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The uptake of amino acids is mediated by amino acid transporters and the peptide transporter, PepT1. The nuclear receptor PPARα may play a role in the activation of PepT1. The objectives of this study were to evaluate the effect of a fasting/refeeding regimen and gavaging a PPARα ligand on expression of chicken PepT1 and PPARα. During the fasting/refeeding trial, chicks were placed on a 24h fast and then divided into 3 groups (Continuously Fasted (CF), Refed/Food Withdrawn, and Refed Adlib) and sampled 1, 2, 3, 5 and 7h post-fast. Expression of PepT1 and PPARα increased almost 2-fold post-24h fast (P < 0.002). The CF group had highest expression for both genes (P < 0.0001) suggesting a coordinate change in PepT1 and PPARα expression during fasting, indicating a possible regulatory role for PPARα of PepT1. Oral gavage trials utilizing PPARα ligand WY- 14643 were conducted with layers and broilers. For the layer trial birds were gavaged daily for 3 d with 50 mg WY-14643/kg in carrier (Ligand) or with just carrier (No Ligand) and sampled on d4. For the broiler trial, birds were gavaged daily with 25 or 50 mg WY-14643/kg or just carrier and sampled on d2 or d3 post gavage. No difference in PepT1 was observed between the Ligand and No Ligand groups. In the layer trial, PPARα decreased 6-fold (P = 0.005) in the Ligand group. Broilers treated with WY-14643 showed no effect of ligand. These results indicate a difference between layers and broilers in response to WY-14643. / Master of Science

L’hépatokine FGF21 (Fibroblast Growth factor 21) joue un rôle primordial dans le contrôle de l’homéostasie énergétique. Des études chez l’Homme et l’animal mettent en évidence ses effets bénéfiques dans la lutte contre l’hyperglycémie, la dyslipidémie et l’obésité. Connue pour être induite en réponse au jeûne par le récepteur nucléaire PPARα (Proliferator Activated Receptor α), des études récentes suggèrent l’implication du facteur de transcription ChREBP (Carbohydrate Responsive Element Binding Protein) dans la réponse nutritionnelle de FGF21. Dans ce contexte, les objectifs de thèse ont été : 1) d’obtenir une meilleure compréhension de la régulation de FGF21 dans le foie par le jeûne et le glucose via les acteurs moléculaires ChREBP et PPARα ; 2) de déterminer la relevance physiologique de l’axe ChREBP-PPARα-FGF21 en réponse au glucose. Nos résultats mettent en évidence que l’expression hépatique de ChREBP est nécessaire à l’induction de FGF21 en réponse au glucose in vitro et in vivo. De manière inattendue, lorsque l’expression de PPARα est spécifiquement invalidée dans le foie, la réponse au glucose de FGF21 est diminuée de manière significative car ChREBP ne peut se lier à son élément de réponse de type ChoRE, présent sur le promoteur de fgf21. La réponse synergique de ChREBP et de PPARα sur FGF21 a été également mise en évidence dans des cultures primaires d’hépatocytes humains. Chez les souris invalidées pour PPARα dans le foie, l’absence de FGF21 circulant entraine une augmentation de la préférence au sucrose. Notre étude révèle l’existence d’un dialogue fonctionnel unique entre ChREBP et PPARα pour la régulation de FGF21 en réponse au glucose / The hepatokine FGF21 (Fibroblast Growth factor 21) plays an important role in the control of energy homeostasis. Studies in humans and animals have established FGF21 as an important therapeutic target for its beneficial effects on hyperglycemia, dyslipidemia and obesity. Induced in response to fasting by the PPARα nuclear receptor (Proliferator Activated Receptor α), recent studies suggest the involvement of ChREBP (Carbohydrate Responsive Element Binding) in the nutritional response of FGF21. In this context, the thesis objectives were: 1) to obtain a better understanding of the regulation of