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<p>Selective therapeutic targeting of tumors requires identification of differences between the homeostatic requirements of cancer and host cells. One such difference is the manner in which cancer cells acquire energy. Cancer cells often grow in an environment of local hypoxia; under these conditions tumor cells depend on glycolysis for energy, but are unable to perform oxidative phosphorylation. Many tumor cells, despite normoxic conditions, continue to perform glycolysis without oxidative phosphorylation. The net result of glycolysis without oxidative phosphorylation is twofold: the need to consume a greater amount of glucose than a non-cancerous host cell, and the burden of increased intracellular lactic acid. The proteins responsible for the transport of lactic acid in and out of cells are known as the monocarboxylate transporters (MCTs). Monocarboxylate Transporter 1 (MCT1) and Monocarboxylate Transporter 4 (MCT4) are the MCTs that play a major role in the transport of lactic acid. Tumor cells depend on MCT1 and MCT4 activity to excrete excess intracellular lactic acid to maintain neutral intracellular pH and homeostasis. Using human neuroblastoma and prostate cancer cell lines this work demonstrates that tumor cells can be selectively targeted tumor under conditions of hypoxia or acidosis in vitro with the drug lonidamine, with a small molecule inhibitor selective for MCT1, or with RNA interference of MCT1. Inhibition of MCT1 activity in neuroblastoma cells under acidic extracellular conditions results in intracellular acidification and cell death. MCT1 mRNA is expressed in human neuroblastoma and positively correlated with clinical risk profile. Inhibition of MCT1 activity in hypoxic prostate cancer cells results in a reduction of lactate excretion, decreased intracellular pH, inhibition of ATP production, and subsequent cell death. MCT1 expression in sections of human prostate tumors has been demonstrated to validate MCT1 as a target in prostate cancer.</p> <p>Through the Pasteur and Warburg effects, tumors have an increased demand for glucose. Some cancers store glycogen, but the reasons for this are largely unknown. It is hypothesized that tumor glycogen is used to promote tumor survival during transient hypoxia or low glucose, and that the mechanisms by which glycogen is stored is a potential therapeutic target in cancer. Tumors from human cell lines (WiDr, PC3, FaDu) have been grown in nude mice, sectioned and stained to measure glycogen storage. Using consecutive frozen sections, levels of hypoxia, glucose, lactate, ATP, and CD31, an endothelial cell marker, have been determined. These sections have been employed to elucidate the "architecture" of tumor metabolism in terms of vessel distance. Additionally, PAS-stained EF5 labeled human tumor samples were used to obtain calibrated hypoxia measurements to correlate with PAS. These studies demonstrate a correlation between hypoxia and the formation of glycogen deposits in human tumors and nude mouse xenografts. In cell culture, formation of glycogen deposits after exposure to hypoxia has been demonstrated, in addition to expression of glycogen synthase in human cancer cell lines.</p> <p>The development of novel selective cancer chemotherapeutics will require the identification of differences between cancerous cells and normal host cells to exploit as targets. Several differences in metabolism, including the need to excrete excess lactic acid and store glycogen under hypoxic conditions, are such targets. Novel therapeutics exploiting these targets should be effective against cancer cells and minimally toxic to host cells.</p> / Dissertation

Submitted by INAÊ CRISTINA REGATIERI null (iregatieri@hotmail.com) on 2016-10-25T11:32:21Z No. of bitstreams: 1 Tese_Inae_Cristina_Regatieri.pdf: 1026482 bytes, checksum: 93ce299c664eb44473c4cdf6c6496fb4 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-10-31T17:37:43Z (GMT) No. of bitstreams: 1 regatieri_ic_dr_jabo.pdf: 1026482 bytes, checksum: 93ce299c664eb44473c4cdf6c6496fb4 (MD5) / Made available in DSpace on 2016-10-31T17:37:43Z (GMT). No. of bitstreams: 1 regatieri_ic_dr_jabo.pdf: 1026482 bytes, checksum: 93ce299c664eb44473c4cdf6c6496fb4 (MD5) Previous issue date: 2016-10-19 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O transportador de monocarboxilato isoforma 1 (MCT1), presente na membrana das hemácias, e sua proteína auxiliar CD147 têm como função transportar H+ e lactato do plasma para dentro das hemácias, mantendo assim, a homeostase ácido-base e retardando a acidose sistêmica e fadiga muscular. Dessa forma, o objetivo desse estudo foi comparar as quantidades das proteínas MCT1 e CD147 em hemácias de cavalos Árabes e Quartos de Milha com diferentes níveis de desempenho atlético. Além disso, objetivou-se buscar por polimorfismos para os genes MCT1, CD147, DMRT3 e PDK4, a fim de checar associações entre os polimorfismos e o desempenho nas raças. Cavalos Árabes e Quartos de Milha foram divididos em dois grupos de acordo com o desempenho em provas de enduro e provas de corridas, respectivamente. A quantidade de MCT1 e CD147 na membrana plasmática das hemácias foi determinada por western blotting com unidades arbitrárias de densidade óptica (OD) e anticorpos reagentes à espécie humana anti-MCT1 e anti-CD147. Os dados para as quantidades de proteínas foram analisados pelo PROC MIXED do SAS. O modelo incluiu a idade como covariável e os efeitos fixos de sexo, raça e grupo de desempenho dentro de raça. As correlações foram analisadas pelo teste de Pearson pelo procedimento PROC CORR. P-valores <0,01 foram considerados estatisticamente significantes. Os polimorfismos dos genes foram analisados por sequenciamento (MCT1 e CD147), PCR-RFLP (DMRT3) e ARMS-PCR (PDK4). Os pacotes estatísticos Genetics, Lattice e GenABEL foram utilizados para comparar as frequências dos grupos de desempenho no software R, com o teste exato de Fisher a 5% de significância. As proteínas MCT1 e CD147 foram encontradas nas hemácias de todos os animais. A quantidade de MCT1 foi significativamente (p<0,0001) maior em Quartos de Milha (2,99 ± 0,35 OD) do que em Árabes (1,04 ± 0,08 OD). Quartos de Milha (3,23 ± 0,38 OD) também apresentaram maior conteúdo de CD147 do que Árabes (0,88 ± 0,06 OD). Não houve diferença estatística nas quantidades de proteínas para os grupos de desempenho de ambas as raças. Correlação positiva foi encontrada entre as quantidades de MCT1 e CD147 (r=0,95; p<0,0001). O Alelo A dos polimorfismos Lys457Gln:1573A>C do gene MCT1 e Ile51Val:168A>G do gene CD147 estavam fixados em ambas as raças. Um novo polimorfismo (AY457175.1:c1498G>A) foi encontrado na sequência do gene MCT1. Para o DMRT3, todos os animais apresentaram o alelo C fixado para o polimorfismo. Árabes mostraram maior frequência para o alelo G do que Quartos de Milha (p<0,01) para o polimorfismo no gene PDK4. Entretanto, não houve diferença entre os grupos de desempenho para as duas raças. Dessa forma, conclui-se que Quartos de Milha têm maiores quantidades de MCT1 e CD147 do que Árabes. Não foi possível determinar a influência dos polimorfismos nos genes MCT1, CD147 e DMRT3 no desempenho atlético das duas raças visto que seus alelos estavam fixados. Além disso, houve diferença significativa nas frequências do polimorfismo no gene PDK4 entre Árabes e Quartos de Milha, mas não houve diferença entre os grupos de desempenho. / Monocarboxylate transporter isoform 1 (MCT1), present in the red blood cell membranes and its ancillary protein CD147 have as function transport H+ and lactate ions from the plasma into the red blood cells, thereby maintaining acid/base homeostasis and retarding systemic acidosis and muscular fatigue. Thereby, the aim of this study was to compare the amount of MCT1 and CD147 proteins in the red blood cells of Arabian and Quarter Horses with different levels of athletic ability. Furthermore, we investigated polymorphisms for MCT1, CD147, DMRT3, and PDK4 genes in Arabian and Quarter Horses in order to check associations between the polymorphisms and the performance in these breeds. Arabian horses were divided into two groups according to their performance in endurance competition and Quarter Horses were separated by its performance in races, determined by Speed Index. The amount of MCT1 and CD147 proteins in the plasma membrane of red blood cells was determined by western blotting analysis with arbitrary optical density units (OD), using a human specific anti-MCT1 and anti- CD147 antibody. Data for the amounts of proteins were analyzed using the PROC MIXED procedure of SAS software. The model for the analysis included the effects of sex, breed and performance group within breed as fixed effect and age as covariate. The correlations were