Liver impact of growth hormone (GH) intermittent treatment during the growth period in mice.

Liver impact of prolonged GH-treatment given to non-GH-deficient growing mice between the third and eighth week of life was evaluated in both sexes. Tissues were collected 6 h after last dose or four weeks later. Somatometric, biochemical, histological, immunohistochemical, RT-qPCR and immunoblotting determinations were performed. Five-week GH intermittent administration induced body weight gain and body and bone length increase, augmented organ weight, higher hepatocellular size and proliferation, and increased liver IGF1 gene expression. Phosphorylation of signaling mediators and expression of GH-induced proliferation-related genes was decreased in GH-treated mice liver 6h after last injection, reflecting active sensitization/desensitization cycles. In females, GH elicited EGFR expression, associated to higher EGF-induced STAT3/5 phosphorylation. Four weeks after treatment, increased organ weight concomitant to body weight gain was still observed, whereas hepatocyte enlargement reverted. However, basal signaling for critical mediators was lower in GH-treated animals and in male controls compared to female ones, suggesting signaling declination.

Insulin signaling in the heart is impaired by growth hormone: a direct and early event.

Growth hormone (GH) exerts major actions in cardiac growth and metabolism. Considering the important role of insulin in the heart and the well-established anti-insulin effects of GH, cardiac insulin resistance may play a role in the cardiopathology observed in acromegalic patients. As conditions of prolonged exposure to GH are associated with a concomitant increase of circulating GH, IGF1 and insulin levels, to dissect the direct effects of GH, in this study, we evaluated the activation of insulin signaling in the heart using four different models: (i) transgenic mice overexpressing GH, with chronically elevated GH, IGF1 and insulin circulating levels; (ii) liver IGF1-deficient mice, with chronically elevated GH and insulin but decreased IGF1 circulating levels; (iii) mice treated with GH for a short period of time; (iv) primary culture of rat cardiomyocytes incubated with GH. Despite the differences in the development of cardiomegaly and in the metabolic alterations among the three experimental mouse models analyzed, exposure to GH was consistently associated with a decreased response to acute insulin stimulation in the heart at the receptor level and through the PI3K/AKT pathway. Moreover, a blunted response to insulin stimulation of this signaling pathway was also observed in cultured cardiomyocytes of neonatal rats incubated with GH. Therefore, the key novel finding of this work is that impairment of insulin signaling in the heart is a direct and early event observed as a consequence of exposure to GH, which may play a major role in the development of cardiac pathology.

Morphological and molecular effects of overexpressed GH on mice mammary gland.

Growth Hormone (GH) plays crucial roles in mammary gland development and growth, and its upregulation has been associated with breast cancer promotion and/or progression. To ascertain how high GH levels could promote mammary tissue oncogenic transformation, morphological characteristics and the expression of receptors involved in mammary growth, development and cancer, and of mitogenic mediators were analyzed in the mammary gland of virgin adult transgenic mice that overexpress GH. Whole mounting and histologic analysis evidenced that transgenic mice exhibit increased epithelial ductal elongation and enlarged ducts along with deficient branching and reduced number of alveolar structures compared to wild type mice. The number of differentiated alveolar structures was diminished in transgenic mice while the amount of terminal end buds (TEBs) did not differ between both groups of mice. GH, insulin-like growth factor 1 (IGF1) and GH receptor mRNA levels were augmented in GH-overexpressing mice breast tissue, as well as IGF1 receptor protein content. However, GH receptor protein levels were decreased in transgenic mice. Fundamental receptors for breast growth and development like progesterone receptor and epidermal growth factor receptor were also increased in mammary tissue from transgenic animals. In turn, the levels of the proliferation marker Ki67, cFOS and Cyclin D1 were increased in GH-overexpressing mice, while cJUN expression was decreased and cMYC did not vary. In conclusion, prolonged exposure to high GH levels induces morphological and molecular alterations in the mammary gland that affects its normal development. While these effects would not be tumorigenic per se, they might predispose to oncogenic transformation.

Growth Hormone Modulation of Hepatic Epidermal Growth Factor Receptor Signaling.

Epidermal growth factor receptor (EGFR) signaling has a central role in the regenerative response of the liver upon injury and is involved in cellular transformation linked to chronic damage. Hepatic EGFR expression, trafficking, and signaling are regulated by growth hormone (GH). Chronically elevated GH levels are associated with liver cancer development and progression in mice. Studies in different in vivo experimental models indicate that EGF and GH mutually crossregulate in a complex manner. Several factors, such as the extent of exposure to supraphysiological GH levels and the pattern of GH administration, are important variables to be considered in exploring the interplay between the two hormones in connection with the progression of hepatic tumors.

Exposure to growth hormone is associated with hepatic up-regulation of cPLA2α and COX.