FGF21 in the liver by fasting and glucose via the molecular actors ChREBP and PPARα; 2) to determine the physiological relevance of the ChREBP-PPARα-FGF21 axis in response to glucose. Our results demonstrate that hepatic expression of ChREBP is necessary for the induction of FGF21 in response to glucose in vitro and in vivo. Unexpectedly, when PPARα expression is specifically invalidated in the liver, the glucose response of FGF21 is significantly decreased as ChREBP cannot bind to its ChoRE response element present on the fgf21 promoter. The synergistic response of ChREBP and PPARα to FGF21 was also demonstrated in primary cultures of human hepatocytes. In mice deficient for PPARα in the liver, the absence of circulating FGF21 leads to an increase in their preference to sucrose. Our study reveals the existence of a unique functional dialogue between ChREBP and PPARα for the regulation of FGF21 in response to glucose

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第18319号 / 農博第2044号 / 新制||農||1021(附属図書館) / 学位論文||H26||N4826(農学部図書室) / 31177 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 伏木 亨, 教授 金本 龍平 / 学位規則第4条第1項該当

Background & aims Cytochrome P450 2A5 (CYP2A5) is induced by ethanol, and the ethanol induction of CYP2A5 is regulated by nuclear factor-erythroid 2-related factor 2 (NRF2). Cyp2a5 knockout (Cyp2a5−/−) mice develop more severe alcoholic fatty liver than Cyp2a5+/+ mice. Fibroblast growth factor 21 (FGF21), a PPARα-regulated liver hormone, is involved in hepatic lipid metabolism. Alcoholic and non-alcoholic fatty liver are enhanced in Pparα knockout (Pparα−/−) mice. This study investigates the relationship between the PPARα-FGF21 axis and the enhanced alcoholic fatty liver in Cyp2a5−/− mice. Methods Mice were fed the Lieber-Decarli ethanol diet to induce alcoholic fatty liver. Results More severe alcoholic fatty liver disease was developed in Cyp2a5−/− mice than in Cyp2a5+/+ mice. Basal FGF21 levels were higher in Cyp2a5−/− mice than in Cyp2a5+/+ mice, but ethanol did not further increase the elevated FGF21 levels in Cyp2a5−/− mice while FGF21 was induced by ethanol in Cyp2a5+/+ mice. Basal levels of serum FGF21 were lower in Pparα−/− mice than in Pparα+/+ mice; ethanol induced FGF21 in Pparα+/+ mice but not in Pparα−/− mice, whereas ethanol induced hypertriglyceridemia in Pparα−/− mice but not in Pparα+/+ mice. Administration of recombinant FGF21 normalized serum FGF21 and triglyceride in Pparα−/− mice. Alcoholic fatty liver was enhanced in liver-specific Fgf21 knockout mice. Pparα and Cyp2a5 double knockout (Pparα−/−/Cyp2a5−/−) mice developed more severe alcoholic fatty liver than Pparα+/+/Cyp2a5−/− mice. Conclusions These results suggest that CYP2A5 protects against the development of alcoholic fatty liver disease, and the PPARα-FGF21 axis contributes to the protective effects of CYP2A5 on alcoholic fatty liver disease.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-09-22T19:23:19Z No. of bitstreams: 1 thiagodealmeidaoliveira.pdf: 1376099 bytes, checksum: 1223858331fe263a8bdcce2a114266c5 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-09-27T13:42:30Z (GMT) No. of bitstreams: 1 thiagodealmeidaoliveira.pdf: 1376099 bytes, checksum: 1223858331fe263a8bdcce2a114266c5 (MD5) / Made available in DSpace on 2017-09-27T13:42:30Z (GMT). No. of bitstreams: 1 thiagodealmeidaoliveira.pdf: 1376099 bytes, checksum: 1223858331fe263a8bdcce2a114266c5 (MD5) Previous issue date: 2017-06-28 / A modulação da resposta imune inata seria uma ferramenta capaz de elhorar a capacidade do sistema imune de combater a infecção por patógenos e outros estímulos externos. A utilização de vias sinalizadoras com a participação de alguns receptores específicos, como o peroxisome proliferator-activated receptor alpha (PPARα) surgiria como importante hipótese para modular a produção de