Investigamos a distribuição de ghrelina e de seu receptor (GHS-R) na mucosa gástrica de ratos durante a terceira semana de vida pós-natal e avaliamos o efeito do desmame precoce sobre estas moléculas. Estudamos também a participação da ghrelina no controle da proliferação celular do epitélio gástrico, e para tanto utilizamos a administração de um antagonista. Detectamos o aumento do número de células imunomarcadas para ghrelina nos animais desmamados precocemente e observamos que nem a expressão de GHS-R nem a concentração proteica deste receptor foram alteradas pela mudança da dieta. O uso do antagonista [D-Lys-3]-GHRP-6 resultou na diminuição do índice de síntese de DNA no epitélio gástrico. Concluímos que a ghrelina e o GHS-R estão distribuídos no estômago durante a terceira semana de vida pós-natal e que o desmame precoce aumenta os níveis de ghrelina no epitélio gástrico, sem comprometer seu receptor. Por fim, sugerimos que esta modulação pode estar envolvida no controle da proliferação celular que é fundamental para o desenvolvimento do estômago. / In the present study, we investigated the distribution of ghrelin and growth hormone secretagogue receptor (GHS-R) in the rat gastric mucosa during the third postnatal week, and evaluated the effects of early weaning on these molecules. In addition, we studied whether ghrelin is part of cell proliferation control in gastric epithelium, and to that we used an antagonist. We detected an increase of ghrelin immunolabelled cells in animals submitted to early weaning and observed that GHS-R expression and protein levels of this receptor were not altered by dietary change. The antagonist [D-Lys-3]-GHRP-6 reduced DNA synthesis index. We concluded that ghrelin and GHS-R are distributed in the gastric mucosa during the third postnatal week and that early weaning increases hormone levels in the gastric epithelium, without changing its receptor. We can suggest that such modulation might be involved in the control of cell proliferation, which is essential for stomach development.

Growth hormone (GH) is a vital pituitary hormone controlling somatic cell growth and development. GH has a multitude of effects on the body: deficiency can lead to dwarfism while excess can cause conditions such as gigantism. Human patients with mutations in the transcription factor Pit-1 show decreased GH and prolactin levels. However, Pit-1 is known to control multiple pituitary hormones, so what other factors lead to the specificity of transcriptional regulation of the GH gene via its promoter? In order to study this, our lab has been analyzing rat pituitary cell lines to understand the role of ZNF292 (formerly called Zn-16), a selective GH transcription regulator with 16 zinc fingers that bind to the GH promoter DNA. This work has used rat MtT/S cells that are unique in that they exclusively express GH. MtT/S cells were procured from Riken Cell Bank in Japan, cultured, and examined for GH hormone and RNA expression. Results confirmed that the MtT/S cells are terminally differentiated as somatotrophs. To understand the role of ZNF292 in transcription of the GH gene, recent rat genomic data was analyzed to determine the positions of 7 exons upstream from the large exon 8 that contains the important zinc finger-encoding portions of the protein. First, MtT/S RNA was reverse transcribed into DNA, then PCR amplification was performed using primer pairs encompassing various sections of the exon 1 – 7 region. Specific PCR products were found with distinct products ranging in size from 130 to 960, all of which agreed with the predicted sizes of these exons. It had previously been theorized that ZNF292 contained a single large exon; however, these results show that splicing of the primary transcript does occur in this upstream region. Characterizing this exonic region was performed because it has been shown that ZNF292 produces circular RNA (circRNA) of unknown function in human endothelial cells and in certain types of cancer. CircRNAs are thought to be created by the “back-splicing” of exons, so that rather than a linear transcript, the ends are circularized for a portion of the transcript. Having determined the sequence and organization of these upstream exons, we are now testing primer sets that will demonstrate productive amplification only from circRNAs. Further, we are removing linear RNAs using RNAse R treatment to selectively enhance circRNA presence in the reactions. Finally, data from RNA-Seq analysis of the MtT/S cells will be scrutinized to determine if additional exon/intron boundaries or alternative splice sites exist in this upstream 7 exon region. The study of circular RNAs could be very important in understanding its role in acting as a microRNA sponge or RNA-binding protein sponge during their regulation of downstream gene transcription. Also, analysis of this mechanism shows potential as a clinical tool in cancer treatment because ZNF292 functions as a tumor suppressor in colorectal and chronic lymphatic leukemia.

The sex-dependent pituitary growth hormone (GH) secretory profiles, pulsatile in males and persistent in females, regulate sex-biased expression of hundreds of genes in mammalian liver, contributing to sex differences in hepatic metabolism and disease. The sex-biased GH actions in the liver are mediated by STAT5b and enhanced by a network of transcription factors including the male-biased BCL6 and the female-specific CUX2, acting in the context of sex-biased chromatin states. First, the transcriptional and epigenomic changes induced by continuous-GH infusion (cGH) in male mice, which rapidly feminizes the temporal profile of liver STAT5 activity, were examined. RNA-seq analysis determined that cGH repressed the majority of male-biased genes and induced most female-biased genes within 4-days; however, several highly female-specific genes showed partial feminization. Female-biased genes already in an active chromatin state in male liver were induced early; genes in an inactive chromatin state often responded late. Early cGH-responsive genes included Cux2 and Bcl6 and their targets. DNase-seq and ChIP-seq were used to identify changes in sex-specific chromatin accessibility and histone modifications accompanying these cGH-induced gene expression changes. H3-K27me3 is a key sex-biased repressive mark found preferentially at highly female-biased genes in male mouse liver. Consistently, induction of female-biased genes by cGH was associated with loss of H3-K27me3 at their gene bodies. H3K27 methylation is catalyzed by Polycomb Repressive Complex-2 (PRC2) through its homologous catalytic subunits EZH1 and EZH2. An Ezh1-knockout mouse model with a hepatocyte-specific knockout of Ezh2 (DKO) was used to further investigate the role of H3-K27me3 in repressing sex-biased genes in mouse liver. Loss of Ezh1/Ezh2 led to a significant decrease in sex-specific gene expression, with many female-biased genes induced and male-biased genes repressed. These gene responses were more extensive in male than female liver, as was the loss of H3K27me3 sites and the reciprocal increases in active histone marks. There was substantial up-regulation of liver cancer and liver fibrosis-related genes in male and female DKO-mouse liver, with a subset of genes preferentially up-regulated in females. Thus, GH regulated sex-biased liver physiology is dictated by transcription factors arranged in a hierarchical network and by dynamic sex-biased epigenetic states. / 2020-06-12T00:00:00Z