Continuously elevated levels of growth hormone (GH) during life in mice are associated with hepatomegaly due to hepatocytes hypertrophy and hyperplasia, chronic liver inflammation, elevated levels of arachidonic acid (AA) at young ages and liver tumors development at old ages. In this work, the hepatic expression of enzymes involved in AA metabolism, cPLA2α, COX1 and COX2 enzymes, was evaluated in young and old GH-transgenic mice. Mice overexpressing GH exhibited higher hepatic expression of cPLA2α, COX1 and COX2 in comparison to controls at young and old ages and in both sexes. In old mice, when tumoral and non-tumoral tissue were compared, elevated expression of COX2 was observed in tumors. In contrast, exposure to continuous lower levels of hormone for a short period affected COX1 expression only in males. Considering the role of inflammation during liver tumorigenesis, these findings support a role of alterations in AA metabolism in GH-driven liver tumorigenesis.

Mice lacking angiotensin type 2 receptor exhibit a sex-specific attenuation of insulin sensitivity.

The renin-angiotensin system modulates insulin action. Pharmacological stimulation of angiotensin type 2 receptor (AT2R) was shown to have beneficial metabolic effects in various animal models of insulin resistance and type 2 diabetes and also to increase insulin sensitivity in wild type mice. In this study we further explored the role of the AT2R on insulin action and glucose homeostasis by investigating the glycemic profile and in vivo insulin signaling status in insulin-target tissues from both male and female AT2R knockout (KO) mice. When compared to the respective wild-type (WT) group, glycemia and insulinemia was unaltered in AT2RKO mice regardless of sex. However, female AT2RKO mice displayed decreased insulin sensitivity compared to their WT littermates. This was accompanied by a compensatory increase in adiponectinemia and with a specific attenuation of the activity of main insulin signaling components (insulin receptor, Akt and ERK1/2) in adipose tissue with no apparent alterations in insulin signaling in either liver or skeletal muscle. These parameters remained unaltered in male AT2RKO mice as compared to male WT mice. Present data show that the AT2R has a physiological role in the conservation of insulin action in female but not in male mice. Our results suggest a sexual dimorphism in the control of insulin action and glucose homeostasis by the AT2R and reinforce the notion that pharmacological modulation of the balance between the AT1R and AT2R receptor could be important for treatment of metabolic syndrome and type 2 diabetes.

Growth hormone upregulates the pro-tumorigenic galectin 1 in mouse liver.

Transgenic mice overexpressing growth hormone (GH) spontaneously develop liver tumors, including hepatocellular carcinoma (HCC), within a year. The preneoplastic liver pathology in these mice recapitulates that observed in humans at high risk of developing hepatic cancer. Although increased expression of galectin 1 (GAL1) in liver tissue is associated with HCC aggressiveness, a link between this glycan-binding protein and hormone-related tumor development has not yet been explored. In this study, we investigated GAL1 expression during liver tumor progression in mice continuously exposed to high levels of GH. GAL1 expression was determined by Western blotting, RT-qPCR and immunohistochemistry in the liver of transgenic mice overexpressing GH. Animals of representative ages at different stages of liver pathology were studied. GAL1 expression was upregulated in the liver of GH-transgenic mice. This effect was observed at early ages, when animals displayed no signs of liver disease or minimal histopathological alterations and was also detected in young adults with preneoplastic liver pathology. Remarkably, GAL1 upregulation was sustained during aging and its expression was particularly enhanced in liver tumors. GH also induced hepatic GAL1 expression in mice that were treated with this hormone for a short period. Moreover, GH triggered a rapid increment in GAL1 protein expression in human HCC cells, denoting a direct effect of the hormone on hepatocytes. Therefore, our results indicate that GH upregulates GAL1 expression in mouse liver, which may have critical implications in tumorigenesis. These findings suggest that this lectin could be implicated in hormone-driven liver carcinogenesis.

A laboratory work to introduce biochemistry undergraduate students to basic enzyme kinetics-alkaline phosphatase as a model.

Enzyme kinetics is an essential topic in undergraduate Biochemistry courses. A laboratory work that covers the principal basic concepts of enzyme kinetics in steady state is presented. The alkaline phosphatase catalyzed reaction of phenyl-phosphate hydrolysis was studied as a model. The laboratory experience was designed to reinforce the concepts of initial velocity dependence on substrate and enzyme concentration, and to highlight the importance of the accurate determination of initial reaction rate. The laboratory work consists in two parts, in which students first determine the enzyme concentration and the time to be used in the following session to obtain the kinetic parameters (KM and Vmax ) by non-lineal fitting of the Michaelis-Menten equation to the initial velocity dependence with substrate concentration results. The experimental methodology is robust, the cost per student is low and the equipment and reagents used are of easy access. © 2018 International Union of Biochemistry and Molecular Biology, 47(1):93-99, 2018.

Analysis of Different Approaches for the Selection of Reference Genes in RT-qPCR Experiments: A Case Study in Skeletal Muscle of Growing Mice.