proteínas de fase aguda e do complemento em hepatócitos bovinos, órgão principal envolvido nessa síntese. Nesse sentido, o presente trabalho se utilizou de técnicas de cultivo primário de hepatócitos bovinos, provenientes de 5 animais diferentes, que foram isolados, cultivados e estimulados com um ligante desse receptor nuclear, o fenofibrato. Após 48 horas de cultivo as células tiveram seu ácido ribonucleico (RNA) extraído e quantificado, seguindo-se à síntese de ácido desoxirribonucleico complementar (cDNA) e execução das reações em cadeia da polimerase quantitativo (qPCR), a fim de se verificar a expressão e produção de proteínas de fase aguda e do sistema complemento por meio de de reações de qPCR. Os sobrenadantes de cultura também foram avaliados após esse período de tempo, onde foram dosadas as mesmas proteínas, por meio da técnica de Enzyme-linked immunosorbent assay (ELISA). Os resultados demonstraram que o cultivo se provou ineficiente no que diz respeito à viabilidade celular e à quantificação de RNA extraído, indicando que mais estudos devem ser feitos para padronização dessa técnica. Além disso, os resultados demonstraram que o PPARα, ativado frente ao seu ligante específico, o fenofibrato, não foi capaz de modular a expressão e tampouco a produção dos fatores de complemento e das proteínas de fase aguda analisadas, podendo indicar que em hepatócitos bovinos a modulação por esse receptor não ocorre da mesma maneira como em humanos e em murinos. No entanto, devido à grande variabilidade racial dos animais cujos fígados foram coletados e ao pequeno número amostral utilizado, análises mais refinadas são necessárias. Dessa maneira, estudos devem ser realizados visando novas moléculas capazes de modular proteínas relacionadas ao sistema imune em bovinos. / The innate immune modulation is a tool capable of improve the immune system capacity to fight infections by pathogens and other external stimuli. The use of signaling paths with participation of some specific receptors, such as peroxisome proliferator-activated receptor alpha (PPARα) arises as an important hypothesis to modulate the acute phase proteins (APP) and complement proteins in bovine liver cells, where most of these molecules are produced in a systemic level. In this way, this work aimed to establish a protocol of primary culture of bovine hepatocytes suitable to analyze intracellular signaling paths and genetic expression and synthesis of proteins related to innate immune system. For that, bovine hepatocytes from five different animals were isolated, cultivated and activated by a receptor ligand - fenofibrate, both alone and associated with proinflammatory stimuli, in different concentrations. After 48 hours of culture, ribonucleic acid (RNA) was extracted and quantified, complementary deoxyribonucleic acid was synthetized and real-time polymerase chain reactions were setup to verify the acute phase and complement proteins expression and synthesis. The culture supernatant was used to analyze the protein dosage by Enzyme-Linked Immunosorbent Assay (ELISA). The results showed that the hepatocytes isolation and culture were not very effective, since the extracted RNA showed low concentration and bad integrity, indicating that more studies must be performed to standardize this technique. Considering the low quantification and bad quality of RNA, it was not possible to access the role of the receptor PPARα in complement factors and acute phase proteins genetic expression and production, when activated by its specific ligand (fenofibrate). Thus, the development of isolation and culture techniques for bovine hepatocytes must continue to be developed in order to better evaluate the intracellular signaling pathways and the search for new molecules capable to modulate proteins related to the immune system in bovines.

