Sex-different and growth hormone-regulated expression of microRNA in rat liver

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are short non-coding RNAs playing an important role in post-transcriptional regulation of gene expression. We have previously shown that hepatic transcript profiles are different between males and females; that some of these differences are under the regulation of growth hormone (GH); and that mild starvation diminishes some of the differences. In this study, we tested if hepatic miRNAs are regulated in a similar manner.</p> <p>Results</p> <p>Using microarrays, miRNA screening was performed to identify sex-dependent miRNAs in rat liver. Out of 324 unique probes on the array, 254 were expressed in the liver and eight (3% of 254) of those were found to be different between the sexes. Among the eight putative sex-different miRNAs, only one female-predominant miRNA (miR-29b) was confirmed using quantitative real-time PCR. Furthermore, 1 week of continuous GH-treatment in male rats reduced the levels of miR-451 and miR-29b, whereas mild starvation (12 hours) raised the levels of miR-451, miR-122a and miR-29b in both sexes. The biggest effects were obtained on miR-29b with GH-treatment.</p> <p>Conclusion</p> <p>We conclude that hepatic miRNA levels depend on the hormonal and nutritional status of the animal and show that miR-29b is a female-predominant and GH-regulated miRNA in rat liver.</p

Obtención de una sonda para cuantificar el mARN del factor activador de la transcripción STAT5 en rata

The strategy for cloning a PCR-amplified STAT5 fragment into an expression vector is described. By optimising the magnesium level and the annealing temperature in the PCR reaction, target fragment amplification was achieved, using mouse STAT5b cDNA as a template. The PCR product was cloned and probe identity was confirmed by restriction analysis and sequencing. This probe was used in a solution hybridisation-Rnase protection assay to quantify STAT5 mRNA in female rats' livers and thymus lymphocytes. A higher STAT5 mRNA expression was found in liver.Se describe la estrategia empleada para clonar, en un vector de expresión, un fragmento del gen de STAT5 amplificado por la técnica de PCR. Variando la concentración de magnesio y la temperatura de alineación, se logró amplificar el fragmento deseado a partir de un cADN de STAT5b de ratón. El producto de PCR se clonó y se comprobó la identidad de la sonda mediante análisis de restricción y secuenciación. Esta sonda se usó en el ensayo de protección con ribonucleasa A o hibridización en solución, para cuantificar el mARN de STAT5 en hígado y linfocitos de timo de ratas hembras. Se encontró una mayor expresión del mARN de STAT5 en hígado

Analysis of siRNA specificity on targets with double-nucleotide mismatches

Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that ∼35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site

Sex-different hepaticglycogen content and glucose output in rats

<p>Abstract</p> <p>Background</p> <p>Genes involved in hepatic metabolism have a sex-different expression in rodents. To test whether male and female rat livers differ regarding lipid and carbohydrate metabolism, whole-genome transcript profiles were generated and these were complemented by measurements of hepatic lipid and glycogen content, fatty acid (FA) oxidation rates and hepatic glucose output (HGO). The latter was determined in perfusates from <it>in situ </it>perfusion of male and female rat livers. These perfusates were also analysed using nuclear magnetic resonance (NMR) spectroscopy to identify putative sex-differences in other liver-derived metabolites. Effects of insulin were monitored by analysis of Akt-phosphorylation, gene expression and HGO after s.c. insulin injections.</p> <p>Results</p> <p>Out of approximately 3 500 gene products being detected in liver, 11% were significantly higher in females, and 11% were higher in males. Many transcripts for the production of triglycerides (TG), cholesterol and VLDL particles were female-predominant, whereas genes for FA oxidation, gluconeogenesis and glycogen synthesis were male-predominant. Sex-differences in mRNA levels related to metabolism were more pronounced during mild starvation (12 h fasting), as compared to the postabsorptive state (4 h fasting). No sex-differences were observed regarding hepatic TG content, FA oxidation rates or blood levels of ketone bodies or glucose. However, males had higher hepatic glycogen content and higher HGO, as well as higher ratios of insulin to glucagon levels. Based on NMR spectroscopy, liver-derived lactate was also higher in males. HGO was inhibited by insulin in parallel with increased phosphorylation of Akt, without any sex-differences in insulin sensitivity. However, the degree of Thr172-phosphorylated AMP kinase (AMPK) was higher in females, indicating a higher degree of AMPK-dependent actions.</p> <p>Conclusions</p> <p>Taken together, males had higher ratios of insulin to glucagon levels, higher levels of glycogen, lower degree of AMPK phosphorylation, higher expression of gluconeogenic genes and higher hepatic glucose output. Possibly these sex-differences reflect a higher ability for the healthy male rat liver to respond to increased energy demands.</p

