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Many sex differences in liver gene expression originate in the brain, depend on GH secretion and may underlie sex disparities in hepatic disease. Because epigenetic mechanisms may contribute, we studied promoter methylation and microRNA abundance in the liver, associated with expression of sexual dimorphic genes in mice with selective disruption of the dopamine D2 receptor in neurons (neuroDrd2KO), which decreases hypothalamic Ghrh, pituitary GH, and serum IGFI and in neonatally androgenized female mice which have increased pituitary GH content and serum IGFI. We evaluated mRNA levels of the female predominant genes prolactin receptor (Prlr), alcohol dehydrogenase 1 (Adh1), Cyp2a4, and hepatocyte nuclear transcription factor 6 (Hnf6) and the male predominant gene, Cyp7b1. Female predominant genes had higher mRNA levels compared to males, but lower methylation was only detected in the Prlr and Cyp2a4 female promoters. In neuroDrd2KO mice, sexual dimorphism was lost for all genes; the upregulation (feminization) of Prlr and Cyp2a4 in males correlated with decreased methylation of their promoters, and the downregulation (masculinization) of Hnf-6 mRNA in females correlated inversely with its promoter methylation. Neonatal androgenization of females evoked a loss of sexual dimorphism only for the female predominant Hnf6 and Adh1 genes, but no differences in promoter methylation were found. Finally, mmumiR-155-5p, predicted to target Cyp7b1 expression, was lower in males in association with higher Cyp7b1 mRNA levels compared to females and was not modified in neuroDrd2KO or TP mice. Our results suggest specific regulation of gene sexually dimorphic expression in the liver by methylation or miRNAs. Fil: Brie, Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ramirez, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Lacau Mengido, Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Becu Villalobos, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Adipose tissue has emerged as a fundamental player in metabolic and energy processes. Beyond its fat storage capacity, it displays adaptive mechanisms to react to metabolic challenges throughout life. Three types of adipocytes, white, brown and beige, share common and yet fundamentally different roles and molecular regulation. This review focuses on brain and endocrine control of the function and plasticity of brown and beige adipocytes. The involvement of brain circuits controlling brown adipocytes and thermogenesis via the sympathetic nervous system is described. This autonomic control is mainly exerted by the hypothalamus and brainstem, and noradrenaline released from sympathetic efferent postganglionic fibers interacts with brown or beige b3 adrenoreceptors (b3Adr) to trigger a cascade of events favorable to thermogenesis. Metabolic plasticity in response to environmental or hormonal cues, such as high fat diets, temperature, or hormones and aging, has been identified as a hallmark feature of brown/beige adipocytes. Besides the plastic response of WAT beiging, which is the generation of brown-like adipocytes within white adipose depots, diet-induced obesity and other cues evoke vascular remodeling and functional hypoxia in BAT leading to a whitening phenotype, characterized by mitochondrial dysfunction and loss, lipid droplet accumulation, and decreased thermogenesis. A detailed analysis of the participation of autophagy, and the endocrine signals relied by prolactin and growth hormone uncovers potential avenues of intervention to improve the metabolic function of BAT. Because BAT dysfunction in rodents is linked to fat gain, and glucose and lipid disorders, targeting BAT function and WAT beiging holds promise for obesity prevention and metabolic improvement.

