Investigations on the effects of peroxisome proliferator-activated receptors and of nuclear factor kappa B on novel organic cation transporter 2 and carnitine uptake in bovine kidney cells
L-Carnitine is a water soluble metabolite, serving as an essential cofactor for fatty acid beta-oxidation by transferring long-chain fatty acids as acylcarnitine esters across the inner mitochondrial membrane. In the body, carnitine is derived from endogenous synthesis and from the intestinal absorption from the dietary sources. Tissues which cannot provide carnitine via endogenous synthesis, like skeletal muscle or myocardium, are dependent on carnitine uptake from the circulation. Carnitine transport is mediated by OCTN2, which is sodium-dependent and has a high-affinity to carnitine. OCTN2-mediated carnitine transport is also responsible for the tubular reabsorption of carnitine in the kidney and is therefore fundamental to maintaining normal carnitine levels in serum. Recent studies in rodents convincingly demonstrated that PPARalpha, which is a well-known central regulator of lipid metabolism and energy homeostasis, is an important transcriptional regulator of genes encoding OCTN2. Gene transcription by PPARalpha is initiated when a ligand, like fatty acids which are liberated from adipose tissue during energy deprivation and taken up into tissues during this state, or exogenous ligands such as fibrates (WY-14,643) bind to the LBD of this transcription factor. In contrast to rodents, little is known with regard to the regulation of OCTN2 by PPARalpha and its isoforms PPARbeta/delta and their roles for carnitine transport in cattle. PPARbeta/delta and PPARalpha have partially overlapping functions. For instance, they are central regulators of fatty acid catabolism since both subtypes control the expression of genes encoding proteins involved in cellular fatty acid uptake, intracellular fatty acid transport, mitochondrial fatty acid uptake, and mitochondrial and peroxisomal fatty acid oxidation. Although PPARs and NF-kappaB have a negative cross talk, in cattle both PPARs and NF-kappaB are activated during transition period. It has been confirmed that OCTN2 expression and carnitine uptake in human colonic epithelial cells are increased by NF-kappaB inducer TNFalpha. However, it is unclear whether NF-kappaB is able to alter OCTN2 and carnitine uptake in bovine kidney cells. Study 1 aimed to investigate the hypothesis that PPARalpha, as in rodents, regulates OCTN2 involving in carnitine uptake in cattle. MDBK cells were incubated 24 h with 150 myM of PPARalpha agonist WY-14,643 in the absence and presence of 10 myM of PPARalpha antagonist GW6471. WY-14,643 increased mRNA and protein levels of OCTN2, whereas co-treatment of MDBK cells with WY-14,643 and GW6471 blocked the WY-14,643-induced increase of mRNA and protein levels of OCTN2. The treatment of MDBK cells with WY-14,643 stimulated specifically Na+-dependent carnitine uptake in MDBK cells, which is likely the consequence of the increased carnitine transport capacity of cells due to the elevated expression of OCTN2. In addition, WY-14,643-stimulated increase of carnitine uptake was completely blocked by treatment of cells with GW6471. Study 2 aimed to investigate the hypothesis that PPARbeta/delta, like PPARalpha, also regulates bovine OCTN2 involving in carnitine uptake. MDBK cells were incubated for 24 h with 1 myM of PPARbeta/delta agonist GW0742 in the absence and presence of 10 myM of PPARbeta/delta antagonist GSK3787. GW0742 increased mRNA and protein levels of OCTN2, whereas co-treatment of MDBK cells with GW0742 and GSK3787 blocked the GW0742-induced increase of mRNA and protein levels of OCTN2. The treatment of MDBK cells with GW0742 stimulated specifically Na+-dependent carnitine uptake in MDBK cells. In addition, GW0742-stimulated increase of carnitine uptake was completely blocked by treatment of cells with GSK3787. Study 3 aimed to investigate the hypothesis that NF-kappaB has a similar function as PPARs in regulating bovine OCTN2 and carnitine uptake. Dose-dependent test was performed to find the optimized concentration of TNFalpha. 5ng/ml of TNFalpha increased the transcription level of IL-6 and IL-1B, which are the well-known NF-kappaB target genes. 5 ng/ml of TNFalpha stimulated NF-kappaB transactivation in MDBK cells. MDBK cells were incubated 24 h with 5 ng/ml NF-kappaB activator TNFalpha in the absence and presence of 1 myM of NF-kappaB inhibitor BAY 11-7085. TNFalpha increased mRNA and protein levels of OCTN2, whereas co-treatment of MDBK cells with TNFalpha and BAY 11-7085 blocked the TNFalpha-induced increase of mRNA and protein levels of OCTN2. The treatment of MDBK cells with TNFalpha stimulated carnitine uptake in MDBK cells. In addition, TNFalpha-stimulated increase of carnitine uptake was completely blocked by treatment of cells with BAY 11-7085.
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