Central cellular signaling pathways involved with the regulation of lipid metabolism in the liver: a review

The liver is primarilyresponsible for energy homeostasis and the regulation of lipid, carbohydrate and protein metabolism. Lipid metabolism consists of distributing lipids to peripheral tissues or ensuring their return to the liver to be reprocessed. Additionally, cellular metabolism isregulated by several molecules in different signaling pathways. Lipid homeostasis in the liver is mainly regulated by AKT, AMPK, SREBP, PPAR, and JNK. The PI3K/AKT/mTOR signaling pathway results in the biosynthesis of macromolecules and regulates lipogenesis and the expression of lipogenic genes. AMPK is an energy sensor that regulates metabolism and is activated when stored ATP is depleted, and it is responsible for the suppression of several key lipogenic factors in the liver related to cholesterol and fatty acid synthesis. SREBPs control lipogenic geneexpressionandcholesterol metabolism and actin the nutritional regulation of fatty acids and triglycerides. The continued activation of SREBPs is associated with cellular stress, inflammation and ultimately steatosis. PPARs are intrinsically important regulators of lipid metabolism. These genes are essential tovarious metabolic processes, especially lipid and glucose homeostasis, and can play a role in cell differentiation. JNK signaling is related to insulin resistance and its activation results in decreased mitochondrial activity and fat accumulation. Therefore, the study of cell signaling pathways related to lipid metabolism and liver function may help to identify abnormalities and develop strategies to manage and regulate metabolic disorders and resulting complications.

Insulin promotes the expression of the gluconeogenic rate-limiting enzymes phosphoenolpyruvate carboxykinase (Pepck) and glucose 6-phosphatase (G6pase) through PI3k/Akt/mTOR signaling pathway in goose hepatocytes

To identify what makes insulin have an activating or inhibiting role in gluconeogenesis in goose hepatocytes and whether insulin regulates PEPCK and G6Pase through the PI3k/Akt/mTOR pathway or not, goose primary hepatocytes were isolated and cultured in vitro. After 12h cultured in serum-free medium, hepatocytes were incubated for 24 h in the medium with no addition (control) or with the addition of 50, 100, and 150 nM of insulin, 1000 nM NVP-BEZ235, or co-addition of 150nM insulin and 1000nM NVP-BEZ235. Glucose concentration and PEPCK and G6Pase expression were determined. The results showed that PEPCK and G6Pase mRNA levels and activities were up regulated in the 50, 100, and 150nM insulin treatments, while glucose concentration was not significantly altered (p > 0.05). Compared with the activation role of 150nM insulin alone, the co-treatment with1000nM NVP-BEZ235 and 150nM insulin significantly down regulated PEPCK mRNA level and G6Pase protein activity (p < 0.05). However, there is a different result on mRNA level of G6Pase. In conclusion, G6Pase and PEPCK are up regulated by insulin through PI3k/Akt/mTOR pathway in goose hepatocytes. However, G6Pase mRNA and protein levels may be regulated by insulin through different signaling pathways.

Insulin promotes the expression of the gluconeogenic rate-limiting enzymes phosphoenolpyruvate carboxykinase (Pepck) and glucose 6-phosphatase (G6pase) through PI3k/Akt/mTOR signaling pathway in goose hepatocytes

To identify what makes insulin have an activating or inhibiting role in gluconeogenesis in goose hepatocytes and whether insulin regulates PEPCK and G6Pase through the PI3k/Akt/mTOR pathway or not, goose primary hepatocytes were isolated and cultured in vitro. After 12h cultured in serum-free medium, hepatocytes were incubated for 24 h in the medium with no addition (control) or with the addition of 50, 100, and 150 nM of insulin, 1000 nM NVP-BEZ235, or co-addition of 150nM insulin and 1000nM NVP-BEZ235. Glucose concentration and PEPCK and G6Pase expression were determined. The results showed that PEPCK and G6Pase mRNA levels and activities were up regulated in the 50, 100, and 150nM insulin treatments, while glucose concentration was not significantly altered (p > 0.05). Compared with the activation role of 150nM insulin alone, the co-treatment with1000nM NVP-BEZ235 and 150nM insulin significantly down regulated PEPCK mRNA level and G6Pase protein activity (p < 0.05). However, there is a different result on mRNA level of G6Pase. In conclusion, G6Pase and PEPCK are up regulated by insulin through PI3k/Akt/mTOR pathway in goose hepatocytes. However, G6Pase mRNA and protein levels may be regulated by insulin through different signaling pathways.(AU)