analyzed by Pearson correlation test using the PROC CORR procedure of SAS software. P values <0.01 were considered statistically significant. Polymorphisms of the genes were analyzed by sequencing (MCT1 and CD147), PCR-RFLP (DMRT3) and ARMS-PCR (PDK4) techniques. The statistical packages Genetics, Lattice and GenABEL were used to compare the frequencies of the groups using the software R, with the Fisher's exact test being performed with significance level of 5%. MCT1 and CD147 proteins were found in the red blood cell membranes of all studied animals. The amount of MCT1 was significantly (p<0.0001) higher in Quarter Horses (2.99 ± 0.35 OD) than in Arabians (1.04 ± 0.08 OD). Quarter Horses (3.23 ± 0.38 OD) also showed bigger contents of CD147 than Arabians (0.88 ± 0.06 OD). There was not statistical difference in the amounts of MCT1 and CD147 between the performance groups of both breeds. Positive correlation was found between the amounts of MCT1 and CD147 (r=0.95; p<0.0001). The A allele from the polymorphisms Lys457Gln:1573A>C of MCT1 and Ile51Val:168A>G of CD147 gene, were fixed in both breeds. A new polymorphism (AY457175.1:c1498G>A) was found in the MCT1 gene sequence. For DMRT3 mutation, all the animals shown to have the C allele fixed for the polymorphism. Arabians showed significant greater frequency of the G allele than Quarter Horses (p<0.01) for the PDK4 polymorphism. However, there was not difference between the groups of performance for both breeds. In summary, it follows that the Quarter Horses have greater amount of MCT1 and CD147 proteins than Arabian. It was not possible to determine the influence of polymorphisms in MCT1, CD147 and DMRT3 genes in the athletic performance of these breeds since they had alleles fixed. There was a significant difference in the frequencies of the PDK4 polymorphism between Arabians and Quarter Horses, but there was not difference between the performance groups. / FAPESP: 2012/24193-0 / FAPESP: 2012/20697-9

Proton-coupled and sodium-dependent monocarboxylate transporters are encoded by the SLC16A and SLC5A gene family of solute carriers, and are responsible for the transport of essential nutrients such as L-lactate, pyruvate, and ketone bodies. Basigin, or CD147, acts as an ancillary protein for MCT1 and MCT4, and is involved in membrane surface expression of transporters. MCT's are also involved in the shuttling of monocarboxylic xenobiotics across cell membranes, including the drugs valproate and gamma hydroxybutyrate. MCT’s are also important for normal mammalian development, particularly during embryogenesis and early neonatal life. Previous studies have shown that ketogenic diets increase MCT expression in the brain, and the obesity biomarker leptin increases MCT1 and CD147 expression and colocalization in colonocytes. Clinical studies in post-mortem tissue demonstrated that hepatic MCT1 expression changes nonlinearly from birth to adulthood. We hypothesize that age and high fat dietary intake regulate monocarboxylate transporter and ancillary protein expression in the liver, and other organs of drug disposition during childhood obesity. The purpose of this study was to elucidate just how diet and ontogeny regulate MCT1, MCT4, CD147, and SMCT1 mRNA and protein expression in the liver, kidney, and ileum. Timed-pregnant rats were fed either normal or high fat diet, and tissue was harvested from the progeny of both cohorts at predetermined postnatal timepoints. Serum leptin levels were measured, and MCT1, MCT4, CD147, and SMCT1 transcripts were evaluated using real time quantitative PCR. Whole cell and total membrane proteins were extracted and transporter expression was analyzed via western blot. In summary, we have demonstrated age, diet, and sex dependent regulation of MCT1, MCT4, CD147, and SMCT1 expression in the liver, kidneys, and intestine, and that these effects are tissue specific. Pediatric drug-dosing is both a pressing and understudied clinical field, with the possibility of altered pharmacokinetics in obese children. Changes in hepatic, renal, and intestinal monocarboxylate transporter expressions during mammalian development may affect functional activity of these transporters and lead to altered metabolism and drug disposition. Further studies of this animal model can shine new light on the dynamic and highly-variable nature of drug pharmacokinetics in pediatric obesity.