To study the pathological effects of continuous hyperprolactinemia on food intake mechanisms we used female mice that lack dopamine D2 receptors in lactotropes (lacDrd2KO). These mice had lifelong hyperprolactinemia, increased food intake, and gradual development of obesity from 5 to 10 months of age. Ongoing endogenous prolactin signaling in lacDrd2KO mice was evidenced by increased basal phosphorylation of STAT5b in hypothalamic areas related to food intake, such as the arcuate (ARN), dorsomedial (DMN), and ventromedial nuclei. In the ARN of young lacDrd2KO mice there were higher Prlr mRNA levels and in obese 10‐month‐old lacDrd2KO mice increased expression of the orexigenic genes Neuropeptide Y (Npy ) and Agouti‐related peptide, compared to controls. Furthermore, Npy expression was increased in the DMN, probably contributing to increased food intake and decreased expression of Uncoupling protein‐1 in brown adipose tissue, both events favoring weight gain. Leptin resistance in obese lacD2RKO mice was evidenced by its failure to lower food intake and a dampened response of STAT3 phosphorylation, specifically in the mediobasal hypothalamus. Our results suggest that pathological chronically high prolactin levels, as found in psychiatric treatments or patients with prolactinomas, may impact on specific hypothalamic nuclei altering gene expression, leptin response, and food intake. Fil: López Vicchi, María Felicitas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ladyman, Sharon R.. Maurice Wilkins Centre; Nueva Zelanda. University of Otago; Nueva Zelanda Fil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Gustafson, Papillon. University of Otago; Nueva Zelanda Fil: Knowles, Penelope. University of Otago; Nueva Zelanda Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Grattan, David R.. Maurice Wilkins Centre; Nueva Zelanda. University of Otago; Nueva Zelanda Fil: Becu Villalobos, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

A multistep signaling cascade originates in brain centers that regulate hypothalamic growth hormone-releasing hormone (Ghrh) and somatostatin expression levels and release to control the pattern of GH secretion. This process is sexually fine-tuned, and relays important information to the liver where GH receptors can be found. The temporal pattern of pituitary GH secretion, which is sex-specific in many species (episodic in males and more stable in females), represents a major component in establishing and maintaining the sexual dimorphism of hepatic gene transcription. The liver is sexually dimorphic exhibiting major differences in the profile of more than 1000 liver genes related to steroid, lipid, and foreign compound metabolism. Approximately, 90% of these sex-specific liver genes were shown to be primarily dependent on sexually dimorphic GH secretory patterns. This proposes an interesting scenario in which the central nervous system, indirectly setting GH profiles through GHRH and somatostatin control, regulates sexual dimorphism of liver activity in accordance with the need for sex-specific steroid metabolism and performance. We describe the influence of the loss of sexual dimorphism in liver gene expression due to altered brain function. Among other many factors, abnormal brain sexual differentiation, xenoestrogen exposure and D2R ablation from neurons dysregulate the GHRH?GH axis, and ultimately modify the liver capacity for adaptive mechanisms. We, therefore, propose that an inefficient brain control of the endocrine growth axis may underlie alterations in several metabolic processes through an indirect influence of sexual dimorphism of liver genes. Fil: Brie, Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ramirez, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: de Winne, Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: López Vicchi, María Felicitas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Villarruel, Luis Abelardo. Comision Nacional de Energia Atomica. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia.; Argentina Fil: Sorianello, Eleonora Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Catalano, Paolo Nicolás. Comision Nacional de Energia Atomica. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia.; Argentina Fil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Becu Villalobos, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Preclinical and clinical studies support that Notch signaling may play an important oncogenic role in cancer, but there is scarce information for pituitary tumors. We therefore undertook a functional study to evaluate Notch participation in pituitary adenoma growth. Tumors generated in nude mice by subcutaneous GH3 somatolactotrope cell injection were treated in vivo with DAPT, a γ-secretase inhibitor, thus inactivating Notch signaling. This treatment led to pituitary tumor reduction, lower prolactin and GH tumor content, and a decrease in angiogenesis. Furthermore, in silico transcriptomic and epigenomic analyses uncovered several tumor suppressor genes related to Notch signaling in pituitary tissue, namely Btg2, Nr4a1, Men1, Zfp36, and Cnot1. Gene evaluation suggested that Btg2, Nr4a1 and Cnot1 may be possible players in GH3 xenograft growth. Btg2 mRNA expression was lower in GH3 tumors compared to the parental line, and DAPT increased its expression levels in the tumor in parallel with the inhibition of its volume. Cnot1 mRNA levels were also increased in the pituitary xenografts by DAPT treatment. And the Nr4a1 gene was lower in tumors compared to the parental line, though not modified by DAPT. Finally, because DAPT in vivo may be also acting on tumor microenvironment, we determined the direct effect of DAPT on GH3 cells in vitro. We found that DAPT decreases the proliferative, secretory and migration potential of GH3 cells. These results position selective interruption of Notch signaling as a potential therapeutic tool in adjuvant treatments for aggressive or resistant pituitary tumors. Fil: Zubeldia Brenner, Lautaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: de Winne, Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Perrone, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina Fil: Rodríguez Seguí, Santiago Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Willems, Christophe. Katholikie Universiteit Leuven; Bélgica Fil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Lacau Mengido, Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Vankelecom, Hugo. Katholikie Universiteit Leuven; Bélgica Fil: Cristina, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina Fil: Becu, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