AVALIAÇÃO DA RESPOSTA IN VITRO AO TRATAMENTO COM INIBIDOR DE RECEPTOR TIROSINA QUINASE (PALLADIA ®) E MTORC1 (RAPAMICINA) EM CÉLULAS DE CARCINOMA PROSTÁTICO CANINO

O carcinoma prostático canino possui prognóstico desfavorável e modalidades terapêuticas limitadas, principalmente em casos avançados. Devido à falta de tratamento eficaz para os carcinomas prostáticos caninos, esse estudo objetivou avaliar o efeito do toceranib (inibidor de receptor tirosina quinase) e da rapamicina (inibidor de mTORC1) em células de carcinoma prostático canino. As duas células de cultura primária advindas de carcinoma prostático canino, denominadas PC1 e PC2, apresentaram expressão proteica de VEGFR2 e PDGFR- e ausência da proteína c-KIT, os quais são alvos do fosfato de toceranib. Adicionalmente, após comparação do transcriptoma das células não tratadas e tratadas com fosfato de toceranib, observaram-se alterações em genes relacionados com a via PDGFR, angiogênese e resistência terapêutica. Além disso, realizou-se o tratamento das células PC1 e PC2 com rapamicina (inibidor de mTORC1) após a comparação do transcriptoma das células tumorais com células de próstata normal e identificação de genes envolvidos na via PI3K/AKT/mTOR. O tratamento com rapamicina diminuiu a expressão gênica de mTOR e 4E-BP1 e aumentou de AKT, podendo representar mecanismos de resistência. Após tratamento com fosfato de toceranib, apenas a PC1 reduziu a viabilidade celular, porém após tratamento com rapamicina, as células PC1 e PC2 reduziram a viabilidade celular de maneira dose dependente, havendo resposta biológica em ambas as terapias. Sendo assim, o bloqueio de múltiplos receptores tirosina quinase e inibição de mTOR representam alvos terapêuticos no carcinoma prostático canino, entretanto a resistência terapêutica ainda é desafiadora e, portanto, a medicina de precisão seria uma forma de superar o problema.

Canine prostatic carcinoma has a poor prognosis and limited therapeutic modalities, mainly in advanced cases. The aim of this study was to evaluate the effect of toceranib phosphate (receptor tyrosine kinase inhibitor) and rapamycin (mTORC1 inhibitor) in canine prostatic carcinoma cells due to the lack of efficient therapies. Two primary canine prostate cancer cell lines, PC1 and PC2, had protein expression of VEGFR2 and PDGFR- and absent c-KIT expression, which are target toceranib phosphate targets. Additionally, we compared transcriptome before and after toceranib phosphate treatment and we observed gene alterations related to PDGFR pathway, angiogenesis and therapeutic resistance. Besides, we treated PC1 and PC2 cells with rapamycin after transcriptome analysis comparing canine prostatic cells from normal prostate and neoplastic cells and identification of genes involved in PI3K/AKT/mTOR pathway. Gene expression of mTOR and 4E-BP1 decreased and AKT increased after rapamycin treatment, and could represent a resistency mechanism. A decrease only in PC1 cell viability was observed after toceranib phosphate treatment, however decreased cell viability in a dose-dependent manner was observed in PC1 and PC2 cells after rapamycin treatment. Therefore, we could observe biological response of canine prostatic carcinoma cells to both therapies. Thus, multiple receptor tyrosine kinase inhibition and mTOR inhibition represent target therapies to canine prostatic carcinoma. However, therapeutic resistance still remains a challenge and precision medicine could be a way to overcome this problem.

