<p>Table S1: Genesets enriched in PC3.pGIPZ xenografts. Table S2: Genesets enriched in PC3.shCtBP1 xenografts. Supplemental Figure 1: (A) Crystal violet stained NIH3T3 cells foci transfected with pcDNA3.CtBP1 or beta-gal expression vectors. (B) CtBP1 RT-qPCR from PC3.CtBP1, PC3.shCtBP1 and PC3.pGIPZ stable cells using specific CtBP1 and Actin beta primers. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation of one representative experiment.***p < 0.001. Supplemental Figure 2: Box plots for (A) triglycerides, (B) glycemia and (C) estradiol serum levels for CD or HFD fed mice were shown. Boxes represent the interquartile range, the horizontal line within each box represents the median, and the upper and lower whiskers represent the standard deviation of one independent experiment. (D-E) Representative photograph of H&E staining from kidney and liver of mice fed with CD or HFD (Magnification 400x & 250x respectively). Supplemental Figure 3: (A) Testosterone and (B) estradiol levels from CD or HFD xenografts mice. Plotted boxes represent the interquartile range, the horizontal line within each of the boxes represents the median, and the upper and lower whiskers represent the standard deviation. * p < 0.05 (C) Specific CtBP1 RT-qPCR from tumors of CD or HFD fed mice. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation from 3 independent mice. * p < 0.05. Supplemental Figure 4: (A) GSEA from microarray data from HFD tumor xenografts. enrichment plots from genesets enriched in PC3.shCtBP1 xenografts. We showed the most significant over-representive cell adhesion categories selected from GSEA analysis from microarray data. (B) CDH1 RT-qPCR from the HFD fed mice tumors was performed. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation from 3 mice. * p < 0.05 (C) GSEA from microarray data from HFD tumor xenografts. The most significant enrichment plots for hormone pathways genesets in PC3.shCtBP1 xenografts were shown. Supplemental Figure 5: (A) Enrichment plots from genesets enriched in PC3.pGIPZ xenografts. Enrichment plots for the most significant over-representive categories selected from GSEA analysis from microarray data were shown. Listed above we found enrichment plots associated to cell cycle regulation processes and proliferation. Supplemental Figure 6: (A-B) BRCA1 RT-qPCR from (A) 22Rv1 or (B) LNCaP cells grew in charcoal FBS-medium and exposed to testosterone or vehicle for 24h. Fold induction was calculated normalizing data to Actin beta and control. (C-D) BRCA1 RT-qPCR from (C) 22Rv1 or (D) LNCaP cells, pre-treated for an hour with letrozole, were then grown as indicated before. Fold induction was calculated normalizing data to Actin beta and control. Data represents the average and standard deviation of one representative experiment. * p < 0.05.</p>
<p>PDF file - 556K, Table S1: Genesets enriched in PC3.pGIPZ xenografts. Table S2: Genesets enriched in PC3.shCtBP1 xenografts. Supplemental Figure 1: (A) Crystal violet stained NIH3T3 cells foci transfected with pcDNA3.CtBP1 or beta-gal expression vectors. (B) CtBP1 RT-qPCR from PC3.CtBP1, PC3.shCtBP1 and PC3.pGIPZ stable cells using specific CtBP1 and Actin beta primers. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation of one representative experiment.***p < 0.001. Supplemental Figure 2: Box plots for (A) triglycerides, (B) glycemia and (C) estradiol serum levels for CD or HFD fed mice were shown. Boxes represent the interquartile range, the horizontal line within each box represents the median, and the upper and lower whiskers represent the standard deviation of one independent experiment. (D-E) Representative photograph of H&E staining from kidney and liver of mice fed with CD or HFD (Magnification 400x & 250x respectively). Supplemental Figure 3: (A) Testosterone and (B) estradiol levels from CD or HFD xenografts mice. Plotted boxes represent the interquartile range, the horizontal line within each of the boxes represents the median, and the upper and lower whiskers represent the standard deviation. * p < 0.05 (C) Specific CtBP1 RT-qPCR from tumors of CD or HFD fed mice. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation from 3 independent mice. * p < 0.05. Supplemental Figure 4: (A) GSEA from microarray data from HFD tumor xenografts. enrichment plots from genesets enriched in PC3.shCtBP1 xenografts. We showed the most significant over-representive cell adhesion categories selected from GSEA analysis from microarray data. (B) CDH1 RT-qPCR from the HFD fed mice tumors was performed. Fold induction was calculated normalizing data to Actin beta and control. Bars represent the average and standard deviation from 3 mice. * p < 0.05 (C) GSEA from microarray data from HFD tumor xenografts. The most significant enrichment plots for hormone pathways genesets in PC3.shCtBP1 xenografts were shown. Supplemental Figure 5: (A) Enrichment plots from genesets enriched in PC3.pGIPZ xenografts. Enrichment plots for the most significant over-representive categories selected from GSEA analysis from microarray data were shown. Listed above we found enrichment plots associated to cell cycle regulation processes and proliferation. Supplemental Figure 6: (A-B) BRCA1 RT-qPCR from (A) 22Rv1 or (B) LNCaP cells grew in charcoal FBS-medium and exposed to testosterone or vehicle for 24h. Fold induction was calculated normalizing data to Actin beta and control. (C-D) BRCA1 RT-qPCR from (C) 22Rv1 or (D) LNCaP cells, pre-treated for an hour with letrozole, were then grown as indicated before. Fold induction was calculated normalizing data to Actin beta and control. Data represents the average and standard deviation of one representative experiment. * p < 0.05.</p>
Prenatal hyperandrogenism is hypothesized as one of the main factors contributing to the development of polycystic ovary syndrome (PCOS). PCOS patients have high risk of developing fatty liver and steatosis. This study aimed to evaluate the role of prenatal hyperandrogenism in liver lipid metabolism and fatty liver development. Pregnant rats were hyperandrogenized with testosterone. At pubertal age, the prenatally hyperandrogenized (PH) female offspring displayed both ovulatory (PHov) and anovulatory (PHanov) phenotypes that mimic human PCOS features. We evaluated hepatic transferases, liver lipid content, the balance between lipogenesis and fatty acid oxidation pathway, oxidant/antioxidant balance and proinflammatory status. We also evaluated the general metabolic status through growth rate curve, basal glucose and insulin levels, glucose tolerance test, HOMA-IR index and serum lipid profile. Although neither PH group showed signs of liver lipid content, the lipogenesis and fatty oxidation pathways were altered. The PH groups also showed impaired oxidant/antioxidant balance, a decrease in the proinflammatory pathway (measured by prostaglandin E2 and cyclooxygenase-2 levels), decreased glucose tolerance, imbalance of circulating lipids and increased risk of metabolic syndrome. We conclude that prenatal hyperandrogenism generates both PHov and PHanov phenotypes with signs of liver alterations, imbalance in lipid metabolism and increased risk of developing metabolic syndrome. The anovulatory phenotype showed more alterations in liver lipogenesis and a more impaired balance of insulin and glucose metabolism, being more susceptible to the development of steatosis.
Besides the hypothalamus and pituitary, melatonin action at the testicular level has been recently suggested. Therefore, we investigated in the Syrian hamster, a well-characterized seasonal breeder, melatonin action on Leydig cells, testicular expression of melatonergic receptors, and possible interactions between melatonin receptors and the previously identified testicular serotoninergic and CRH systems. In isolated Leydig cells from active testes of adult hamsters kept in a long-day (14 h light, 10 h dark) photoperiod and from regressed testes of adult animals exposed to a short-day photoperiod during 16 wk (6 h light, 18 h dark), melatonin significantly reduced human chorionic gonadotropin-stimulated production of cAMP and the main androgens: testosterone and androstane-3alpha,17beta-diol, respectively, and decreased the expression of steroidogenic acute regulatory protein, P450 side chain cleavage, 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase. In Leydig cells exposed to a short-day photoperiod during 16 wk, melatonin stimulated the conversion of testosterone into 5alpha-reduced androgens by inducing 5alpha-reductase isoform 1, and controlled androstane-3alpha,17beta-diol production by inhibiting 3alpha-hydroxysteroid dehydrogenase expression. Melatonin subtype (mel1a) receptors were detected in Leydig cells. Although the local serotonin system did not mediate melatonin action on androgen production, melatonergic effect on steroidogenesis involved the interaction between mel1a receptors and the inhibitory CRH system. Moreover, melatonin significantly increased CRH mRNA levels and production in hamster Leydig cells expressing CRH subtype 1 receptors. Our studies indicate that melatonin may act as a local inhibitor of human chorionic gonadotropin-stimulated cAMP and androgen production through mel1a receptors, down-regulation of steroidogenic acute regulatory protein, and key steroidogenic enzymes expression and its interaction with the local CRH system.