To determine the effect of providing water within the area grazed by dairy cows on milk yield and quality, compared to requiring cows to walk to a distant water trough, on a dairy farm in the Pampa region of Argentina during summer. Holstein dairy cows were allocated to two herds with similar parity, days in milk and milk production. They were grazed in one paddock that was divided in two, with a fixed water trough at one end. Cows were moved twice daily to grazing plots within the paddock. Control cows (n=66) could only access water from the fixed trough, whereas supplemented cows (n=67) also received water from a mobile trough within the grazing plot. Milk production of each cow, and water consumption of the two herds were measured daily over 62 days. Milk composition for each herd was determined weekly from Days 18 to 60 of the study, and grazing behaviour was observed between 08:00 and 16:00 hours on Days 11-15, 19-22 and 39-43. Fil: Miglierina, M. M.. Universidad Nacional de La Plata; Argentina Fil: Bonadeo, Nadia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina Fil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Becu, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Lacau, Isabel María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

We studied the impact of high prolactin titers on liver and adipocyte gene expression related to glucose and insulin homeostasis in correlation with obesity onset. To that end we used mutant female mice that selectively lack dopamine type 2 receptors (D2Rs) from pituitary lactotropes (lacDrd2KO), which have chronic high prolactin levels associated with increased body weight, marked increments in fat depots, adipocyte size, and serum lipids, and a metabolic phenotype that intensifies with age. LacDrd2KO mice of two developmental ages, 5 and 10 mo, were used. In the first time point, obesity and increased body weight are marginal, although mice are hyperprolactinemic, whereas at 10 mo there is marked adiposity with a 136% increase in gonadal fat and a 36% increase in liver weight due to lipid accumulation. LacDrd2KO mice had glucose intolerance, hyperinsulinemia, and impaired insulin response to glucose already in the early stages of obesity, but changes in liver and adipose tissue transcription factors were time and tissue dependent. In chronic hyperprolactinemic mice liver Prlr were upregulated, there was liver steatosis, altered expression of the lipogenic transcription factor Chrebp, and blunted response of Srebp-1c to refeeding at 5 mo of age, whereas no effect was observed in the glycogenesis pathway. On the other hand, in adipose tissue a marked decrease in lipogenic transcription factor expression was observed when morbid obesity was already settled. These adaptive changes underscore the role of prolactin signaling in different tissues to promote energy storage. Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: López Vicchi, María Felicitas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Brie, Belen. Ministerio de Ciencia. Tecnología e Innovación Productiva. Agencia Nacional de Promoción Cientifíca y Tecnológica; Argentina Fil: De Winne, Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Fiore, Esteban Juan. Universidad Austral. Facultad de Cs.biomédicas. Instituto de Investigaciones En Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Investigaciones En Medicina Traslacional; Argentina Fil: Pérez Millán, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Mazzolini Rizzo, Guillermo Daniel. Universidad Austral. Facultad de Cs.biomédicas. Instituto de Investigaciones En Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Investigaciones En Medicina Traslacional; Argentina Fil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Becu, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Development of resistance to anthelmintic drugs has motivated the search for diagnostic methods to identify animals for targeted selective treatments. We compared three methods for the diagnosis of nematode infection in relation to milk production in a fully grazing dairy herd of 150 cows in the humid Pampa (Argentina). Animals had feces, blood and milk sampled during the first postpartum month for EPG, pepsinogen and anti-Ostertagia antibody determination, respectively. With the results obtained two groups of cows, divided in high and low parasite burden, were conformed for each method, and milk production was then compared between groups. When cows were separated by the EPG method (EPG=0 (N=106) vs. EPG0 (N=44)) a difference of nearly 800 l of milk per cow per lactation was found (P0.05). On the other hand, milk production between groups separated by Pepsinogen (mUtyr ≤ 1000 vs. mUtyr1000) or by anti-Ostertagia (ODR ≤ 0.5 vs. ODR0.5) results did not differ. Interestingly, proportion of cows in each group differed between methods (P0.0001), and the anti-Ostertagia method yielded significantly more cows in the high index group compared to results using the EPG or Pepsinogen method. No correlations were found between parasite indexes determined by the different methods. High parasite burden estimation found may be ascribed to the production system, fully grazing all year round, and to the sampling time, at the beginning of lactation with cows in negative energy balance and depressed immunity. The fact that the cows were born and reared outside, on pasture with continuous nematode larvae exposure, may also account for the results obtained. In conclusion, EPG counting during the first postpartum month may be a useful tool for the diagnosis of production impairment induced by high nematode burden in adult grazing dairy cows. The anthelmintic treatment of only the EPG-positive recently calved cows would improve milk production, while reducing selective pressure on nematode population for the development of resistance.