Change of the mtor pathway in tissues of overfed geese

This study aimed at examining the effect of overfeeding on the activity of the mTOR pathway in the liver and muscle tissues of Gang geese. Eighty healthy male Gang geese were reared under the same feeding conditions, and were divided at 14 weeks of age into a control group and an overfed group. All birds were slaughtered after three weeks of over feeding. Gene expression and protein content of several genes involved in the mTOR pathway were evaluated. The results showed that the gene expression of mTOR, raptor, and rictor, and the protein contents of mTOR and PI3K were higher in liver, breast muscle, and leg muscle of the overfed group than that of control group. However, the S6K expression level was clearly lower in the liver of the overfed group than that of control group, and there was no evident difference in both breast muscle and leg muscle between the control group and the overfed group. These results suggest that overfeeding induces the activity of raptor, rictor, and mTOR, and that mTOR signaling pathway was closely linked with PI3K pathway in the evaluated geese.(AU)

Change of the mtor pathway in tissues of overfed geese

This study aimed at examining the effect of overfeeding on the activity of the mTOR pathway in the liver and muscle tissues of Gang geese. Eighty healthy male Gang geese were reared under the same feeding conditions, and were divided at 14 weeks of age into a control group and an overfed group. All birds were slaughtered after three weeks of over feeding. Gene expression and protein content of several genes involved in the mTOR pathway were evaluated. The results showed that the gene expression of mTOR, raptor, and rictor, and the protein contents of mTOR and PI3K were higher in liver, breast muscle, and leg muscle of the overfed group than that of control group. However, the S6K expression level was clearly lower in the liver of the overfed group than that of control group, and there was no evident difference in both breast muscle and leg muscle between the control group and the overfed group. These results suggest that overfeeding induces the activity of raptor, rictor, and mTOR, and that mTOR signaling pathway was closely linked with PI3K pathway in the evaluated geese.

EFEITOS DA RAPAMICINA SOBRE CÉLULAS DE TUMORES DE MAMA CANINO PRIMÁRIO E METASTÁTICO CULTIVADAS IN VITRO

As neoplasias estão se tornando cada vez mais comuns entre os animas domésticos, seja devido a maior expectativa de vida ou pelo avanço dos métodos diagnósticos e tratamentos. As neoplasias mamárias são o tipo mais comum de neoplasias entre as fêmeas caninas. A procura por novos tratamentos e/ou tratamentos específicos para os animais é cada vez mais comum frente à esse quadro. A rapamicina é um fármaco antifúngico com atividade antitumoral, que atua inibindo o complexo mTOR, já testada para o tratamento de neoplasias em diferentes órgãos em humanos, podendo ser uma alternativa no tratamento de neoplasias em cães. Devido ao grande número de repetições que os ensaios com fármacos necessitam, o cultivo celular in vitro é uma alternativa inicial para a descoberta de novos medicamentos eficazes no tratamento dessa espécie, além de também se tornar um ensaio pré-clínico. O objetivo desse estudo foi avaliar o efeito da rapamicina em células de tumores mamários primários e suas respectivas metástases de cadelas, cultivadas in vitro. Foram utilizados dois tipos histológicos tumores de mama, provenientes do tumor primário e suas respectivas metástases. Após a colheita, as amostras foram classificadas por histopatologia como carcinoma sólido grau III e carcinoma adenoescamoso, como tumores triplo negativos não basal pela imuno-histoquímica e tanto os tumores primários como suas metástases apresentaram expressão positiva para as proteínas PTEN, AKT, mTOR e 4EBP1 na imunofluorescência. Parte do tecido tumoral foi cultivado in vitro e as células foram avaliadas quanto a morfologia e fenótipo celular, expressão gênica (qPCR) e viabilidade celular (MTT). Os genes validados pela qPCR foram AKT, mTOR e PTEN, relacionados com a via AKT-mTOR. A expressão do gene PTEN estava diminuída e do mTOR aumentada nas células de tumores primários, quando comparadas as suas metastases. O tratamento com rapamicina e ensaio MTT foram realizados nas concentrações de 2, 5, 10 e 12 M, por 24, 48 e 72 h. O ensaio de MTT mostrou que para uma diminuição de 50% da viabilidade celular, a concentração de rapamicina deve ser 2 M para o carcinoma adenoescamoso e 10 M para metástase. Para o carcinoma sólido grau III e sua respectiva metástase, a concentração utilizada foi de 12 M. A rapamicina teve um resultado satisfatório na inibição do crescimento dos dois tipos celulares utilizados nesse estudo.