Proton- and sodium-dependent monocarboxylate transporters (MCTs (SLC16A) and SMCTs (SLC5A)) transport monocarboxylates such as ketone bodies, lactate and pyruvate, as well as drugs such as gamma-hydroxybutyric acid. CD147 acts as an ancillary protein for MCT1 and MCT4, and is involved in membrane trafficking. Previously, it has been shown that MCT expression changes under different sex hormone conditions in skeletal muscle and Sertoli cells. However, it is unknown if MCTs, SMCTs or CD147 demonstrate sex differences in tissues where they play an important role in drug disposition. Monocarboxylate transporter substrates GHB and valproic acid have demonstrated sex differences in pharmacokinetic profiles. We hypothesize that sex hormones regulate monocarboxylate transporters and CD147 expression in drug disposition tissues, including the liver, intestine and kidney. The purpose of the current study is to evaluate sex and sex hormone dependent regulation of MCT1, MCT4, SMCT1 and CD147 mRNA and protein expression in drug disposition tissues. Liver, kidney and intestinal segments (duodenum, jejunum and ileum) were harvested from estrus cycle staged female rats, ovariectomized (OVX) females, males and castrated (CST) male rats. Hormone replacement experiments were performed to investigate testosterone and 17β-estradiol dependent regulation of renal MCTs, SMCT1 and CD147 in OVX females and CST males. mRNA of MCT1, MCT4, SMCT1 and CD147 was evaluated by real time quantitative PCR. Whole cell protein and membrane protein was extracted, target protein expression was evaluated by western blot. We have demonstrated sex and sex hormone dependent regulation of MCT1, MCT4, SMCT1 and CD147 in the liver, intestine regions and kidney occurs in a tissue specific manner. mRNA, protein expression and membrane localization of monocarboxylate transporters and CD147 were regulated differently by sex hormones. Sex differences in MCTs and SMCTs expression are important determinants of drug disposition in the body and sex differences in their regulation may contribute to differences in drug pharmacokinetics.

O objetivo desse trabalho foi avaliar a expressão de genes envolvidos no transporte de monocarboxilatos (Mct1, Mct2, Mct3 e Mct4) e de genes específicos da oogênese (Bmp15, Gdf9 e Has2) em complexos cumulus-oócito (CCOs) selecionados pelo teste BCB. Após seleção morfológica com base no grau de compactação das células do cumulus (CCs) e no grau de homogeneidade do citoplasma, os CCOs foram corados com 26 μM BCB (azul cresil brilhante) por 90 min e divididos em dois grupos: BCB+, que apresentavam o ooplasma corado de azul, e BCB-, com ooplasma não corado. Foram utilizados dois grupos controles não expostos ao BCB: o grupo holding foi submetido às mesmas condições que os grupos corados e o outro grupo controle foi diretamente submetido à maturação in vitro (MIV), após a seleção morfológica dos CCOs. A expressão gênica relativa foi determinada por RT-PCR em CCOs coletados antes e ao final da maturação. A expressão também foi avaliada, separadamente, em oócitos desnudos (ODs) e células do cumulus (CCs) antes e após a maturação. A análise dos transcritos demonstrou que houve aumento significativo (p < 0,05) na expressão relativa de Gdf9 e Bmp15 nos grupos BCB+, BCB- e holding antes da MIV, enquanto Has2 teve aumento significativo (p < 0,01) após a MIV apenas no grupo controle. Os outros genes analisados (Mct1, Mct2 e Mct4) mantiveram-se estáveis durante a maturação. O aumento na abundância relativa de alguns transcritos durante a MIV pode ser atribuído as condições de incubações durante o teste BCB. Nossos resultados demonstraram, pela primeira vez, a expressão de Mct1, 2 e 4 em CCOs bovinos. Enquanto o mRNA de Mct1 e Mct4 estava presente em ODs e em CC, o Mct2 foi detectado somente em CCs. Não detectamos a expressão de transcritos de Mct3 em CCOs. As diferenças na expressão dessas três isoformas sugerem um papel único para esses transportadores durante a maturação. / The aim of this study was to determine the relative expression of genes involved in transport of monocarboxylates (Mct1, Mct2, Mct3 e Mct4) and oogenesis specific genes (Bmp15, Gdf9 and Has2) in immature and mature bovine cumulus-oocyte complexes (COC) after selection by BCB. Immature COCs underwent morphological selection and were stained with 26 mM BCB for 90 min. Based on ooplasm staining, oocytes were distributed in two groups BCB+ (blue color) and BCB- (non-stained). The holding control group was exposed to the same incubation conditions as stained COCs, but without BCB. Control group was submitted to in vitro maturation (IVM) immediately after morphological selection. mRNA expression was investigated by RT-PCR in COCs before and after IVM. No relationship was observed in the relative expression of Has2, Gdf9, Bmp15 or Mct1, 2 and 4 transcripts between BCB- and BCB+ COCs. Transcripts analysis showed that Gdf9 and Bmp15 in BCB+, BCB- and holding groups were upregulated (p < 0.05) before IVM, while Has2 was up-regulated (p < 0.01) after IVM in the control group. Others genes remained stable during maturation (Mct1, 2 and 4). The increase in relative abundance of some transcripts during IVM may be attributed to incubation conditions during the BCB test. Our results showed, for the first time, Mct1, 2 and 4 expression in bovine COCs. Mct1 and Mct4 transcripts were present in denuded oocytes and cumulus cell, while Mct2 was detected only in cumulus cells. These differences between the three isoforms in localization suggest unique roles for each in monocarboxylate transport during maturation.

