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The transcription factor, Pit-1, is involved in the transcriptional regulation of the mammalian prolactin (Prl), growth hormone (GH) and thyroid stimulating hormone b-subunit genes (TSHb) as well as its own gene. The role of Pit-1 in avian species is unknown. Three turkey (t) Pit-1 isoforms have been identified that arise from alternative transcription initiation and alternative splicing. Splicing of exon 1 to an alternative acceptor splice site in exon 2 results in a 28 amino acid insertion in tPit-1β* relative to tPit-1*. Both isoforms initiate transcription at exon 1. A tPit-1 transcript unique to the turkey has been identified and arises following transcription initiation upstream of the alternative acceptor splice site in exon 2. Western blot analysis of pituitary extracts has revealed two isoforms of 37 and 40 kDa. The ability of Pit-1 to transactivate the Prl, GH, and Pit-1 promoters was determined with cotransfection assays. The tPrl, tGH, tPit-1 and rat (r) Prl promoters were cloned upstream of the luciferase gene in a reporter construct. Turkey Pit-1 isoforms and rPit-1 were expressed under the control of the Avian Sarcoma Virus Long Terminal Repeat (ASVLTR) promoter. Cotransfection analyses in mouse L cells indicate that tPit-1* activates the tPrl, tGH, tPit-1 and rPrl promoters 4.6-, 3.8-, 1.7-, and 29.0-fold, respectively. Similar results were observed when cotransfection assays were performed in a turkey pituitary-derived cell line and in primary turkey pituitary cells. These results indicate that tPit-1 is not a strong activator of the tPrl, tGH, or tPit-1 genes, whereas Pit-1 does activate these genes in mammals. A point mutation at amino acid position 176 (ser ⟹ leu) in the POU-homeodomain results in a mutant tPit-1 that shows decreased activity on all promoters tested. Turkey Pit-1* (ser-176) activates the rPrl promoter 14-fold lower than the wild type tPit-1* (leu-176). / Master of Science

Mammary tissue from nine Holstein cows was collected within one week of parturition, at 60 and 180 days postpartum. Blood samples were collected at 6-hr intervals from two days prior to . until two days after surgery. A membrane-enriched fraction of tissue homogenates was prepared by differential centrifugation. Displacement curve data was analyzed by a microcomputer program. Mean prolactin (Prl) during the periparturient period was greater than either postpartum period, but not prior to biopsy. Dissociation constants (Kd) estimated with NIH-bPRL-6 as competitor were not different among stages of lactation, and averaged 8.97 x 10⁻⁸M. Receptor concentrations were less during _the periparturient period than later lactation. The Kd was 100-fold greater when estimated with human growth hormone as competitor. It is concluded that lactogenic hormone receptor concentrations in bovine mammary tissue increase with the onset of lactation, following a pattern similar to that observed in non-ruminants. Three experiments were conducted to investigate endocrine metabolic hormone profiles in Holstein cattle of differing genetic merit at several ages. Control animals were randomly bred to non-AI sires originating in the Virginia Tech Dairy herd. Selected animals were offspring of commercially available AI sires. In one experiment, mean plasma Prl was greater in control animals after feeding and insulin injection, while growth hormone (GH) was greater in selected animals at all ages. Free fatty acids were greater in selected animals at 6 and 24 months of age, while glucose (Glc) and urea were unaffected by genetic merit. In a second experiment, Holstein bull calves were administered Glc and thyrotropin releasing hormone (TRH) on different days. Plasma GH was greater in selected animals. Plasma Prl was greater in control animals after TRH. In the third experiment, Holstein cows received TRH at 30, 90 and 200 days postpartum (DPP). Net energy balance was negative at 30, while positive at 90 and 200 DPP. Plasma GH before and after TRH was greater in selected animals, and greater during early than later lactation. Thus, the results of the three experiments indicate that increased plasma GH may be associated with selection for increased milk yield. / Ph. D.