Parasitism in cattle is known to impair growth and development. Recent findings suggest that productivity of adult animals is also affected, but little is known about the physiological mechanisms involved. Furthermore, development of nematode resistance to drugs makes imperative the search of management practices that avoid whole herd treatment. We undertook an epidemiological and endocrine study in a grass based dairy farm in Argentina to study the effect of parasites on milk production and the underlying mechanisms involved, and identify individual animals that would benefit from antiparasitic treatment. All the cows in the dairy were followed monthly for egg parasite output in feces. Samples were cultured for genera determination. Milk production and reproductive results were recorded and periodical bleedings for hormone determination were performed. Nematode egg output (EPG) was maximal in late Summer and Autumn and minimal in Spring in coincidence with the Ostertagia inhibition-disinhibition cycle as this genus had the highest prevalence in all the study. The highest proportion of positive samples was found in the high producing herd and maximal counts were found in the peripartal period. Milk production did not correlate with EPG mean values but, when cows were grouped by EPG positivity around parturition, a significant difference in total milk production between EPG null and positive cows was observed. Positive cows produced 7%, 12% or 15% less milk than null EPG cows, depending on the sampling month/s chosen for classification. The highest difference was seen when both prepartum and postpartum samples were taken into account. No difference in lactation length and a marginal effect on partum to first service interval were encountered. Endocrine studies revealed a decrease in serum growth hormone (GH), type I insulin-like growth factor (IGF-I) and prolactin during lactation in cows with positive EPG in the first postpartum sample with respect to null EPG cows at that time. GH levels decreased and prolactin and IGF-I levels increased in both groups of cows from month 0 to 6 in milk. Serum insulin levels remained stable throughout lactation and were similar in both groups of cows. In conclusion, EPG around parturition may be a useful tool for identifying cows that will have a decrease in productivity due to parasite effects and would possibly benefit from an antiparasitic treatment. Besides, our results suggest that detrimental effect of parasites on milk production may be mediated by GH, IGF-I and prolactin serum levels.

