline than males, which results in a greater rate of de novo arginine synthesis. Males, however, are able to support higher arginine fluxes than females due to a reduction in the hydrolysis of arginine into ornithine (arginase activity). Furthermore, some of this disposal of arginine takes place even before this amino acid enters the circulation. The measurement of arginine and related amino acids fluxes and interconversions were able to capture these complex interactions between sex and genetic background; the arginine metabolome analysis, however, failed to do so.

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