INTRODUÇÃO: A prolactina é um hormônio polipeptídico, que possui reconhecida ação sistêmica, principalmente na fisiologia da reprodução, porém, seu desequilíbrio, em especial a hiperprolactinemia, é cada vez mais frequente na prática clínica. Apesar de ser um distúrbio relativamente comum, ainda existem dúvidas quanto aos efeitos moleculares da hiperprolactinemia no trato genital, particularmente no útero, e também na hipófise. O presente estudo teve por objetivo verificar os efeitos da hiperprolactinemia induzida pela metoclopramida na expressão gênica da prolactina e de seus receptores no útero e na hipófise de camundongo fêmea. MÉTODOS: Utilizaram-se 49 camundongos fêmeas (Wistar), randomicamente divididas em 7 grupos contendo 7 animais cada: 1) SS não ovariectomizadas que receberam solução salina (veículo); 2) M não ovariectomizadas tratadas com metoclopramida; 3) OSS ovariectomizadas tratadas com solução salina (veículo); 4) OM ovariectomizadas tratadas com metoclopramida; 5) OME ovariectomizadas tratadas com metoclopramida e 17-estradiol; 6) OMP ovariectomizadas tratadas com metoclopramida e progesterona micronizada; 7) OMEP ovariectomizadas tratadas com metoclopramida, 17-estradiol e progesterona micronizada. Após 50 dias os animais foram sacrificados sendo retirados o útero e a hipófise de cada animal para extração do ácido ribonucleico total, que foi utilizado para a síntese de ácido desoxirribonucleico complementar e avaliação da expressão gênica da prolactina e das diferentes isoformas de seus receptores, por reação em cadeia da polimerase em tempo real. RESULTADOS: Na hipófise, em animais não ovariectomizados, o tratamento com metoclopramida aumentou a expressão do gene que codifica a prolactina em relação ao tratamento apenas com o veículo. Nos animais castrados, a progesterona isoladamente ou associada ao estrogênio determinou o incremento do RNA mensageiro da prolactina em relação aos outros animais castrados que receberam outras combinações de tratamento. Este efeito foi semelhante ao da metoclopramida em animais com os ovários intactos. Em relação ao receptor de prolactina, o estrogênio e a progesterona, isoladamente, foram responsáveis pelo incremento da isoforma S2. No útero houve aumento na expressão de RNA mensageiro de prolactina após tratamento com metoclopramida ou com tratamento isolado ou combinado de estrogênio e progesterona. A ovariectomia determinou a redução da expressão das isoformas S1 e S2 do receptor de prolactina de todas as isoformas estudadas. Já o tratamento estroprogestativo determinou elevação da formas S3 e L do receptor, enquanto com a progesterona isoladamente causou apenas o incremento da forma L do receptor da prolactina no útero dos animais castrados. CONCLUSÕES: Nossos dados sugerem que o tratamento com metoclopramida altera de forma diferente a expressão de prolactina e de seus receptores quando se compara o resultado da hipófise em relação ao útero em camundongos fêmeas castrados e tratados com esteróides sexuais / INTRODUCTION: Prolactin is a polypeptide hormone with a recognized systemic action mainly on reproductive physiology. However, prolactin imbalance, particularly hyperprolactinemia, is increasingly more frequent in clinical practice. Although it is a comparatively common disorder, there are still doubts about the molecular effects of hyperprolactinemia on the genital tract especially in the uterus and the pituitary. The present study aimed at verifying the effects of metoclopramide-induced hyperprolactinemia on the gene expression of prolactin and its receptors in the uterus and pituitary of the female mouse. METHODS: Forty-nine female Wistar mice were randomized to 7 equal-sized groups as follows: 1) SS nonoophorectomized mice treated with saline solution (vehicle); 2) M nonoophorectomized mice treated with metoclopramide; 3) OSS oophorectomized mice treated with saline solution (vehicle); 4) OM oophorectomized mice treated with metoclopramide; 5) OME oophorectomized mice treated with metoclopramide and 17-estradiol; 6) OMP oophorectomized mice treated with metoclopramide and micronized progesterone; 7) OMEP oophorectomized mice treated with metoclopramide, 17-estradiol, and micronized progesterone. The animals were sacrificed 50 days after the end of the treatment, and the uterus and pituitary of each animal were removed for extraction of total ribonucleic acid, which was then used for synthesizing complementary deoxyribonucleic acid and for evaluating the gene expression of prolactin and the different isoforms of its receptors by