The relationship between antidopaminergic drugs and glucose has not been extensively studied, even though chronic neuroleptic treatment causes hyperinsulinemia in normal subjects or is associated with diabetes in psychiatric patients. We sought to evaluate dopamine D2 receptor (D2R) participation in pancreatic function. Glucose homeostasis was studied in D2R knockout mice (Drd2(-/-)) mice and in isolated islets from wild-type and Drd2(-/-) mice, using different pharmacological tools. Pancreas immunohistochemistry was performed. Drd2(-/-) male mice exhibited an impairment of insulin response to glucose and high fasting glucose levels and were glucose intolerant. Glucose intolerance resulted from a blunted insulin secretory response, rather than insulin resistance, as shown by glucose-stimulated insulin secretion tests (GSIS) in vivo and in vitro and by a conserved insulin tolerance test in vivo. On the other hand, short-term treatment with cabergoline, a dopamine agonist, resulted in glucose intolerance and decreased insulin response to glucose in wild-type but not in Drd2(-/-) mice; this effect was partially prevented by haloperidol, a D2R antagonist. In vitro results indicated that GSIS was impaired in islets from Drd2(-/-) mice and that only in wild-type islets did dopamine inhibit GSIS, an effect that was blocked by a D2R but not a D1R antagonist. Finally, immunohistochemistry showed a diminished pancreatic beta-cell mass in Drd2(-/-) mice and decreased beta-cell replication in 2-month-old Drd2(-/-) mice. Pancreatic D2Rs inhibit glucose-stimulated insulin release. Lack of dopaminergic inhibition throughout development may exert a gradual deteriorating effect on insulin homeostasis, so that eventually glucose intolerance develops Fil: Garcia Tornadu, Isabel Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina Fil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina Fil: Chamson Reig, Astrid. Lawson Health Research Institute; Canadá Fil: Wheeler, Michael B.. University of Toronto. Departments of Physiology and Medicine ; Canadá Fil: Hill, David J.. Lawson Health Research Institute; Canadá Fil: Arany, Edith. Lawson Health Research Institute; Canadá Fil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiologia, Biologia Molecular y Celular. Laboratorio de Fisiologia y Biologia Molecular; Argentina Fil: Becu, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina

Dopamine D2 receptor (D2R) participation in prolactin regulation is well documented, but the role of D2Rs in the control of other hormones involved in growth, food intake and glucose metabolism has not been extensively studied. The study of D2R knockout mice (Drd2–/–) puts forward new insights into the role of the D2R in growth hormone (GH)-releasing hormone-GH regulation, peptides involved in food intake, glucose homeostasis, as well as in prolactinoma development. The expected phenotype of chronic hyperprolactinemia and prolactinoma development was found in the Drd2–/– mouse, and this model constitutes a valuable tool in the study of dopamine-resistant prolactinomas. Unexpectedly, these mice were growth retarded, and the importance of functional hypothalamic D2Rs in the neonatal period was revealed. In the Drd2–/– mouse there was a failure of high neonatal GH levels and therefore the expansion of pituitary somatotropes was permanently altered. These mice also had increased food intake, and a sexually dimorphic participation of the D2R in food intake regulation is suggested. The effect described is probably secondary to D2R regulation of prolactin secretion. Furthermore, the negative modulation of D2Rs on α-melanocyte-stimulating hormone release and positive action on the hypothalamic expression of orexins reveals the complex D2R regulation of food intake. Finally, pancreatic D2Rs inhibit glucose-stimulated insulin release. Lack of dopaminergic inhibition throughout development in the Drd2–/– mouse may exert a gradual deteriorating effect on insulin homeostasis, so that eventually glucose intolerance develops. These results highlight the complex endocrine actions of the D2Rs at different levels, hypothalamus, pituitary or pancreas, which function to improve fitness, reproductive success and survival.

Liver sexual gene dimorphism, which depends mainly on specific patterns of GH secretion, may underlie differential susceptibility to some liver diseases. Because GH and prolactin secretion are regulated by dopaminergic pathways, we studied the participation of brain and lactotrope dopamine 2 receptors (D2Rs) on liver gene sexual dimorphism, to explore a link between the brain and liver gene expression. We used global D2R knockout mice (Drd2(-/-)) and conducted a functional dissection strategy based on cell-specific Drd2 inactivation in neurons (neuroDrd2KO) or pituitary lactotropes. Disruption of neuronal D2Rs (which impaired the GH axis) decreased most of male or female-predominant class I liver genes and increased female-predominant class II genes in males, consistent with the positive (class I) or negative (class II) regulation of these genes by GH. Notably, sexual dimorphism