The activation of pituitary GABA<sub>B</sub> receptors by the specific agonist baclofen inhibits pituitary hormone secretion in vitro. Here we studied the mechanism of action of GABA<sub>B</sub> receptors in rat adenohypophysis. Anterior pituitary cells were obtained by trypsinization and were either plated for hormonal studies and cAMP determination or incubated in FURA 2AM for calcium measurements. Baclofen (BACL: 1·10<sup>–5</sup> <i>M</i>) significantly inhibited basal and thyrotropic releasing hormone (TRH)-stimulated (1·10<sup>–7</sup> <i>M</i>) PRL secretion in anterior pituitary cells from proestrous rats. In the presence of pertussis toxin (PTX: 150 ng/ml, 20 h), which leads to the uncoupling of the G<sub>i/o</sub>-protein from the receptor, both effects of BACL were abolished while the effect of dopamine (DA: 1·10<sup>–8</sup> <i>M</i>), used as an inhibitory control, was reduced from 70 to 25%. PTX also reversed BACL-induced inhibition of gonadotropin-releasing hormone (GnRH)-elicited luteinizing hormone (LH) secretion in anterior pituitary cells from 15-day-old female rats. In addition, though working in a pituitary mixed cell population, in which only some cell types possess GABA<sub>B</sub> receptors, BACL (1·10<sup>–5</sup> <i>M</i>) attenuated the forskolin-induced (0.5 µ<i>M</i>) increase in cAMP. This effect was prevented by co-incubation with the antagonist 2 hydroxysaclofen and by preincubation with PTX. BACL (5· 10<sup>–5</sup> <i>M</i>) and DA (5·10<sup>–7</sup> <i>M</i>) inhibited basal intracellular calcium concentrations ([Ca<sup>2+</sup>]<sub>i</sub>) in pituitary cells and the effect of the latter was significantly stronger. The effect of BACL on [Ca<sup>2+</sup>]<sub>i</sub> was abolished after preincubation with PTX. In the presence of the potassium channel blocking agents barium (200 µ<i>M </i>and 1 m<i>M</i>) and tetraethylammonium (10 m<i>M</i>), BACL was still able to inhibit [Ca<sup>2+</sup>]<sub>i</sub>. Blockade of voltage-sensitive calcium channels (VSCC) with either verapamil (5·10<sup>–6</sup> <i>M</i>) or nifedipine (1·10<sup>–6</sup> <i>M</i>) completely abolished the effect of BACL on [Ca<sup>2+</sup>]<sub>i</sub>. In the presence of 12.5 m<i>M</i> potassium concentration baclofen significantly inhibited [Ca<sup>2+</sup>]<sub>i</sub>. In conclusion, our results describe the negative coupling of adenohypophyseal GABA<sub>B</sub> receptors to VSCC through PTX-sensitive G-proteins. These characteristics suggest a resemblance of these receptors to the typical presynaptic GABA<sub>B</sub> sites described in the central nervous system.
Hypercortisolism induces a state of insulin resistance that can occur concurrently with fasting hyperglycaemia, dyslipidaemia and diabetes mellitus. Metformin reduces hepatic glucose production and insulin resistance of the skeletal muscle and adipose tissue. The aim of this study was to evaluate the effects of metformin on the control of metabolic disorders of dogs with hyperadrenocorticism (HAC). Twenty-three dogs with HAC were randomly divided into two groups, consisting of a control group and a metformin group (10 mg metformin/kg/12 h). Both groups received the same treatment for HAC. At baseline and 3 months, blood glucose, total cholesterol, triglycerides and insulin concentrations, in addition to urinary cortisol:creatinine ratio, Homeostatic Model Assessment (HOMA) for insulin sensitivity and β-cell function were measured. Dogs treated with metformin showed significantly reduced glycaemia, cholesterolaemia and triglyceridaemia. They also presented reduced hyperinsulinism and insulin resistance, as well as improved pancreatic β-cell function. The implementation of metformin as an adjuvant therapy is effective for the normalisation of metabolic disorders of dogs with HAC.Keywords: Dyslipidaemia, Hyperadrenocorticism, Insulin resistance, Metformin.