Although it was once thought that neurons solely rely on glucose as a substrate for cellular energy production, it is now known that small monocarboxylate molecules, like pyruvate, lactate, and ketone bodies, are also utilized. Monocarboxylates are transported across plasma membranes via facilitated diffusion using a family of transport proteins known as monocarboxylate transporters (MCTs). Four MCTs (MCT1, MCT2, MCT3, and MCT4) are expressed within neural tissues. Expression of the MCTs has been tied to co-expression of a cell adhesion molecule belonging to the Basigin subset of the immunoglobulin superfamily (IgSF). Basigin gene products are known to interact with MCT1 and MCT4 in the mammalian neural retina and this association is essential to support the cellular energy needs of photoreceptors. A previous study indicated that Basigin gene products use hydrophobic amino acids within specific regions of the transmembrane domain to interact with MCT1. In the present study, it is hypothesized that the same amino acids within the transmembrane domain are used to interact with MCT4, but that no association exists with MCT2, which typically interacts with a different member of the IgSF subset. Therefore, the purpose of the present study was to assess the association between Basigin gene products and MCT4, and with MCT2. Recombinant proteins corresponding to the transmembrane domain of Basigin gene products were used in in vitro binding assays with endogenous MCT2 and MCT4 from mouse brain protein lysates. Contrary to the hypothesis, it was determined that the transmembrane domain of Basigin gene products binds to both MCT2 and MCT4 in vitro. Different amino acids within the transmembrane domain of Basigin gene products are used for each association and the pattern is different from that used in the association with MCT1. The data suggest that Basigin plays multiple roles in the nervous system.

O objetivo desse trabalho foi avaliar a expressão de genes envolvidos no transporte de monocarboxilatos (Mct1, Mct2, Mct3 e Mct4) e de genes específicos da oogênese (Bmp15, Gdf9 e Has2) em complexos cumulus-oócito (CCOs) selecionados pelo teste BCB. Após seleção morfológica com base no grau de compactação das células do cumulus (CCs) e no grau de homogeneidade do citoplasma, os CCOs foram corados com 26 μM BCB (azul cresil brilhante) por 90 min e divididos em dois grupos: BCB+, que apresentavam o ooplasma corado de azul, e BCB-, com ooplasma não corado. Foram utilizados dois grupos controles não expostos ao BCB: o grupo holding foi submetido às mesmas condições que os grupos corados e o outro grupo controle foi diretamente submetido à maturação in vitro (MIV), após a seleção morfológica dos CCOs. A expressão gênica relativa foi determinada por RT-PCR em CCOs coletados antes e ao final da maturação. A expressão também foi avaliada, separadamente, em oócitos desnudos (ODs) e células do cumulus (CCs) antes e após a maturação. A análise dos transcritos demonstrou que houve aumento significativo (p < 0,05) na expressão relativa de Gdf9 e Bmp15 nos grupos BCB+, BCB- e holding antes da MIV, enquanto Has2 teve aumento significativo (p < 0,01) após a MIV apenas no grupo controle. Os outros genes analisados (Mct1, Mct2 e Mct4) mantiveram-se estáveis durante a maturação. O aumento na abundância relativa de alguns transcritos durante a MIV pode ser atribuído as condições de incubações durante o teste BCB. Nossos resultados demonstraram, pela primeira vez, a expressão de Mct1, 2 e 4 em CCOs bovinos. Enquanto o mRNA de Mct1 e Mct4 estava presente em ODs e em CC, o Mct2 foi detectado somente em CCs. Não detectamos a expressão de transcritos de Mct3 em CCOs. As diferenças na expressão dessas três isoformas sugerem um papel único para esses transportadores durante a maturação. / The aim of this study was to determine the relative expression of genes involved in transport of monocarboxylates (Mct1, Mct2, Mct3 e Mct4) and oogenesis specific genes (Bmp15, Gdf9 and Has2) in immature and mature bovine cumulus-oocyte complexes (COC) after selection by BCB. Immature COCs underwent morphological selection and were stained with 26 mM BCB for 90 min. Based on ooplasm staining, oocytes were distributed in two groups BCB+ (blue color) and BCB- (non-stained). The holding control group was exposed to the same incubation conditions as stained COCs, but without BCB. Control group was submitted to in vitro maturation (IVM) immediately after