Einleitung: Monocarboxylattransporter (MCT) 1 & 4 sind in zahlreichen Geweben als Kotransporter für Monocarboxylate und Protonen beschrieben. Auch im Pansenepithel werden MCT benötigt, um kurzkettige Fettsäuren (SCFA) aus dem Pansenlumen in die Pansenepithelzelle aufzunehmen (MCT4) und um SCFA und deren Metabolite aus der Pansenepithelzelle in das Blut auszuschleusen (MCT1). Die transepitheliale Permeation von SCFA über die Pansenwand ist von enormer Bedeutung, da sie die wichtigste Energiequelle der Wiederkäuer darstellen. Die beteiligten Transportprozesse müssen dementsprechend einer Anpassung an variierende Mengen von SCFA unterliegen. Bisherige Studien bei anderen Spezies deuten auf eine Regulation des MCT1 auf mRNA Ebene über den Peroxisom-Proliferator-aktivierten Rezeptor α (PPARα) hin. Ziele der Untersuchung: Das Ziel dieser Arbeit war herauszufinden, ob MCT1 in ovinen Pansenepithelzellen über PPARα reguliert wird und ob auch MCT4 dieser Regulation unterliegt. Eine gleichzeitige Regulation beider Transporter läge nahe, da sie gemeinsam an der transepithelialen Permeation beteiligt sind. Die Auswirkungen solch einer Regulation auf die Proteinexpression und die Transportleistung der MCT sollte charakterisiert werden. Ebenfalls war das Potenzial der bei erhöhter Kraftfutterfütterung vermehrt anfallenden SCFA Butyrat auf die MCT1 Expression zu untersuchen. Material & Methoden: Aus dem Vorhof von Schafen wurden Pansenepithelzellen gewonnen und entsprechend einer bereits etablierten Methode kultiviert. Nach einer Subkultivierung wurden die Zellen immunzytochemisch mit Antikörpern gegen MCT1, MCT4 und Na+/K+-ATPase untersucht, um deren Lokalisation in den kultivierten Pansenepithelzellen zu bestimmen. Weiterhin erfolgte eine Behandlung mit WY 14.643, einem spezifischen, synthetischen PPARα Agonisten, sowie mit GW 6471, einem Antagonisten des PPARα. Mittels qPCR wurden die relativen mRNA Mengen von MCT1, MCT4, ACO, CPT1A und CACT bestimmt und auf die Referenzgene GAPDH und Na+/K+-ATPase normalisiert. Die Proteinexpression von MCT1 und MCT4 wurde mittels Western Blot bestimmt. Zur funktionellen Quantifizierung wurde der intrazelluläre pH-Wert der Zellen mittels Spektrofluorometrie gemessen und der laktatabhängige Protonentransport als Vergleichswert zwischen den Behandlungen genutzt. Um den MCT-abhängigen Teil des Transportes zu bestimmen, wurde ein spezifischer MCT1 & 4 Inhibitor, die p-Hydroxymercuribenzensulfonsäure (pHMB) eingesetzt. Die Zellen wurden mit Butyrat über einen Zeitraum von 6 und 48 h induziert. Die Erfassung der MCT1 Expression erfolgte mittels semiquantitativer PCR. Ergebnisse: MCT1 & 4 sind sowohl in der Zellmembran als auch intrazellulär in den Pansenepithelzellen lokalisiert. Die mRNA Expressionsdaten konnten zeigen, dass MCT1 und die PPARα Zielgene durch WY 14.643 hochreguliert werden konnten, wohingegen die MCT4 Expression keine eindeutige Antwort auf die Stimulation zeigt. Die Behandlung mit den Antagonisten zeigt eine Abhängigkeit der MCT1 Expression von PPARα, die MCT4 Expression konnte dagegen nicht beeinflusst werden. Mittels pHMB gelang es, den laktatabhängigen Protonenexport fast vollständig zu blocken. Sowohl laktatabhängiger Protonenexport als auch die Proteinexpression zeigten keine Änderung durch WY 14.643 Stimulation. Die Butyratexposition veränderte die Morphologie der Pansenepithelzellen und schien nicht geeignet für Untersuchungen der mRNA Expression zu sein. Schlussfolgerungen: Es konnte in dieser Arbeit erstmals gezeigt werden, dass MCT1 in Pansenepithelzellen über PPARα reguliert wird, nicht aber MCT4. PPARα scheint demnach einer der entscheidenden Angriffspunkte für die Regulation des SCFA Transportes zu sein, dessen natürliche