the real-time polymerase chain reaction. RESULTS: In the pituitary of the nonoophorectomized mice, the treatment with metoclopramide against that with vehicle alone increased the expression of the prolactin-encoding gene. In the castrated animals, progesterone by itself or in conjunction with estrogen determined a raise in prolactin messenger RNA as opposed to the two other treatments with different combinations. This effect was similar to that produced by metoclopramide in animals with intact ovaries. Estrogen and progesterone, acting independently of each other, were responsible for the increase in the S2 isoform of the prolactin receptor. In the uterus, there was heightened expression of prolactin messenger RNA under the effect of the treatment with metoclopramide or with estrogen and/or progesterone. Oophorectomy caused a greater reduction in expression of the prolactin receptor S1 and S2 isoforms than in the other isoforms. However, the combined estrogen plus progesterone treatment led to an increase in the S3 and L forms of the receptor, while progesterone alone resulted solely in a higher expression of the L form of the prolactin receptor in the endometrium of the castrated mice. CONCLUSION: Our data suggest that metoclopramide treatment induces different changes in the expression of prolactin and its receptors according to whether the effect occurs in the pituitary or the uterus of castrated female mice treated with sex steroids

Atualmente, é crescente o interesse em estudar o potencial do Sistema Nervoso Central (SNC) como alvo de medicamentos antidiabéticos, uma vez que ele possui receptores de insulina e desempenha papel crítico na regulação da homeostase glicêmica. Nesse sentido, o Cycloset® (mesilato de bromocriptina de liberação rápida), um medicamento de ação central aprovado nos Estados Unidos para o tratamento do DMT2, atende a essa tendência atual. Trabalhos prévios mostram efeitos benéficos da bromocriptina (Bromo) sobre a hiperglicemia e hiperlipidemia em modelos de animais obesos tratados com essa droga. Por ser um agonista dopaminérgico, um dos possíveis mecanismos de ação dessa droga pode ser bloqueando a liberação e produção de prolactina (Prl). Níveis elevados de prolactina na circulação sanguínea, observados tanto em indivíduos com prolactinomas como em pessoas tratadas com medicamentos que causam hiperprolactinemia, geram anormalidades no metabolismo de carboidratos e lipídeos, o que pode levar a um quadro de síndrome metabólica. Na presente tese, testamos a hipótese de que ao menos parte dos efeitos antidiabéticos da Bromo seja mediada pela inibição da secreção de prolactina. Avaliamos os efeitos do tratamento com Bromo em camundongos machos e fêmeas geneticamente obesos e resistentes à insulina (ob/ob), bem como testamos se os efeitos benéficos do medicamento seriam revertidos com a reposição de Prl. Machos tratados com Bromo apresentaram maior sensibilidade à insulina, enquanto que a reposição de Prl manteve os animais menos sensíveis, tais como os animais do grupo controle. As fêmeas tratadas com Bromo apresentaram tendência à melhora de sensibilidade à insulina, bem como foram mais tolerantes à glicose, sendo que a reposição de Prl em animais tratados com Bromo também reverteu o efeito benéfico do medicamento. Dessa forma, demonstramos que ao menos parte dos efeitos antidiabéticos da Bromo é mediada pela inibição da secreção basal de Prl. Em um segundo conjunto de experimentos, testamos se a administração de antagonistas de prolactina (G129R-hPrlR) em machos ob/ob, por vias centrais ou periféricas, produziria efeito antidiabético. Observamos que tanto o tratamento periférico como o central diminui a curva glicêmica dos animais em testes de tolerância à glicose e melhoram a sensibilidade à insulina, embora ainda não tenhamos obtido valores significativos devido a nossa amostragem. Por fim, investigamos se a ação da Prl sobre o metabolismo ocorre por meio da interação com o receptor de estrógeno alfa (ERα). Verificamos que receptores de prolactina e de ERα são expressos em áreas comuns no SNC e que variações nos níveis circulantes de estrógeno causam mudanças na sensibilidade à prolactina. Portanto, no presente trabalho, identificamos o possível mecanismo pelo qual a Bromocriptina promove melhorias no controle glicêmico e, de forma inédita, produzimos evidências que o uso de antagonistas de prolactina pode ter potencial no tratamento do DMT2. / Type 2 Diabetes mellitus (T2DM) is a syndrome characterized by dysfunctions in the metabolism of glucose, amino acids and