morphological selection. mRNA expression was investigated by RT-PCR in COCs before and after IVM. No relationship was observed in the relative expression of Has2, Gdf9, Bmp15 or Mct1, 2 and 4 transcripts between BCB- and BCB+ COCs. Transcripts analysis showed that Gdf9 and Bmp15 in BCB+, BCB- and holding groups were upregulated (p < 0.05) before IVM, while Has2 was up-regulated (p < 0.01) after IVM in the control group. Others genes remained stable during maturation (Mct1, 2 and 4). The increase in relative abundance of some transcripts during IVM may be attributed to incubation conditions during the BCB test. Our results showed, for the first time, Mct1, 2 and 4 expression in bovine COCs. Mct1 and Mct4 transcripts were present in denuded oocytes and cumulus cell, while Mct2 was detected only in cumulus cells. These differences between the three isoforms in localization suggest unique roles for each in monocarboxylate transport during maturation.

O objetivo desse trabalho foi avaliar a expressão de genes envolvidos no transporte de monocarboxilatos (Mct1, Mct2, Mct3 e Mct4) e de genes específicos da oogênese (Bmp15, Gdf9 e Has2) em complexos cumulus-oócito (CCOs) selecionados pelo teste BCB. Após seleção morfológica com base no grau de compactação das células do cumulus (CCs) e no grau de homogeneidade do citoplasma, os CCOs foram corados com 26 μM BCB (azul cresil brilhante) por 90 min e divididos em dois grupos: BCB+, que apresentavam o ooplasma corado de azul, e BCB-, com ooplasma não corado. Foram utilizados dois grupos controles não expostos ao BCB: o grupo holding foi submetido às mesmas condições que os grupos corados e o outro grupo controle foi diretamente submetido à maturação in vitro (MIV), após a seleção morfológica dos CCOs. A expressão gênica relativa foi determinada por RT-PCR em CCOs coletados antes e ao final da maturação. A expressão também foi avaliada, separadamente, em oócitos desnudos (ODs) e células do cumulus (CCs) antes e após a maturação. A análise dos transcritos demonstrou que houve aumento significativo (p < 0,05) na expressão relativa de Gdf9 e Bmp15 nos grupos BCB+, BCB- e holding antes da MIV, enquanto Has2 teve aumento significativo (p < 0,01) após a MIV apenas no grupo controle. Os outros genes analisados (Mct1, Mct2 e Mct4) mantiveram-se estáveis durante a maturação. O aumento na abundância relativa de alguns transcritos durante a MIV pode ser atribuído as condições de incubações durante o teste BCB. Nossos resultados demonstraram, pela primeira vez, a expressão de Mct1, 2 e 4 em CCOs bovinos. Enquanto o mRNA de Mct1 e Mct4 estava presente em ODs e em CC, o Mct2 foi detectado somente em CCs. Não detectamos a expressão de transcritos de Mct3 em CCOs. As diferenças na expressão dessas três isoformas sugerem um papel único para esses transportadores durante a maturação. / The aim of this study was to determine the relative expression of genes involved in transport of monocarboxylates (Mct1, Mct2, Mct3 e Mct4) and oogenesis specific genes (Bmp15, Gdf9 and Has2) in immature and mature bovine cumulus-oocyte complexes (COC) after selection by BCB. Immature COCs underwent morphological selection and were stained with 26 mM BCB for 90 min. Based on ooplasm staining, oocytes were distributed in two groups BCB+ (blue color) and BCB- (non-stained). The holding control group was exposed to the same incubation conditions as stained COCs, but without BCB. Control group was submitted to in vitro maturation (IVM) immediately after morphological selection. mRNA expression was investigated by RT-PCR in COCs before and after IVM. No relationship was observed in the relative expression of Has2, Gdf9, Bmp15 or Mct1, 2 and 4 transcripts between BCB- and BCB+ COCs. Transcripts analysis showed that Gdf9 and Bmp15 in BCB+, BCB- and holding groups were upregulated (p < 0.05) before IVM, while Has2 was up-regulated (p < 0.01) after IVM in the control group. Others genes remained stable during maturation (Mct1, 2 and 4). The increase in relative abundance of some transcripts during IVM may be attributed to incubation conditions during the BCB test. Our results showed, for the first time, Mct1, 2 and 4 expression in bovine COCs. Mct1 and Mct4 transcripts were present in denuded oocytes and cumulus cell, while Mct2 was detected only in cumulus cells. These differences between the three isoforms in localization suggest unique roles for each in monocarboxylate transport during maturation.