Liganden im Pansen aber noch nicht bekannt sind. Damit legt diese Arbeit den Grundstein für regulative Studien am intakten Pansenepithel. / Introduction: Monocarboxylate transporters (MCT) 1 & 4 are cotransporters of monocarboxylates and protons in a variety of mammalian cell types. In the ruminal epithelium MCT are necessary to transport short-chain fatty acids (SCFA) from the lumen into the ruminal epithelial cell (MCT4) and to discharge SCFA and their metabolites from the cell into the blood (MCT1). Transepithelial permeation of SCFA is of great importance, because they are the main source of energy for ruminants. The regulation of appropriate transport proteins should thus be subject to the adaptation to varying SCFA amounts. Previous studies in other species suggested that gene expression of MCT1 is regulated by peroxisome proliferator-activated receptor α (PPARα), a ligand-activated nuclear receptor. Aims: The aim of the study was to examine if MCT1 in ruminal epithelial cells is regulated by PPARα and furthermore if MCT4 can be regulated by PPARα, as well. A simultaneous regulation seems likely, because both are acting jointly in the transepithelial transporting of SCFA. The implications of such a regulation on protein expression and transport capacity of MCT should be characterized. The effect of butyrate, a SCFA which increases under concentrate feeding, on MCT1 expression was determined. Materials & Methods: Ruminal epithelial cells of sheep were cultivated according to methods previously established. After subcultivation, immunocytochemistry with antibodies against MCT1, MCT4 and Na+/K+-ATPase was performed to determine their localization in ruminal epithelial cells. For studying the influence of PPARα, WY 14.643, a synthetic and selective ligand of PPARα, and GW 6471, a synthetic antagonist of PPARα, were applied to the culture medium of the cells. After processing the specimens, the relative amount of mRNA of MCT1, MCT4 and the target genes ACO, CPT1A and CACT were analyzed by qPCR and normalized on the reference genes GAPDH and Na+/K+-ATPase. Protein abundance of MCT1 & 4 was measured by using the Western Blot method. Functional quantification was measured by the intracellular pH (pHi) of cells using spectrofluorometry as well as comparing the effect of WY 14.643 treatment on lactate-dependent proton export. To determine the MCT-dependent part of the pHi recovery, p-hydroxymercuribenzoic acid (pHMB), a specific inhibitor of MCT1 & 4, was applied. Cells were also treated with butyrate for 6 h and 48 h and the mRNA abundance of MCT1 was analyzed by semiquantitative PCR. Results: Both MCT1 and MCT4 were localized in the cell membrane as well as in the cytoplasm of ruminal epithelial cells. By qPCR it could be demonstrated that the mRNA abundance of MCT1 and PPARα target genes in the ruminal epithelial cells was increased by WY 14.643 in comparison to untreated cells, whereas the response of MCT4 did not yield distinct results. Treatment with the PPARα antagonist pointed out, that MCT1 is influenced by PPARα, but not MCT4. Lactate-dependent proton export was blocked almost completely by pHMB. Both lactate-dependent proton export and protein expression were not altered by WY 14.643 treatment. Butyrate exposure changed the morphology of ruminal epithelial cells and seemed unsuitable for the analysis of mRNA expression. Conclusion: For the first time, it could be demonstrated, that MCT1 in ruminal epithelial cells is regulated by PPARα, but not MCT4. PPARα seems to be a vital target in the rumen for SCFA transport regulation, whose natural triggers have yet to be identified. Furthermore, this study provides the basis for regulative studies on intact ruminal epithelium.