Neoplasms are becoming increasingly common among animals, whether due to longer life expectancy or new diagnostic methods and treatments. Breast neoplasms are the most common type of neoplasm among canine females. The search for new treatments and / or specific treatments for animals is increasingly common in this context. Rapamycin is an antifungal drug with antitumor activity, which acts to inhibit the mTOR comple. It was already tested for the treatment of neoplasms in different organs in humans and may be an alternative in the treatment of neoplasms in dogs. Due to the large number of replicates required by drug assays, in vitro cell culture is an early alternative for the discovery of new drugs that might be effective in treatment of this species, as well as becoming a preclinical assay. The aim of this study was to evaluate the effect of rapamycin on primary and metastatic mammary dog tumor on in vitro cultured cells. Two histological types of neoplastic cells from the primary tumor and their respective metastases were used. After collection, the samples were classified by histopathology as grade III solid carcinoma and adenosquamous carcinoma, as non-basal triple negative tumors by immunohistochemistry, and both the primary tumors and their metastases presented positive expression for PTEN, AKT, mTOR and 4EBP1 proteins in immunofluorescence. A fragment of the tumor tissue was cultured in vitro and the cells were evaluated for cell morphology and phenotype, gene expression (qPCR) and cell viability (MTT). The genes validated by qPCR were AKT, mTOR and PTEN, related to the AKT-mTOR pathway. PTEN gene expression was decreased and mTOR increased in primary tumor cells when compared to metastatic tumors. Treatment with rapamycin and MTT assay were performed at concentrations of 2, 5, 10 and 12 M for 24, 48 and 72 h. The MTT assay showed that for a 50% decrease in cell viability, the concentration of rapamycin should be 2 M for adenosquamous carcinoma and 10 M for its metastasis. For grade III solid carcinoma and its respective metastasis, the concentration used was 12 M. The treatment with rapamicyn inhibited the cell growth in the two cell types cultured in vitro.

SUPLEMENTAÇÃO DIETÉTICA DE 25-HIDROXICOLECALCIFEROL (25-0HD3) EM DIFERENTES FASES DE CRIAÇÃO NO CRESCIMENTO ÓSSEO E MUSCULAR DE FRANGOS DE CORTE