Einleitung: Kurzkettige Fettsäuren (SCFA) stellen das hauptsächliche Energiesubstrat für Wiederkäuer dar. In Anbetracht des - bedingt durch höhere Milch-, Mast und Reproduktionsleistung - steigenden Energiebedarfs von Hauswiederkäuern wie Milchkuh und Mastbulle ist es von zentraler Bedeutung, die Mechanismen zur Resorption dieser Energielieferanten bzw. Ansatzpunkte für die Beeinflussung dieser Transportprozesse genau zu kennen. Dieses Wissen kann möglicherweise dabei helfen, zukünftig die Energieaufnahme der Tiere zu unterstützen bzw. sogar effizienter zu gestalten. Ziele der Untersuchungen: Deshalb war es Ziel der vorliegenden Arbeit, die Mechanismen zur Resorption von SCFA zu charakterisieren, wobei der Schwerpunkt auf den Transport aus den Pansenepithelzellen ins Blut gelegt wurde, da hierzu im Gegensatz zu ihrer Aufnahme aus dem Pansenlumen in die Epithelzellen noch sehr wenig bekannt war. In einem zweiten Schritt sollte untersucht werden, inwiefern die nachgewiesenen Mechanismen einer Regulation unterliegen und über welche Signalwege diese vermittelt werden könnte. Materialien und Methoden: Zur Charakterisierung der beteiligten Resorptionsmechanismen wurden Epithelstücke aus dem ventralen Pansensack von Schafen in Ussing-Kammern eingespannt und mit Hilfe radioaktiv markierten Azetats, Butyrats und L-Laktats der Transport dieser Substrate unter verschiedenen Bedingungen sowie verschiedenen Hemmstoffeinflüssen untersucht. Zur Charakterisierung regulativer Einflüsse wurden die Epithelstücke über sechs bzw. 24 Stunden mit Butyrat inkubiert und anschließend RNA bzw. Totalprotein extrahiert. Hiermit konnten Veränderungen in mRNA- und Proteinexpression mittels quantitativer Echtzeit-PCR bzw. Western Blot nachgewiesen werden. Ergebnisse: Die Untersuchungen der vorliegenden Arbeit konnten zeigen, dass der Transport von SCFA über die basolaterale Membran des Pansenepithels hauptsächlich proteinvermittelt erfolgt. Eine signifikante Beteiligung lipophiler Diffusion, d.h. ein passiver Transport, kann weitgehend ausgeschlossen werden. Der aktive Transport wies eine bikarbonatabhängige und eine bikarbonatunabhängige Komponente auf. Der Einsatz von Hemmstoffen verschiedener Transportproteine ergab deutliche Hinweise darauf, dass der Monocarboxylattransporter (MCT) 1 eine Rolle beim bikarbonatgekoppelten Transport von Azetat bzw. allgemein unmetabolisierten SCFA spielt. Diese Hinweise wurden untersetzt durch die Beobachtung, dass MCT 1, aber auch der apikal bzw. intrazellulär lokalisierte MCT 4 durch langfristige Inkubation des Epithels mit Butyrat sowohl auf mRNA- als auch auf Proteinebene signifikant erhöht exprimiert wurden, was als Anpassungsreaktion an eine Substratakkumulation interpretiert werden kann. Außerdem wurde auch die mRNA-Expression des Putativen Anionentransporters (PAT) 1 durch Inkubation mit Butyrat erhöht, was für eine Beteiligung auch dieses Transportproteins am SCFA-Transport über das Pansenepithel spricht. Allerdings ist im Gegensatz zu MCT 1 die Lokalisation des PAT 1 in der basolateralen Membran noch fraglich. Die Expressionssteigerung von Zielgenen des Nukleären Faktors ĸB und des Peroxisomenproliferator-aktivierten Rezeptors α sowie des Hypoxie-induzierbaren Faktors selbst deuten weiterhin darauf hin, dass die Steigerung der Transportkapazitäten von MCT 1 und 4 und auch PAT 1 über diese Signalwege vermittelt wird. Schlussfolgerungen: Zusammenfassend konnte in dieser Arbeit erstmals der Transport von SCFA über die basolaterale Membran des Pansenepithels näher charakterisiert werden, sodass es nun möglich ist, zusammen mit den bereits vorliegenden Befunden für die apikale Membran ein komplettes Modell dafür zu erstellen. Auch wurden Erkenntnisse zu regulativen