was lost for class I and II genes in neuroDrd2KO mice. Disruption of lactotrope D2Rs did not modify class I or II genes in either sex, because GH axis was preserved. But surprisingly, 1 class II gene (Prlr) and female-predominant class I genes were markedly up-regulated in lacDrd2KO females, pointing to direct or indirect effects of prolactin in the regulation of selected female-predominant liver genes. This suggestion was strengthened in the hyperprolactinemic Drd2(-/-) female mouse, in which increased expression of the same 4 liver genes was observed, despite a decreased GH axis. We hereby demonstrate endocrine-mediated D2R actions on sexual dimorphic liver gene expression, which may be relevant during chronic dopaminergic medications in psychiatric disease. Fil: Ramirez, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Pérez Millán, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Garcia Tornadu, Isabel Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Rubinstein, Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina Fil: Becu, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina

Prolactin, a pleiotropic hormone secreted by lactotropes, has reproductive and metabolic functions. Chronically elevated prolactin levels increase food intake, but in some hyperprolactinemic states such as in the global dopamine D2 receptor (D2R) knockout mouse, food intake is not increased. Here, we conduct a cell-specific genetic dissection study using conditional mutant mice that selectively lack D2Rs from pituitary lactotropes (lacDrd2KO) to evaluate the role of elevated prolactin levels without any confounding effect of central D2Rs on motor and reward mechanisms related to food intake. LacDrd2KO female mice exhibited chronic hyperprolactinemia, pituitary hyperplasia, and a preserved GH axis. In addition, lacDrd2KO female but not male mice evidenced increased food intake by three months of age and, from five months onwards their body weights were heavier. A marked increment in fat depots, adipocyte size, serum triglyceride and non-esterified fatty acid levels, and a decrease in lipolytic enzymes in adipose tissue were evidenced. Furthermore, lacDrd2KO female mice had glucose intolerance but a preserved response to insulin. In the hypothalamus Npy mRNA expression was increased, and Pomc and Ppo mRNA levels were unaltered (in contrast to results in global D2R knockout mouse). Thus, the orexigenic effect of prolactin, and its action on hypothalamic Npy expression were fully evidenced, leading to increased food intake and adiposity. Our results highlight the metabolic role of prolactin and illustrate the value of studying cell-specific mutant mice to disentangle patho-physiological mechanisms otherwise masked in null allele mutants or in animals treated with pervasive pharmacological agents. Fil: Pérez Millán, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ramirez, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Noain, Daniela Maria Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Becu, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Parasitism during development impairs normal growth and delays the onset of puberty through altered hormone profiles, including insulin-like growth factor one (IGF-1). Asmammary gland development during prepuberty is strongly dependent on IGF-1, we determined if antiparasitic treatment during this stage of growth improved mammary gland development. One group of Holstein heifers was treated monthly, rotationally with antiparasitic drugs from birth to 70 weeks of age, a second group was untreated. Treated heifer calves had between 56% and 65% less EPG counts than untreated ones. Presence of Ostertagia, Cooperia, Haemonchus and Trichostrongylus was demonstrated. Treatment effectively advanced the onset of puberty and increased IGF-1 levels. At 20, 30, 40 and 70 weeks of age biopsies from the mammary gland were taken and histological sections were prepared and stained with hematoxylin–eosin. Pictures were analyzed to compare parenchyma area in relation to total mammary tissue between groups. Mammary samples from treated heifers had higher ratios of parenchyma/total area than untreated ones. As mammary development during prepuberty is crucial for mammary performance during lactation, these results add new evidence to the importance of gastrointestinal parasite control in heifers., Escuela de Agricultura y Ganadería "María Cruz y Manuel L. Inchausti", Facultad de Ciencias Veterinarias

