PSI-22 Effects of modifying PPAR activity on carnitine uptake in swine mammary organoids.

Abstract Carnitine is essential in growth and development of newborn pigs due to its role in energy production and detoxification via removing acyl-residues. The primary source of carnitine after birth is sow’s milk because newborn pigs have a limited capacity to synthesize carnitine. The carnitine concentration in sow milk varies with dietary carnitine and lactation stage in which the expression of mammary organic cation transporter (OCTN) may play a decisive role as the mediator of carnitine homeostasis. Regulation of peroxisome proliferator–activated receptor (PPAR) influences expression of genes associated with carnitine synthesis and OCTN activity in liver, kidney and enterocyte, but the regulatory role in mammary tissue is not clear especially as lactation leads to down-regulation of PPARα. In this study, the effects of altering PPARα and PPARγ expression on carnitine uptake was examined in vitro in organoids cultured from mammary tissue of pregnant sows. The organoids were incubated with PPARα agonist clofibrate (10 µM), PPARγ agonist thiazolidinedione (TDZ, 10 µM) and antagonist GW9662 (5 µM) in a 3-dimentional environment. The expression of genes PPARα and PPARγ, and genes associated with carnitine uptake OCTN1 (SLC22A4) and OCTN2 (SLC22A5), and genes associated with fatty acid oxidation, carnitine palmitoyltransferase (CPTIα), CPTIβ and acyl-CoA oxidase (ACOX) were determined after 48 hours using RT-PCR. Carnitine uptake rate was measured by adding 16 μM of 3H-labeled carnitine (1µCi/µmol) to cultures for 30 minutes. Clofibrate stimulated expression of PPARα, SLC22A4, SLC22A5 and CPTIβ (p < 0.05) with no significant changes in PPARγ, CPTIα and ACOX (p > 0.1). However, the stimulation did not change carnitine uptake. TDZ had no impact on the expression of all examined genes but GW-9662 significantly increased CPTIα expression by 4-fold. Consistent with CPTIα expression, carnitine transfer rate increased by 70%. Correlation analysis between the gene expression and carnitine uptake rate indicated that the elevation of carnitine uptake was correlated with CPTIα expression (p < 0.005, Corr=0.97) or the ratio of CPTIα/CPTIβ, (p < 0.005, Corr=0.99). We conclude that up-regulation of PPARα expression increases expression of genes associated with mammary carnitine transporters and fatty acid oxidation, but carnitine uptake is subject to the metabolic status of mammary tissue. Supported by Animal Health and Production and Animal Products (grant no. 2023-67015-39663/1030033) from the USDA National Institute of Food and Agriculture, and by the North Carolina Agricultural Research Hatch projects 1016618 and 02780. Keyword: carnitine, mammary, organoids