Einleitung: Monocarboxylattransporter (MCT) 1 & 4 sind in zahlreichen Geweben als Kotransporter für Monocarboxylate und Protonen beschrieben. Auch im Pansenepithel werden MCT benötigt, um kurzkettige Fettsäuren (SCFA) aus dem Pansenlumen in die Pansenepithelzelle aufzunehmen (MCT4) und um SCFA und deren Metabolite aus der Pansenepithelzelle in das Blut auszuschleusen (MCT1). Die transepitheliale Permeation von SCFA über die Pansenwand ist von enormer Bedeutung, da sie die wichtigste Energiequelle der Wiederkäuer darstellen. Die beteiligten Transportprozesse müssen dementsprechend einer Anpassung an variierende Mengen von SCFA unterliegen. Bisherige Studien bei anderen Spezies deuten auf eine Regulation des MCT1 auf mRNA Ebene über den Peroxisom-Proliferator-aktivierten Rezeptor α (PPARα) hin. Ziele der Untersuchung: Das Ziel dieser Arbeit war herauszufinden, ob MCT1 in ovinen Pansenepithelzellen über PPARα reguliert wird und ob auch MCT4 dieser Regulation unterliegt. Eine gleichzeitige Regulation beider Transporter läge nahe, da sie gemeinsam an der transepithelialen Permeation beteiligt sind. Die Auswirkungen solch einer Regulation auf die Proteinexpression und die Transportleistung der MCT sollte charakterisiert werden. Ebenfalls war das Potenzial der bei erhöhter Kraftfutterfütterung vermehrt anfallenden SCFA Butyrat auf die MCT1 Expression zu untersuchen. Material & Methoden: Aus dem Vorhof von Schafen wurden Pansenepithelzellen gewonnen und entsprechend einer bereits etablierten Methode kultiviert. Nach einer Subkultivierung wurden die Zellen immunzytochemisch mit Antikörpern gegen MCT1, MCT4 und Na+/K+-ATPase untersucht, um deren Lokalisation in den kultivierten Pansenepithelzellen zu bestimmen. Weiterhin erfolgte eine Behandlung mit WY 14.643, einem spezifischen, synthetischen PPARα Agonisten, sowie mit GW 6471, einem Antagonisten des PPARα. Mittels qPCR wurden die relativen mRNA Mengen von MCT1, MCT4, ACO, CPT1A und CACT bestimmt und auf die Referenzgene GAPDH und Na+/K+-ATPase normalisiert. Die Proteinexpression von MCT1 und MCT4 wurde mittels Western Blot bestimmt. Zur funktionellen Quantifizierung wurde der intrazelluläre pH-Wert der Zellen mittels Spektrofluorometrie gemessen und der laktatabhängige Protonentransport als Vergleichswert zwischen den Behandlungen genutzt. Um den MCT-abhängigen Teil des Transportes zu bestimmen, wurde ein spezifischer MCT1 & 4 Inhibitor, die p-Hydroxymercuribenzensulfonsäure (pHMB) eingesetzt. Die Zellen wurden mit Butyrat über einen Zeitraum von 6 und 48 h induziert. Die Erfassung der MCT1 Expression erfolgte mittels semiquantitativer PCR. Ergebnisse: MCT1 & 4 sind sowohl in der Zellmembran als auch intrazellulär in den Pansenepithelzellen lokalisiert. Die mRNA Expressionsdaten konnten zeigen, dass MCT1 und die PPARα Zielgene durch WY 14.643 hochreguliert werden konnten, wohingegen die MCT4 Expression keine eindeutige Antwort auf die Stimulation zeigt. Die Behandlung mit den Antagonisten zeigt eine Abhängigkeit der MCT1 Expression von PPARα, die MCT4 Expression konnte dagegen nicht beeinflusst werden. Mittels pHMB gelang es, den laktatabhängigen Protonenexport fast vollständig zu blocken. Sowohl laktatabhängiger Protonenexport als auch die Proteinexpression zeigten keine Änderung durch WY 14.643 Stimulation. Die Butyratexposition veränderte die Morphologie der Pansenepithelzellen und schien nicht geeignet für Untersuchungen der mRNA Expression zu sein. Schlussfolgerungen: Es konnte in dieser Arbeit erstmals gezeigt werden, dass MCT1 in Pansenepithelzellen über PPARα reguliert wird, nicht aber MCT4. PPARα scheint demnach einer der entscheidenden Angriffspunkte für die Regulation des SCFA Transportes zu sein, dessen natürliche Liganden im Pansen aber noch