Neonatal androgenization masculinizes the GH axis and thus may impact on liver gene regulation. Neonatal testosterone administration to female mice decreased (defeminized) female predominant GH-dependent liver gene expression (Hnf6, Adh1, Prlr, Cyp3a41) and did not modify male predominant genes (Cyp7b1, Cyp4a12, Slp). Female predominance of Cis mRNA, an inhibitor of episodic GH signaling pathway, was unaltered. At birth, Cyp7b1 promoter exhibited a higher methylation status in female livers, while the Hnf6 promoter was equally methylated in both sexes; no differences in gene expression were detected at this age. In adulthood, consistent with sex specific predominance, lower methylation status was determined for the Cyp7b1 promoter in males, and for the Hnf6 promoter in females, and this last difference was prevented by neonatal androgenization. Therefore, early steroid treatment or eventually endocrine disruptor exposure may alter methylation status and sexual dimorphic expression of liver genes, and consequently modify liver physiology in females. Fil: Ramirez, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Fil: Zubeldia Brenner, Lautaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Fil: Wargon, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Fil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Fil: Becu, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina

Dopamine and estradiol interact in the regulation of lactotroph cell proliferation and prolactin secretion. Ablation of the dopamine D2 receptor gene (Drd2−/−) in mice leads to a sexually dimorphic phenotype of hyperprolactinemia and pituitary hyperplasia, which is stronger in females. TGF-β1 is a known inhibitor of lactotroph proliferation. TGF-β1 is regulated by dopamine and estradiol, and it is usually down-regulated in prolactinoma experimental models. To understand the role of TGF-β1 in the gender-specific development of prolactinomas in Drd2−/− mice, we compared the expression of different components of the pituitary TGF-β1 system, including active cytokine content, latent TGF-β–binding protein isoforms, and possible local TGF-β1 activators, in males and females in this model. Furthermore, we evaluated the effects of dopamine and estradiol administration to elucidate their role in TGF-β1 system regulation. The expression of active TGF-β1, latent TGF-β–binding protein isoforms, and several putative TGF-β1 activators evaluated was higher in male than in female mouse pituitary glands. However, Drd2−/− female mice were more sensitive to the decrease in active TGF-β1 content, as reflected by the down-regulation of TGF-β1 target genes. Estrogen and dopamine caused differential regulation of several components of the TGF-β1 system. In particular, we found sex- and genotype- dependent regulation of active TGF-β1 content and a similar expression pattern for 2 of the putative TGF-β1 activators, thrombospondin-1 and kallikrein-1, suggesting that these proteins could mediate TGF-β1 activation elicited by dopamine and estradiol. Our results indicate that (1) the loss of dopaminergic tone affects the pituitary TGF-β1 system more strongly in females than in males, (2) males express higher levels of pituitary TGF-β1 system components including active cytokine, and (3) estradiol negatively controls most of the components of the system. Because TGF-β1 inhibits lactotroph proliferation, we propose that the higher levels of the TGF-β1 system in males could protect or delay the development of prolactinomas in Drd2−/− male mice.

Prolactin-secreting adenomas are the most frequent type among pituitary tumors, and pharmacological therapy with dopamine agonists remains the mainstay of treatment. But some adenomas are resistant, and a decrease in the number or function of dopamine D2 receptors (D2Rs) has been described in these cases. D2R knockout [Drd2(-/-)] mice have chronic hyperprolactinemia and pituitary hyperplasia and provide an experimental model for dopamine agonist-resistant prolactinomas. We described previously that disruption of D2Rs increases vascular endothelial growth factor (VEGF) expression. We therefore designed two strategies of antiangiogenesis using prolactinomas generated in Drd2(-/-) female mice: direct intra-adenoma mVEGF R1 (Flt-1)/Fc chimera (VEGF-TRAP) injection for 3 weeks [into subcutaneously transplanted pituitaries from Drd2(-/-) mice] and systemic VEGF neutralization with the specific monoclonal antibody G6-31. Both strategies resulted in substantial decrease of prolactin content and lactotrope area, and a reduction in tumor size was observed in in situ prolactinomas. There were significant decreases in vascularity, evaluated by cluster of differentiation molecule 31 vessel staining, and proliferation (proliferating cell nuclear antigen staining) in response to both anti-VEGF treatments. These data demonstrate that the antiangiogenic approach was effective in inhibiting the growth of in situ dopamine-resistant prolactinomas as well as in the transplanted adenomas. No differences in VEGF protein expression were observed after either anti-VEGF treatment, and, although serum VEGF was increased in G6-31-treated mice, pituitary activation of the VEGF receptor 2 signaling pathway was reduced. Our results indicate that, even though the role of angiogenesis in pituitary adenomas is contentious, VEGF might contribute to adequate vascular supply and represent a supplementary therapeutic target in dopamine agonist-resistant prolactinomas. Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Pérez Millán, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Cristina, Carolina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; Argentina Fil: Becu, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina