Recipient Organization
CAL POLY CORPORATION
(N/A)
SAN LUIS OBISPO,CA 93407
Performing Department
(N/A)
Non Technical Summary
Animal productivity is highly dependent upon the efficient utilization of amino acids, particularly for those amino acids that are first limiting for growth. In poultry, lysine is often the first or second limiting amino acid for growth. The importance of this amino acid in poultry nutrition is clear and dietary and feeding strategies aimed to maximize lysine utilization are well-established. What is less clear and well-established, however, is an understanding of the basic mechanisms regulating lysine utilization at the cellular and molecular level. Lysine utilization is first controlled at the level of the plasma membrane through the catalytic functions of lysine transporters. We have shown that CAT-2 isoforms are the predominant lysine transport system expressed in chicken liver and skeletal muscle. The objective of this proposal, therefore, is to determine the function and relative importance of CAT-2 isoforms in lysine transport and utilization in these tissues.
These studies will increase our understanding of the mechanisms that regulate lysine utilization at the cellular and molecular level which are important for understanding lysine's role in limiting growth and protein accretion in poultry.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
The long-term objective of this research is to identify the molecular determinants regulating lysine utilization in chicken tissues. The specific goal of this proposal is to determine how lysine utilization is regulated by lysine transport systems, particularly CAT-2 isoforms, that are the primary lysine transporters expressed in chicken liver and skeletal muscle. The specific objectives of the proposed research are: 1) to determine the function of chicken CAT-2 proteins by defining their cellular and subcellular localization and transport kinetics, and 2) to determine the relative importance of chicken CAT-2 isoforms in lysine transport by overexpressing CAT-2 proteins and decreasing CAT-2 gene expression in the chicken LMH hepatocyte cell line.
Project Methods
A combination of genetic, biochemical and nutritional approaches will be utilized to determine the function and relative importance of chicken CAT-2 isoforms in lysine transport and utilization. The function of chicken CAT-2 proteins will be studied by expressing these proteins in CHO-K1 cells. The cellular and subcellular localization of CAT-2 proteins will be determined by immunofluorescence staining. Total amounts and rates of lysine uptake will be measured to determine the transport kinetics of CAT-2 proteins. The importance of CAT-2 proteins in lysine transport will be determined by either overexpressing CAT-2 proteins or decreasing CAT-2 gene expression in the chicken LMH hepatocyte cell line. Hepatocytes will be transfected with an isoform specific CAT-2 expression vector and CAT-2 protein levels will be monitored by Western blot and immunofluorescence. Total amounts and rates of lysine uptake will be measured over various lysine concentrations to determine the
effect of elevated CAT-2 protein levels on lysine uptake. CAT-2 protein expression will be reduced by transfecting hepatocytes with CAT-2 small interfering RNA to knock-down CAT-2 mRNA abundance. CAT-2 protein levels will be monitored by Western blot and immunofluorescence and CAT-2 mRNA levels will be monitored by quantitative real-time PCR. Total amounts and rates of lysine uptake will be measured over various lysine concentrations to determine the effect of decreased CAT-2 protein levels on lysine uptake. The mRNA levels of other lysine transport systems will also be measured by quantitative real-time PCR to determine if their transcription is altered by CAT-2 protein levels.