Mutations in metabolic pathways, what role does genetic background play?

Authors: Marini, Juan; EREZ, A - BAYLOR COLLEGE MED; LEE, B - BAYLOR COLLEGE MED

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 6/8/2007
Publication Date: 9/9/2007
Citation: Marini, J.C., Erez, A., Lee, B. 2007. Mutations in metabolic pathways, what role does genetic background play [abstract]? In: 2nd International Symposium of Energy and Protein Metabolism and Nutrition, September 9-13, 2007, Vichy, France. 124:245.

Interpretive Summary:

Technical Abstract: Ornithine transcarbamylase (OTC) is a key enzyme for the synthesis of urea and the endogenous synthesis of arginine. OTC is present in hepatocytes and enterocytes and catalyzes the synthesis of citrulline. Although the spf-ash mutation results in a reduction in enzyme abundance, ureagenesis is maintained if urea cycle intermediates (UCI) are provided. However, preliminary work has shown that UCI were not necessary to maintain ureagenesis in a new line of spf-ash mutant mice. The objective of the present work was to determine the effect of genetic background in the manifestation of the spf-ash mutation regarding urea, UCI, and nitric oxide production. The OTC(spf-ash) mutation, originally present in B6EiC3Sn (B6) mice, was bred into an outbred mouse line (ICR). After 10 generations of backcrossing, mice were studied utilizing a multiple tracer protocol during an unbalanced nitrogen load (glycine-alanine, 6.1 mmol/kg/h). A primed continuous infusion of (13)C(18)O urea, (15)N(15)N arginine, and (13)C(2)H(4) citrulline was conducted for 4 h to determine the entry rate of these compounds. Additionally, the conversion of arginine to citrulline (a measurement of NO production) and citrulline to arginine were calculated. Western analysis to determine the abundance of OTC protein was performed in hepatic and intestinal tissue; a-tubulin was utilized to normalize the OTC signal. A reduction in hepatic and intestinal OTC abundance was observed as a consequence of the spf-ash mutation (127 vs. 40 arbitrary units, and 185 vs. 30 au, respectively; SE=8.6 au). Although no difference in the expression of OTC was found in mutant mice of the two genetic backgrounds, B6 control animals had a higher expression of the enzyme in the liver, but a lower expression in the intestine when compared to ICR controls. The reduction in intestinal OTC activity resulted in a reduced citrulline flux in mutant mice from both genetic backgrounds (176 vs. 81 mmol/kg/h; SE=7.1 mmol/kg/h). Arginine flux, however, was only reduced in B6 spf-ash mice (376 vs. 502 mmol/kg/h; SE=23.2 mmol/kg/h). Nitric oxide (arginine to citrulline flux) production was higher in ICR mice than in animals of the B6 genetic background (10.5 vs. 4.9 mmol/kg/h; SE=1.1 mmol/kg/h); furthermore, a trend (P < 0.10) for higher nitric oxide production in ICR mutant mice was observed when compared to B6 controls, despite the reduction in OTC enzyme activity which was thought to limit arginine availability for nitric oxide synthesis. ICR mice were able to convert a greater proportion of the citrulline flux into arginine than B6 mice (72 vs. 63%, P < 0.001), which might explain these observations. The reduction in hepatic OTC abundance and activity seen in spf-ash mice resulted in a reduced urea flux only in mice of the B6 background (5525 vs. 3418 mmol/kg/h; SE=250 mmol/kg/h). The inability to sustain ureagenesis under the unbalanced nitrogen load resulted in the development of hyperammonemia in B6 mutant animals. Although genetic background did not affect the abundance of OTC enzyme in mutant mice, the fluxes through this enzyme were very different. Mice of the B6 background produced less citrulline and urea, which translated into a reduced arginine flux and hyperammonemia. The choice of appropriate genetic background for the expression of mutations in metabolic pathways is crucial for the study of these complex processes.