About 50% of the Na+ reabsorbed in thick ascending limbs traverses the paracellular pathway. Nitric oxide (NO) reduces the permselectivity of this pathway via cGMP, but its effects on absolute Na+ ([Formula: see text]) and Cl- ([Formula: see text]) permeabilities are unknown. To address this, we measured the effect of l-arginine (0.5 mmol/l; NO synthase substrate) and cGMP (0.5 mmol/l) on [Formula: see text] and [Formula: see text] calculated from the transepithelial resistance (Rt) and [Formula: see text]/[Formula: see text] in medullary thick ascending limbs. Rt was 7,722 ± 1,554 ohm·cm in the control period and 6,318 ± 1,757 ohm·cm after l-arginine treatment (P < 0.05). [Formula: see text]/[Formula: see text] was 2.0 ± 0.2 in the control period and 1.7 ± 0.1 after l-arginine (P < 0.04). Calculated [Formula: see text] and [Formula: see text] were 3.52 ± 0.2 and 1.81 ± 0.10 × 10-5 cm/s, respectively, in the control period. After l-arginine they were 6.65 ± 0.69 (P < 0.0001 vs. control) and 3.97 ± 0.44 (P < 0.0001) × 10-5 cm/s, respectively. NOS inhibition with Nω-nitro-l-arginine methyl ester (5 mmol/l) prevented l-arginine's effect on Rt Next we tested the effect of cGMP. Rt in the control period was 7,592 ± 1,470 and 4,796 ± 847 ohm·cm after dibutyryl-cGMP (0.5 mmol/l; db-cGMP) treatment (P < 0.04). [Formula: see text]/[Formula: see text] was 1.8 ± 0.1 in the control period and 1.6 ± 0.1 after db-cGMP (P < 0.03). [Formula: see text] and [Formula: see text] were 4.58 ± 0.80 and 2.66 ± 0.57 × 10-5 cm/s, respectively, for the control period and 9.48 ± 1.63 (P < 0.007) and 6.01 ± 1.05 (P < 0.005) × 10-5 cm/s, respectively, after db-cGMP. We modeled NO's effect on luminal Na+ concentration along the thick ascending limb. We found that NO's effect on the paracellular pathway reduces net Na+ reabsorption and that the magnitude of this effect is similar to that due to NO's inhibition of transcellular transport.
'/%3e%3cuse xlink:href='%23a' transform='rotate(45 400 250)'/%3e%3cuse xlink:href='%23a' transform='rotate(67.5 400 250)'/%3e%3cpath id='b' fill='%2385340a' d='M404.31 274.41c.453 9.054 5.587 13.063 4.579 21.314 2.213-6.525-3.124-11.583-2.82-21.22m-7.649-23.757 19.487 42.577-16.329-43.887'/%3e%3cuse xlink:href='%23b' transform='rotate(22.5 400 250)'/%3e%3cuse xlink:href='%23b' transform='rotate(45 400 250)'/%3e%3cuse xlink:href='%23b' transform='rotate(67.5 400 250)'/%3e%3c/g%3e%3cuse xlink:href='%23c' transform='rotate(90 400 250)'/%3e%3cuse xlink:href='%23c' transform='rotate(180 400 250)'/%3e%3cuse xlink:href='%23c' transform='rotate(270 400 250)'/%3e%3ccircle cx='400' cy='250' r='27.778' fill='%23f6b40e' stroke='%2385340a' stroke-width='1.5'/%3e%3cpath