ARS | Publication request: Aminoadipate semialdehyde synthase mRNA knockdown reduces the lysine requirement of a mouse hepatic cell line

Submitted to: Journal of Nutrition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 12, 2008
Publication Date: N/A

Interpretive Summary: Lysine is an essential amino acid in animals that is frequently the first or second limiting amino acid for growth in cereal grain-based diets for human, swine, poultry, and fish. Meeting the lysine requirement is imperative in humans for optimal health and in livestock production to avoid the monetary losses associated with suboptimal animal performance. Additionally, improving lysine nutriture would improve health in humans consuming a lysine limiting diet and decrease the feed costs in animal production systems, which generally account for between fifty and seventy-five percent of production costs. A disease found in humans, familial hyperlysinemia, is characterized by a 90% reduction in the activity of aminoadipate semialdehyde synthase (AASS), an enzyme that degrades free lysine. Interestingly, individuals with familial hyperlysinemia have a lower lysine requirement that is caused by a reduction in lysine catabolism through the AASS pathway. Our objective was to reduce the lysine requirement of a mouse hepatic cell line, a suitable model system for economically important animals, by utilizing RNA interference (RNAi) to decrease AASS mRNA. Here we report that murine hepatic cells with reduced AASS expression and lower LKR activity and lysine oxidation have a lower lysine requirement than the wild type cells. Therefore, the results provide the initial proof in principle that RNAi can be used to decrease nutrient requirements. Extrapolation of these results to livestock and aquaculture production signifies the incredible potential for improved economic gain and reduced environmental impact.

Technical Abstract: a-Aminoadipate d-semialdehyde synthase (AASS) is the bifunctional enzyme containing the lysine a-ketoglutarate reductase (LKR) and saccharopine dehydrogenase activities responsible for the first two steps in the irreversible catabolism of lysine. A rare disease in humans, familial hyperlysinemia, can be caused by very low LKR activity and, as expected, reduces the lysine ¿requirement¿ of the individual. This concept was applied to a murine hepatic cell line (ATCC, FL83B) utilizing RNA interference to achieve AASS mRNA knockdown. Cells were antibiotic selected for stable transfection of two plasmids that express different short hairpin RNA sequences for AASS knockdown. Compared to the wild type cell line, AASS mRNA abundance was reduced 79.0 +/- 6.4% (P<0.05), resulting in a 29.8 +/- 5.2% (P<0.05) reduction in AASS protein abundance, 41.3 +/- 10.0% (P<0.05) less LKR activity, and a reduction in lysine oxidation by 50.7 +/- 11.8%. To determine the effect of AASS knockdown on the lysine requirement, cells were grown in media containing 12.5, 25.0, 50.0, 100, or 200 uM lysine. Using a segmented model approach for growth rate analysis, the lysine requirement of the cell line with AASS silencing was 43.4 +/- 1.7 uM, approximately 26% lower (P<0.05) than the lysine requirement of the wild type cell line. These results indicate AASS knockdown decreases the lysine requirement of the cell via a reduction of lysine catabolism through the saccharopine pathway, providing the initial proof in principle that RNA interference can be used to reduce the nutrient requirement of a system.