Einflüssen auf diesen Transport gewonnen, die es zukünftig ermöglichen könnten, die Resorption der SCFA aus dem Pansen nutritiv oder eventuell pharmakologisch zu beeinflussen. / Introduction: The main energy source for ruminants are short chain fatty acids (SCFA). Considering the ever increasing energy requirements of cattle due to increasing milk yield and meat production, it is crucial to identify the mechanisms for the resorption of these energy sources as well as possibilities to influence these transport mechanisms. This knowledge could help support the animals’ energy uptake or even making it more efficient. Aim: Thus, the aim of the present study was to characterise mechanisms for the resorption of SCFA focusing on their transport from the epithelial cells into the blood. In particular, since – compared to the research findings on the uptake of SCFA from ruminal lumen into the cells – so far only very little was known regarding this side of the epithelium. In a second step, the study aimed to elucidate whether the mechanisms observed are subject to regulatory processes and which signalling pathways are involved. Materials and methods: To characterise the transport mechanisms involved, epithelial pieces from the ventral sac of ovine rumen were mounted in Ussing chambers. Using radioactively labelled acetate, butyrate and L-lactate, the transport of these substrates was investigated under different conditions and by applying different inhibitors for potential SCFA transport proteins. To characterise regulatory influences, epithelial pieces were incubated with butyrate for six and 24 hours, respectively. Subsequently, total RNA and protein were extracted to detect changes in mRNA and protein expression using quantitative real time PCR and western blot, respectively. Results: The present study could show that transport of SCFA across the basolateral membrane of rumen epithelium is mainly realised by protein-mediated mechanisms. A significant participation of lipophilic diffusion, i.e. a passive transport, can almost entirely be excluded. The active transport could be divided into a bicarbonate-dependent and a bicarbonate-independent part. The experiments with inhibitors of different transport proteins showed clear evidence of an involvement of monocarboxylate transporter (MCT) 1 in the bicarbonate-dependent transport of acetate and non-metabolised SCFA in general. This evidence was supported by the finding that the expression of MCT 1 but also of the apically and intracellularly localised MCT 4 was increased significantly on both mRNA- and protein-level after long-term incubation of the epithelium with butyrate. This can be interpreted as an adaptation to a substrate accumulation. Additionally, butyrate incubation led to an increased mRNA expression of putative anion transporter (PAT) 1, which makes an involvement of this transport protein in SCFA transport across ruminal epithelium likely as well. However, in contrast to MCT 1 the localisation of PAT 1 in the basolateral membrane is still questionable. The increased expression of target genes of nuclear factor ĸB and peroxisome-proliferator activated receptor α as well as of hypoxia inducible factor strongly point to an involvement of these pathways in the increased expression of MCT 1 and 4 as well as PAT 1. Conclusions: In summary, this study could characterise the transport of SCFA across the basolateral membrane of ruminal epithelium in detail for the first time. This enables us to draw a complete model of ruminal SCFA transport. Also, evidence for regulatory influence on this transport processes was found, perhaps making it possible to influence resorption of SCFA from rumen by nutritive or pharmacological means in the future.