free fat acids. Although most of the drugs currently used to treat T2DM targets peripheral organs, a growing interest in studying the Central Nervous System (CNS) as a potential target of antidiabetic drugs is appearing. The CNS possesses insulin receptors and plays a critical role in regulating glucose homeostasis. In this sense, Cycloset® (quick release bromocriptine mesylate) a drug that acts on CNS, was recently approved in United States to treat T2DM. Previous studies have shown beneficial effects of bromocriptine (Bromo) on hyperglycemia and hyperlipidemia in obese animal models. As a dopaminergic agonist, a possible mechanism of action of this drug could be caused by a decreased prolactin (Prl) production and release. High serum prolactin levels, as observed in patients bearing prolactinomas or individuals using drugs that induce hyperprolactinemia, generate abnormalities in carbohydrate and lipid metabolism, which can lead to metabolic syndrome. In the current thesis, we tested the hypothesis that part of bromocriptine antidiabetic effects is due to an inhibition of prolactin secretion. We evaluated Bromo effects in genetically obese and insulin resistant male and female mouse (ob/ob), as well as we tested whether replacing Prl could reverse the beneficial effects of Bromo. Males treated with Bromo showed lower insulin resistence, whereas Prl replacement decreased insulin sensitivity. Females treated with Bromo showed tendency towards an improvement in their insulin sensitivity and glucose tolerance. Prl replacement also reversed the beneficial effects of Bromo in this group. Thus, we demonstrated that at least part of the antidiabetic effects of Bromo is due to inhibition of Prl secretion. In another set of experiments, we tested whether central or peripheral treatment with prolactin antagonists (G129R-hPrlR) causes antidiabetic effects in ob/ob male mice. Both peripheral and central treatment decreased the glycemic curve during glucose and insulin tolerance tests, although we still did not obtain statistically significant values with our sample size. Lastly, we investigated whether metabolic Prl action occurs due to a putative interaction with estrogen receptor alpha (ERα). We found a wide co-expression between Prl receptor and ERα in the CNS. Additionally, changes in estrogen levels decrease prolactin sensitivity. Therefore, in the present study we identified the possible mechanism by which bromocriptine promotes improvements in glycemic control, and for the first time, we obtained evidence that the use of prolactin antagonists can have a potential effect in the treatment of T2DM.

A macroprolactinemia é condição freqüente na hiperprolactinemia e em geral, sem impacto clínico. Os dados sobre a atividade biológica da macroprolactina (bbPRL) são controversos e baseados em bioensaio heterólogo com células de rato Nb2. A atividade biológica da bbPRL é observada in vitro e não in vivo, provavelmente porque seu alto peso molecular evita sua passagem pelos capilares. A bioatividade da bbPRL talvez varie de acordo com a especificidade do receptor de prolactina (PRLR). Avaliamos a bioatividade da bbPRL de indivíduos macroprolactinêmicos (Grupo I, n = 18) e da PRL monomérica (mPRL) de pacientes hiperprolactinêmicos sem bbPRL (Grupo II, n = 5) em Nb2 e em células Ba/F-LLP, transfectadas com o PRLR humano. Enquanto ambos ensaios apresentam resultados similares para a atividade de mPRL, nossos resultados indicam que a atividade da bbPRL é presente em ensaio heterólogo e não em ensaio homólogo. O ensaio Ba/F-LLP é sensível e apresenta melhor correlação com a atividade in vivo da bbPRL / Macroprolactinemia is a frequent finding in hyperprolactinemic individuals, usually without clinical impact. Data on biological activity of macroprolactin (bbPRL) is mostly based on a heterologous bioassay (Nb2 cell). Biological activity of bbPRL observed in vitro but not in vivo maybe due to its high molecular weight preventing its passage through capillary barrier. Alternatively, bbPRL bioactivity may differ depending on the PRL receptor species specificity. BbPRL from macroprolactinemic individuals and monomeric PRL (mPRL) from hyperprolactinemic patients without macroprolactinemia were tested in two bioassays: Nb2 and in Ba/F-LLP, which expresses human prolactin receptor. While both bioassays achieve similar results considering mPRL activity, our results indicate that bbPRL displays activity in a heterologous but not in a homologous bioassay, consistently with the apparent absence of bbPRL bioactivity in vivo