Receptor-mediated nuclear translocation of growth hormone

We have previously shown that the growth hormone (GH) receptor-binding protein is associated with the nucleus. me show here both by electron microscopy and nuclear isolation that GIT is subject to rapid nuclear translocation. The intracellular fate of intravenously injected I-125-bovine growth hormone (bGH) was examined in the rat hepatocyte by electron microscopic autoradiography. The hormone appeared rapidly at the plasma membrane, then sequentially in lysosomal and multivesicular bodies and/or the nuclear membrane before final translocation to the nuclear matrix. Maximal translocation to the nuclear matrix occurred within 30 min of injection. Nuclear translocation of I-125-hGH was also studied by isolation of nuclei from cells stably transfected with cDNAs encoding the GH receptor, GH-binding protein, and a membrane bound but cytoplasmic domain-deficient receptor. Specific internalization and nuclear translocation of hormone only occurred in cells transfected with the full-length receptor. The translocation was rapid and became saturated within 1 h after addition of hormone to the culture media. SDS-polyacrylamide gel electrophoresis of isolated nuclei showed that GH is transported to the nucleus as the intact molecule. Pretreatment of cells with lysosomotropic agents (chloroquine, ammonium chloride, and bacitracin) decreased hormone degradation and increased nuclear translocation of GH. The nuclear translocation of GH was achieved independent of the cytoskeletal system (microtubular, microfilament, and intermediate filament networks). Thus, GH is subject to rapid receptor-dependent nuclear translocation via the endosomal pathway

Nuclear translocation and anchorage of the growth hormone receptor

The extracellular domain of the rabbit growth hormone (GH) receptor has previously been shown to be associated with the nucleus, However, in this species the GH binding protein (BP) is derived by proteolytic cleavage of the full-length receptor, and thus distinction between the receptor and EP is difficult, The intracellular domain of the GH receptor is required for GH-stimulated function, Thus a direct nuclear function of GH would presumably require the receptor intracellular domain in the nucleus, We have therefore characterized the rat nuclear GH receptor and BP based on their distinct antigenic identity, We show, in vivo, that the full-length receptor is associated with the nucleus, including the respective subnuclear fractions (nucleoplasm, outer nuclear membranes, inner nuclear membranes, and chromatin). In vivo, the receptor is also subject to ligand-dependent nuclear translocation

Effects of prolactin on ventricular myocyte shortening and calcium transport in the streptozotocin-induced diabetic rat

© 2020 Cardiology; Cell biology; Endocrinology; Molecular Biology; Pathophysiology; Pharmacology; Physiology; prolactin; Diabetes mellitus; heart; Ventricular myocytes; Contraction; Calciu

MicroRNAs: Novel Regulators Involved in the Pathogenesis of Psoriasis?

MicroRNAs are a recently discovered class of posttranscriptional regulators of gene expression with critical functions in health and disease. Psoriasis is the most prevalent chronic inflammatory skin disease in adults, with a substantial negative impact on the patients' quality of life. Here we show for the first time that psoriasis-affected skin has a specific microRNA expression profile when compared with healthy human skin or with another chronic inflammatory skin disease, atopic eczema. Among the psoriasis-specific microRNAs, we identified leukocyte-derived microRNAs and one keratinocyte-derived microRNA, miR-203. In a panel of 21 different human organs and tissues, miR-203 showed a highly skin-specific expression profile. Among the cellular constituents of the skin, it was exclusively expressed by keratinocytes. The up-regulation of miR-203 in psoriatic plaques was concurrent with the down-regulation of an evolutionary conserved target of miR-203, suppressor of cytokine signaling 3 (SOCS-3), which is involved in inflammatory responses and keratinocyte functions. Our results suggest that microRNA deregulation is involved in the pathogenesis of psoriasis and contributes to the dysfunction of the cross talk between resident and infiltrating cells. Taken together, a new layer of regulatory mechanisms is involved in the pathogenesis of chronic inflammatory skin diseases