id='h' fill='%23843511' d='M409.47 244.06c-1.897 0-3.713.822-4.781 2.531 2.136 1.923 6.856 2.132 10.062-.219a7.333 7.333 0 0 0-5.281-2.312zm-.031.438c1.846-.034 3.571.814 3.812 1.656-2.136 2.35-5.55 2.146-7.687.437.935-1.495 2.439-2.067 3.875-2.094z'/%3e%3cuse xlink:href='%23d' transform='matrix(-1 0 0 1 800.25 0)'/%3e%3cuse xlink:href='%23e' transform='matrix(-1 0 0 1 800.25 0)'/%3e%3cuse xlink:href='%23f' transform='translate(18.862)'/%3e%3cuse xlink:href='%23g' transform='matrix(-1 0 0 1 800.25 0)'/%3e%3cpath fill='%2385340a' d='M395.75 253.84c-.913.167-1.563.977-1.563 1.906 0 1.062.878 1.906 1.938 1.906a1.89 1.89 0 0 0 1.563-.812c.739.556 1.764.615 2.312.625.084.002.193 0 .25 0 .548-.01 1.573-.069 2.313-.625.36.516.935.812 1.562.812 1.06 0 1.938-.844 1.938-1.906 0-.929-.65-1.74-1.563-1.906.513.18.844.676.844 1.219a1.28 1.28 0 0 1-1.281 1.281c-.68 0-1.242-.54-1.282-1.219-.208.417-1.034 1.655-2.656 1.719-1.622-.064-2.447-1.302-2.656-1.719-.04.679-.6 1.219-1.281 1.219a1.28 1.28 0 0 1-1.281-1.281c0-.542.33-1.038.843-1.219zm2.09 5.69c-2.138 0-2.983 1.937-4.906 3.219 1.068-.427 1.91-1.27 3.406-2.125 1.496-.855 2.772.187 3.625.187h.031c.853 0 2.13-1.041 3.625-.187 1.497.856 2.369 1.698 3.438 2.125-1.924-1.282-2.8-3.219-4.938-3.219-.426 0-1.271.23-2.125.656h-.031c-.853-.426-1.698-.656-2.125-.656z'/%3e%3cpath fill='%2385340a' d='M397.12 262.06c-.844.037-1.96.207-3.563.688 3.848-.855 4.697.437 6.407.437h.03c1.71 0 2.56-1.292 6.407-.438-4.274-1.282-5.124-.437-6.406-.437h-.031c-.802 0-1.437-.312-2.844-.25z'/%3e%3cpath fill='%2385340a' d='M393.75 262.72c-.248.003-.519.005-.813.031 4.488.428 2.331 3 7.032 3h.03c4.702 0 2.575-2.572 7.063-3-4.7-.426-3.214 2.344-7.062 2.344h-.031c-3.608 0-2.496-2.421-6.22-2.375zm10.1 6.94a3.848 3.848 0 0 0-3.846-3.846 3.848 3.848 0 0 0-3.847 3.846 3.955 3.955 0 0 1 3.847-3.04 3.952 3.952 0 0 1 3.846 3.04z'/%3e%3cpath id='e' fill='%2385340a' d='M382.73 244.02c4.915-4.273 11.11-4.915 14.53-1.709.837 1.121 1.373 2.32 1.593 3.57.43 2.433-.33 5.062-2.236 7.756.215-.001.643.212.856.427 1.697-3.244 2.297-6.577 1.74-9.746a13.815 13.815 0 0 0-.67-2.436c-4.7-3.845-11.11-4.272-15.81 2.138z'/%3e%3cpath id='d' fill='%2385340a' d='M390.42 242.74c2.777 0 3.419.642 4.7 1.71 1.284 1.068 1.924.854 2.137 1.068.213.215 0 .854-.426.64s-1.284-.64-2.564-1.708c-1.283-1.07-2.563-1.069-3.846-1.069-3.846 0-5.983 3.205-6.41 2.991-.426-.214 2.137-3.632 6.41-3.632z'/%3e%3cuse xlink:href='%23h' transform='translate(-19.181)'/%3e%3ccircle id='f' cx='390.54' cy='246.15' r='1.923' fill='%2385340a'/%3e%3cpath id='g' fill='%2385340a' d='M385.29 247.44c3.633 2.778 7.265 2.564 9.402 1.282 2.136-1.282 2.136-1.709 1.71-1.709-.427 0-.853.427-2.564 1.281-1.71.856-4.273.856-8.546-.854z'/%3e%3c/svg%3e)