nicht bekannt sind. Damit legt diese Arbeit den Grundstein für regulative Studien am intakten Pansenepithel. / Introduction: Monocarboxylate transporters (MCT) 1 & 4 are cotransporters of monocarboxylates and protons in a variety of mammalian cell types. In the ruminal epithelium MCT are necessary to transport short-chain fatty acids (SCFA) from the lumen into the ruminal epithelial cell (MCT4) and to discharge SCFA and their metabolites from the cell into the blood (MCT1). Transepithelial permeation of SCFA is of great importance, because they are the main source of energy for ruminants. The regulation of appropriate transport proteins should thus be subject to the adaptation to varying SCFA amounts. Previous studies in other species suggested that gene expression of MCT1 is regulated by peroxisome proliferator-activated receptor α (PPARα), a ligand-activated nuclear receptor. Aims: The aim of the study was to examine if MCT1 in ruminal epithelial cells is regulated by PPARα and furthermore if MCT4 can be regulated by PPARα, as well. A simultaneous regulation seems likely, because both are acting jointly in the transepithelial transporting of SCFA. The implications of such a regulation on protein expression and transport capacity of MCT should be characterized. The effect of butyrate, a SCFA which increases under concentrate feeding, on MCT1 expression was determined. Materials & Methods: Ruminal epithelial cells of sheep were cultivated according to methods previously established. After subcultivation, immunocytochemistry with antibodies against MCT1, MCT4 and Na+/K+-ATPase was performed to determine their localization in ruminal epithelial cells. For studying the influence of PPARα, WY 14.643, a synthetic and selective ligand of PPARα, and GW 6471, a synthetic antagonist of PPARα, were applied to the culture medium of the cells. After processing the specimens, the relative amount of mRNA of MCT1, MCT4 and the target genes ACO, CPT1A and CACT were analyzed by qPCR and normalized on the reference genes GAPDH and Na+/K+-ATPase. Protein abundance of MCT1 & 4 was measured by using the Western Blot method. Functional quantification was measured by the intracellular pH (pHi) of cells using spectrofluorometry as well as comparing the effect of WY 14.643 treatment on lactate-dependent proton export. To determine the MCT-dependent part of the pHi recovery, p-hydroxymercuribenzoic acid (pHMB), a specific inhibitor of MCT1 & 4, was applied. Cells were also treated with butyrate for 6 h and 48 h and the mRNA abundance of MCT1 was analyzed by semiquantitative PCR. Results: Both MCT1 and MCT4 were localized in the cell membrane as well as in the cytoplasm of ruminal epithelial cells. By qPCR it could be demonstrated that the mRNA abundance of MCT1 and PPARα target genes in the ruminal epithelial cells was increased by WY 14.643 in comparison to untreated cells, whereas the response of MCT4 did not yield distinct results. Treatment with the PPARα antagonist pointed out, that MCT1 is influenced by PPARα, but not MCT4. Lactate-dependent proton export was blocked almost completely by pHMB. Both lactate-dependent proton export and protein expression were not altered by WY 14.643 treatment. Butyrate exposure changed the morphology of ruminal epithelial cells and seemed unsuitable for the analysis of mRNA expression. Conclusion: For the first time, it could be demonstrated, that MCT1 in ruminal epithelial cells is regulated by PPARα, but not MCT4. PPARα seems to be a vital target in the rumen for SCFA transport regulation, whose natural triggers have yet to be identified. Furthermore, this study provides the basis for regulative studies on intact ruminal epithelium.

No description available.

No description available.