O experimento teve por objetivo avaliar a inclusão de 25-hidroxicolecalciferol (25-OHD3) em dietas inicias, de crescimento ou abate suplementadas com vitamina D3 sobre o desempenho produtivo, bem-estar, retenção mineral, características de carcaça, parâmetros ósseos e sanguíneos, qualidade da carne, miopatias e expressão gênica do músculo do peito de frangos de corte de 1 a 46 dias de idade. Para isso, foram utilizados 1584 pintos de corte Ross®, machos. O delineamento experimental adotado foi inteiramente casualizado com 4 tratamentos de 9 repetições com 44 aves cada (12,5 aves/m²). Os níveis de suplementação de vitamina D foram fornecidos em concentrações iguais (3,000 UI/kg de ração) em todas as dietas. A dieta controle (DC) continha 3,000 UI D3 por kg de ração, oferecida no período de 1 a 46 dias, enquanto as dietas experimentais continham 3000 UI D3 + 2760 UI de 25-OHD3 por kg de ração, oferecidas em três programas de alimentação: 1 a 21, 1 a 35 e 1 a 46 dias. A inclusão de 25-OHD3 nas rações para frangos de corte em diferentes períodos de criação não alterou (p>0,05) os parâmetros de desempenho produtivo, gait score, rendimento de carcaça e qualidade de carne. Para as medidas ósseas, houve correlação positiva (r=0,291) entre a densitometria óssea e os níveis séricos de 25-OHD3 quando suplementado durante todo o período de criação. Foi observado efeito quadrático (p<0,05) crescente da inclusão de 25-OHD3 a partir da suplementação de 1 a 21 dias sobre os níveis séricos de 25-OHD3, expressão gênica da proteína mTOR e teor de proteína no peito. Enquanto o rendimento do peito aumentou linearmente (p<0,05) de acordo com o período de suplementação. As aves suplementadas com 25-OHD3 por 46 dias apresentaram concentrações circulantes de 25-OHD3 significativamente maiores (p < 0,05) em comparação com a dieta controle e com as suplementações de 21 e 35 dias. Os níveis séricos 25-OHD3 avaliados possuem correlação positiva (r=0,58) com a expressão da via mTOR, que por sua vez, resulta em estimulação da síntese proteica no peito das aves. A avaliação histológica do peito mostrou diminuição (p<0,05) da incorporação de colágeno e gordura nas fibras musculares de acordo com o período de suplementação de 25-OHD3. O programa vitamínico proposto com a suplementação de 25-OHD3 associado a vitamina D3 durante toda a vida produtiva das aves contribuiu positivamente para o aumento dos níveis séricos de vitamina D e expressão da via mTOR, sendo uma estratégia interessante para aumentar o rendimento de peito, apesar de não resultar em benefícios sobre o desempenho produtivo, a estrutura óssea e a qualidade da carne.

The objective of this experiment was to evaluate the inclusion of 25-hydroxycholecalciferol (25-OHD3) in diets, supplemented with vitamin D3, on yield, well-being, mineral retention, carcass characteristics, bone parameters and blood pressure, meat quality, myopathies and gene expression of the breast muscle of broiler chickens from 1 to 46 days of age. For this, 1584 Ross® male broilers were used. The experimental design was completely randomized with 4 treatments of 9 replicates with 44 birds each (12.5 birds / m²). Vitamin D supplementation levels were given in equal concentrations (3,000 IU / kg of feed) in all diets. The control diet (DC) contained 3,000 IU D3 per kg of feed, offered in the period from 1 to 46 days, while the experimental diets contained 3000 IU D3 + 2760 IU of 25-OHD3 per kg of feed, offered in three programs of feed: 1 to 21, 1 to 35 and 1 to 46 days. The inclusion of 25-OHD3 in the diets for broiler chickens in different breeding seasons did not change (p> 0.05) the parameters of productive performance, gait score, carcass yield and meat quality. For bone measurements, there was a positive correlation (r = 0.291) between bone densitometry and serum levels of 25-OHD3 when supplemented throughout the breeding period. Increasing quadratic (p <0.05) effect of 25-OHD3 inclusion from 1 to 21 day supplementation on serum levels of 25-OHD3, gene expression of mTOR protein, and protein content in chest. While the breast yield increased linearly (p <0.05) according to the period of supplementation. The birds supplemented with 25-OHD3 for 46 days had significantly higher circulating concentrations of 25-OHD3 (p <0.05) compared to the control diet and supplements of 21 and 35 days. The serum 25-OHD3 levels were positively correlated (r = 0.58) with expression of the mTOR pathway, which in turn resulted in stimulation of protein synthesis in the breast of the birds. Histological evaluation of the chest showed a decrease (p <0.05) in the incorporation of collagen and fat in muscle fibers according to the 25-OHD3 supplementation period. The vitamin program proposed with vitamin D3 supplementation of 25-OHD3 during the whole productive life of the birds contributed positively to the increase of serum vitamin D levels and expression of the mTOR pathway, being an interesting strategy to increase the yield of breast, although it does not result in benefits on the productive performance, the bony structure and the quality of the meat.