Background Hedgehog signaling plays an important role in embryonic development, organogenesis and cancer. In the adult liver, Hedgehog signaling in non-parenchymal cells has been found to play a role in certain disease states such as fibrosis and cirrhosis. However, whether the Hedgehog pathway is active in mature healthy hepatocytes and is of significance to liver function are controversial. Findings Two types of mice with distinct conditional hepatic deletion of the Smoothened gene, an essential co-receptor protein of the Hedgehog pathway, were generated for investigating the role of Hedgehog signaling in mature hepatocytes. The knockout animals (KO) were inconspicuous and healthy with no changes in serum transaminases, but showed a slower weight gain. The liver was smaller, but presented a normal architecture and cellular composition. By quantitative RT-PCR the downregulation of the expression of Indian hedgehog (Ihh) and the Gli3 transcription factor could be demonstrated in healthy mature hepatocytes from these mice, whereas Patched1 was upregulated. Strong alterations in gene expression were also observed for the IGF axis. While expression of Igf1 was downregulated, that of Igfbp1 was upregulated in the livers of both genders. Corresponding changes in the serum levels of both proteins could be detected by ELISA. By activating and inhibiting the transcriptional output of Hedgehog signaling in cultured hepatocytes through siRNAs against Ptch1 and Gli3, respectively, in combination with a ChIP assay evidence was collected indicating that Igf1 expression is directly dependent on the activator function of Gli3. In contrast, the mRNA level of Igfbp1 appears to be controlled through the repressor function of Gli3, while that of Igfbp2 and Igfbp3 did not change. Interestingly, body weight of the transgenic mice correlated well with IGF-I levels in both genders and also with IGFBP-1 levels in females, whereas it did not correlate with serum growth hormone levels. Conclusions Our results demonstrate for the first time that Hedgehog signaling is active in healthy mature mouse hepatocytes and that it has considerable importance for IGF-I homeostasis in the circulation. These findings may have various implications for mouse physiology including the regulation of body weight and size, glucose homeostasis and reproductive capacity.

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One of the oldest, extant, lineages of vertebrates, the sea lamprey, was used to clarify the evolutionary origin and divergence of the growth hormone receptor (GHR) family. A single, full-length, cDNA, and a second, partial, cDNA were identified and shown to encode proteins that share amino acid identity with GHRs and prolactin receptors (PRLR s) previously identified. The complexity of the dynamic signaling system, with special emphasis on this system in fish and in the context of the evolution of this system, is discussed in the first chapter. The second chapter integrates the new insights gained by these studies. Included is a newly proposed phylogenetic analysis and revised nomenclature-system for vertebrate GHRs that better represents the evolutionary history of the receptor family. The molecular evolution of the receptors is, furthermore, highlighted as the backdrop for the continued discussion regarding how the GH-family of hormones exhibit such coordinated and pleiotropic actions.

The magnitude of improvements in muscular strength and size are influenced by the volume and intensity of a resistance training program. While it is clearly advantageous for resistance-trained individuals to utilize programming specific to these goals, it not clear which is more important. Therefore the purpose of the present investigation was to determine the effect of focusing on training volume versus intensity on changes in muscle size and strength. Changes in muscular strength and size were examined in 29 resistance-trained men following 8 weeks of resistance training. Participants were randomly assigned to either a high volume (VOL, n = 14, 4 x 10 – 12RM, 1min rest) or high intensity (INT, n = 15, 4 x 3 – 5RM, 3min rest) resistance training program. Lean body mass, lean arm and leg mass, were assessed by dual energy X-ray absorptiometry, while ultrasound images (VL-vastus lateralis, RF-rectus femoris, PM-pectoralis major, and TB-triceps brachii) were used to assess changes in muscle cross-sectional area (CSA) and thickness (MT). Strength was measured by one repetition-maximum (1RM) squat (SQ) and bench press (BP). Changes in muscular (RF & VL) activation in response to increases in submaximal SQ intensity (40-, 60-, 80-, & 100%-1RM) were assessed via surface electromyography. Blood samples were collected at baseline, immediately post, 30min post, and 60min post-exercise at week 3 (WK3) and week 10 (WK10), to assess plasma/serum testosterone, growth hormone (GH), insulin-like growth factor-1 (IGF1), cortisol (CORT), and insulin. Area under the curve analysis revealed a greater (p < 0.05) increase for VOL (WK3: GH & CORT; WK10: CORT) compared to INT. Compared to WK3, WK10 showed reduced responses for VOL (GH and CORT) and INT (IGF1). Significant group differences were observed for changes in lean arm mass (INT: 5.2 ± 2.9%, VOL: 2.2 5.6%) and BP 1RM (INT: 14.8 ± 9.7%, VOL: 6.9 ± 9.0%). Over the course of 8 weeks, our data indicate that trained men would benefit more when focusing on training intensity, rather than volume, for strength and size improvements.