AVALIAÇÃO DA EXPRESSÃO GÊNICA E PROTÉICA DA VIA mTOR/4EBP1/eIF4E NOS CARCINOMAS PROSTÁTICOS CANINOS

O câncer é uma doença complexa que precisa de um microambiente favorável para seu crescimento e progressão. Esse microambiente tumoral esta constituido por células neoplásicas, vasos sanguíneos, células imunes, fibroblastos e a matriz extracellular (MEC). Geralmente, as células neoplásicas apresentam modificações nas suas vias de sinalização. No homem, a via mTOR/4E-BP1/eIF4E foi descrita como alterada em diferentes tumores, incluindo o câncer de próstata (CP).Além do homem, o cão é espécie doméstica que desenvolve com mais frequência o CP, sendo considerada um potencial modelo para estudos na área de Oncologia Comparada. Devido à limitada informação sobre a via mTOR/4E-BP1/eIF4E e os componentes da MEC nos CP caninos, o objetivo deste estudo foi avaliar a expressão gênica e protéica de mTOR, 4E-BP-1 e eIF4E neste tipo de tumor. Outrossim, avaliar a expressão dos colágenos (C-I e C-III) pela técnica Picrosirius (PSR) e imuno-histoquímica no tecido prostático normal e neoplásico. Foram utilizadas 35 amostras de tecido prostático caninos. Identificou-se alta expressão protéica de p-mTOR e eIF4E nos CP caninos com alto GS ( 8), assim como, correlação positiva entre essas proteínas. Nos colágenos não foi observada diferença de expressão quando comparadas amostras de próstata normal e CP canino. De forma similar com o CP humano, estes resultados sugerem que p-mTOR e eIF4E podem ser bons marcadores para o processo carcinogênico prostático canino e estão correlacionados com alto GS. Além disso, a distribuição de colágenos foi similar no tecido prostático normal e neoplásico.

Cancer is a complex disease that needs a favorable microenvironment for its growth and progression. This tumor microenvironment consists of neoplastic cells, blood vessels, immune cells, fibroblasts and extracellular matrix (ECM). Generally, neoplastic cells show modification in their signaling pathways. In men, the mTOR/4E-BP1/eIF4E pathway has been described as altered in different tumors, including prostate cancer (PC). Apart from men,the dog is the only species that develops with high frequencythePC, being considered a potential model for comparative oncology initiatives. Due to limited information on this pathway and ECM components in canine tumors, this study aimed to investigate mTOR, 4E-BP1 and eIF4E gene and protein expression in canine PC. Additionally, to evaluate the expression of collagens (C-I and C-III) by Picrosirius Red and Immunohistochemistry in normal samples and canine PC. Were used a total of 35 formalin-fixed paraffin-embedded (FFPE) samples from canine prostatic tissue. We identified higher p-mTOR and eIF4E protein levels in the canine PC with higher GS ( 8), as well as, significant positive correlation between these proteins. No difference statistical was observed in the collagen expression between normal samples and canine PC. Similar to human PC; our data suggested that p-mTOR and eIF4E good markers for canine prostatic carcinogenic process and are correlated with higher GS. Also, the distribution and collagen levels are similar in normal and neoplastic canine prostate.

Uterine environment and conceptus development in ruminants

Interferon tau (IFNT), the pregnancy recognition signal from trophectoderm cells of ruminant conceptuses abrogates the uterine luteolytic mechanism to ensure maintenance of functional corpora lutea for production of progesterone (P4). IFNT acts in concert with P4 to induce expression of genes for transport and/or secretion of histotroph that includes nutrients such as glucose and arginine that activate the mechanistic target of rapamycin (MTOR) nutrient sensing cell signaling pathway to stimulate proliferation, migration, differentiation and translation of mRNAs essential for growth and development of the conceptus. Arginine, leucine, glutamine and glucose increase in the uterine lumen during the peri-implantation period of pregnancy due to increased expression of their transporters by uterine luminal epithelium (LE) and superficial glandular epithelium (sGE) in response to P4 and IFNT. In day 16 ovine conceptus explant cultures, arginine increases GTP cyclohydrolase 1 mRNA, and IFNT, while arginine and glucose increase ornithine decarboxylase, nitric oxide synthase 2, and GCH1. Arginine can be metabolized to nitric oxide (NO) and polyamines which stimulate proliferation of ovine trophectoderm (oTr) cells. Secreted phosphoprotein 1 (SPP1, also known as osteopontin) in uterine histotroph increases focal adhesion assembly as a prerequisite for adhesion and migration of oTr cells through activation and cross-talk between MTOR, MAPK, and myosin II motor pathways. Glucose, arginine, leucine and glutamine stimulate MTOR signaling, proliferation and mRNA translation by oTr cells. Further, glucose and fructose were equivalent in stimulating proliferation and synthesis of hyaluronic acid via the hexosamine pathway in oTr and pig Tr cells. These mechanisms allow select nutrients and SPP1 to act coordinately to affect synthesis of proteins involved in cell signaling affecting conceptus growth, development, and survival during the peri-implantation period of pregnancy.

Uterine environment and conceptus development in ruminants

Interferon tau (IFNT), the pregnancy recognition signal from trophectoderm cells of ruminant conceptuses abrogates the uterine luteolytic mechanism to ensure maintenance of functional corpora lutea for production of progesterone (P4). IFNT acts in concert with P4 to induce expression of genes for transport and/or secretion of histotroph that includes nutrients such as glucose and arginine that activate the mechanistic target of rapamycin (MTOR) nutrient sensing cell signaling pathway to stimulate proliferation, migration, differentiation and translation of mRNAs essential for growth and development of the conceptus. Arginine, leucine, glutamine and glucose increase in the uterine lumen during the peri-implantation period of pregnancy due to increased expression of their transporters by uterine luminal epithelium (LE) and superficial glandular epithelium (sGE) in response to P4 and IFNT. In day 16 ovine conceptus explant cultures, arginine increases GTP cyclohydrolase 1 mRNA, and IFNT, while arginine and glucose increase ornithine decarboxylase, nitric oxide synthase 2, and GCH1. Arginine can be metabolized to nitric oxide (NO) and polyamines which stimulate proliferation of ovine trophectoderm (oTr) cells. Secreted phosphoprotein 1 (SPP1, also known as osteopontin) in uterine histotroph increases focal adhesion assembly as a prerequisite for adhesion and migration of oTr cells through activation and cross-talk between MTOR, MAPK, and myosin II motor pathways. Glucose, arginine, leucine and glutamine stimulate MTOR signaling, proliferation and mRNA translation by oTr cells. Further, glucose and fructose were equivalent in stimulating proliferation and synthesis of hyaluronic acid via the hexosamine pathway in oTr and pig Tr cells. These mechanisms allow select nutrients and SPP1 to act coordinately to affect synthesis of proteins involved in cell signaling affecting conceptus growth, development, and survival during the peri-implantation period of pregnancy.(AU)

Caracterização de um novo modelo de maturação de oócito in vitro e participação do mtor na ovulação em bovinos.

O primeiro estudo caracterizou um modelo in vitro de bloqueio do reinício da meiose de oócitos bovinos. Em um primeiro momento, demonstramos que o uso de um inibidor dos EGFR (AG1478; 5µM) em um sistema de cultivo com metades foliculares (FHS) foi eficiente para manter 89,3% dos oócitos em vesícula germinativa durante 15 h. O sistema de bloqueio foi reversível, tendo em vista que os oócitos completaram a maturação após um período adicional de 18 e 20 h, e suportaram o desenvolvimento embrionário subsequente após fertilização in vitro. Quanto ao perfil molecular, no oócito não foi verificado efeito do tratamento na expressão dos genes avaliados. Entretanto, nas células do cumulus, enquanto a expressão de EGR1, TNFAIP6 e HAS2 foi diminuída pelo tratamento com AG1478, a expressão de CX43 e IMPDH1 foi diminuída pela influência das FHS. Além disso, nas células da granulosa observamos uma diminuição nos níveis de expressão de PGR e ADAMTS1 pelo tratamento com AG1478. Os dados de Western blot nos mostraram que a abundância de p-ERK1/2 diminui em decorrência do tratamento com AG1478 associado as FHS. Posteriormente, verificamos que o efeito inibitório do AG1478 juntamente com as FHS não foi revertido pelo tratamento com AngII ou PGs. Em conclusão, este estudo propõem um modelo efetivo e reversível para o bloqueio do reinício da meiose de oócitos bovinos. Em um segundo estudo, investigamos o papel do sistema mTOR durante o período pré-ovulatório em bovinos. Utilizando um modelo in vivo, demonstramos que ocorre um aumento na atividade do mTOR em células da granulosa 3 e 6 h após indução da ovulação com GnRH. Em momentos similares (3 h após GnRH) ocorreu maior abundância proteica para p-ERK1/2, STAR e EGR1. Ao injetar rapamicina no ambiente intrafolicular in vivo, não foram observadas alterações nas taxas de ovulação. Entretanto, o uso da rapamicina em cultivo in vitro de células da granulosa inibiu a expressão de RNAm para EREG induzida pelo LH. Além disso, os dados de cultivo comprovaram o efeito da rapamicina em bloquear a atividade do mTOR, caracterizada pela abundância proteica de p-P70S6K, induzir um provável aumento na abundância de p-AKT e não alterar os níveis de p-ERK1/2 e EGR1. Esses resultados demonstram que o sistema mTOR é regulado positivamente pelo LH em momentos similares a p-ERK1/2, STAR e EGR1. Além disso, os dados de inibição do mTOR contribuem para sugerir uma outra rota para a ovulação controlada pela p-AKT, na qual ocorre ativação de ERK1/2 em uma via independente dos níveis de expressão de EREG, AREG e COX2.

In the first study, we characterized an in vitro culture system able to delaying meiosis resumption of bovine oocytes. Firstly, we demonstrated that the use of an EGFR inhibitor (AG1478; 5M) in a culture system with follicular hemisections (FHS) was effective to maintain 89.3% of the oocytes in germinal vesicle stage (GV) during 15 h. This blocking effect was dependent on the FHS, since in its absence only 40% of the oocytes remain in GV stage. The meiosis blockage was totally reversible, since the oocytes reached matured stages after an additional 18 and 20 h maturation period and were able to support the embryonic development after in vitro fertilization. Regarding the molecular profile of the cells involved in the blocking system, we did not observe treatment effect on mRNA expression of the genes evaluated in oocyte. However, in cumulus cells, whereas the expression of EGR-1, TNFAIP6 and HAS2 was inhibited by AG1478 treatment, the expression of CX43 and IMPDH1 was decreased by FHS influence. Moreover, in the granulosa cells we observed a downregulation in the expression levels of PGR and ADAMTS1 by AG1478 treatment. The Western blot data revealed that the treatment with AG1478 plus FHS induces a downregulation in p-ERK1/2 protein abundance. In the next experiment, we verified that the AngII or PGE2 and PGF2 did not reverse the inhibitory effect of AG1478 plus FHS on meiosis resumption. In conclusion, findings from this study revealed an effective and reversible system to prevent meiosis resumption of bovine oocytes. In the second study, we investigate the role of mTOR system and its relation with LH regulated genes during preovulatory period in cattle. Using an in vivo model, we demonstrated mTOR kinase activity in granulosa cells 3 and 6 h after induction of ovulation with GnRH. In the similar moments (3 h after GnRH), we observed an increase in p-ERK1/2, STAR and EGR1 protein abundance. The inhibition of mTOR kinase activity by intrafollicular injection of rapamycin did not alter the ovulation rate. However, the treatment of granulosa cells in vitro with rapamycin interrupted the LH-induced increase in EREG mRNA levels. Moreover, the effect of rapamycin in culture was proved by inhibiting the p-P70S6K protein levels. In the same Western blot analysis, we verified that rapamycin may be inducing AKT activity and did not alter Phospho-ERK1/2 status and EGR1 protein abundance. These results provided the first evidence in cattle that mTOR system is upregulated by LH at time points similar to p-ERK1/2, STAR and EGR1. In addition, the mTOR inhibition data contribute to suggest an AKT dependent pathway during ovulation process, in which occurs ERK1/2 activation in a pathway independent of EREG, AREG and PTGS2 mRNA levels.