The reliability of reverse transcription-quantitative PCR (RT-qPCR) results in gene expression studies depends on the approaches used to account for non-biological variations. In order to find a proper normalization strategy for the study of genes related to growth hormone signaling in skeletal muscle of growing mice, nine unrelated genes were evaluated as internal controls. According to the most used algorithms-geNorm, the Comparative ΔCq method, NormFinder and BestKeeper-GSK3B, YWHAZ, RPL13A and RN18S were found as the most stable. However, the relative expression levels of eight of the potential reference genes assessed decreased with age in cDNA samples obtained from the same amount of total RNA. In a different approach to analyze this apparent discrepancy, experiments were performed with cDNA obtained from equal amounts of purified mRNA. Since the decline was still observed, the hypothesis of an age-related change in mRNA to total RNA ratio that could account for the systematic decrease was rejected. Differences among experimental groups could be due to a substantial increase with age in highly expressed mRNAs, which would bias the quantitation of the remaining genes. Consequently, those reference genes reflecting this dilution effect, which would have been discarded considering their variable relative expression levels, arose as suitable internal controls.

Attenuation of epidermal growth factor (EGF) signaling by growth hormone (GH).

Transgenic mice overexpressing growth hormone (GH) show increased hepatic protein content of the epidermal growth factor receptor (EGFR), which is broadly associated with cell proliferation and oncogenesis. However, chronically elevated levels of GH result in desensitization of STAT-mediated EGF signal and similar response of ERK1/2 and AKT signaling to EGF compared to normal mice. To ascertain the mechanisms involved in GH attenuation of EGF signaling and the consequences on cell cycle promotion, phosphorylation of signaling mediators was studied at different time points after EGF stimulation, and induction of proteins involved in cell cycle progression was assessed in normal and GH-overexpressing transgenic mice. Results from kinetic studies confirmed the absence of STAT3 and 5 activation and comparable levels of ERK1/2 phosphorylation upon EGF stimulation, which was associated with diminished or similar induction of c-MYC, c-FOS, c-JUN, CYCLIN D1 and CYCLIN E in transgenic compared to normal mice. Accordingly, kinetics of EGF-induced c-SRC and EGFR phosphorylation at activating residues demonstrated that activation of these proteins was lower in the transgenic mice with respect to normal animals. In turn, EGFR phosphorylation at serine 1046/1047, which is implicated in the negative regulation of the receptor, was increased in the liver of GH-overexpressing transgenic mice both in basal conditions and upon EGF stimulus. Increased basal phosphorylation and activation of the p38-mitogen-activated protein kinase might account for increased Ser 1046/1047 EGFR. Hyperphosphorylation of EGFR at serine residues would represent a compensatory mechanism triggered by chronically elevated levels of GH to mitigate the proliferative response induced by EGF.

Mitogenic signaling pathways in the liver of growth hormone (GH)-overexpressing mice during the growth period.

Growth hormone (GH) is a pleiotropic hormone that triggers STATs, ERK1/2 and Akt signaling, related to cell growth and proliferation. Transgenic mice overexpressing GH present increased body size, with a disproportionate liver enlargement due to hypertrophy and hyperplasia of the hepatocytes. We had described enhanced mitogenic signaling in liver of young adult transgenic mice. We now evaluate the activation of these signaling cascades during the growth period and relate them to the morphological alterations found. Signaling mediators, cell cycle regulators and transcription factors involved in cellular growth in the liver of GH-overexpressing growing mice were assessed by immunoblotting, RT-qPCR and immunohistochemistry. Hepatocyte enlargement can be seen as early as 2-weeks of age in GH-overexpressing animals, although it is more pronounced in young adults. Levels of cell cycle mediators PCNA and cyclin D1, and transcription factor c-Jun increase with age in transgenic mice with no changes in normal mice, whereas c-Myc levels are higher in 2-week-old transgenic animals and cyclin E levels decline with age for both genotypes. STAT3, Akt and GSK3 present higher activation in the adult transgenic mice than in the growing animals, while for c-Src and mTOR, phosphorylation in GH-overexpressing mice is higher than in control siblings at 4 and 9 weeks of age. No significant changes are observed for ERK1/2, neither by age or genotype. Thus, the majority of the mitogenic signaling pathways are gradually up-regulated in the liver of GH-transgenic mice, giving rise to the hepatic morphological changes these mice exhibit.

Long-lived hypopituitary Ames dwarf mice are resistant to the detrimental effects of high-fat diet on metabolic function and energy expenditure.

Growth hormone (GH) signaling stimulates the production of IGF-1; however, increased GH signaling may induce insulin resistance and can reduce life expectancy in both mice and humans. Interestingly, disruption of GH signaling by reducing plasma GH levels significantly improves health span and extends lifespan in mice, as observed in Ames dwarf mice. In addition, these mice have increased adiposity, yet are more insulin sensitive compared to control mice. Metabolic stressors such as high-fat diet (HFD) promote obesity and may alter longevity through the GH signaling pathway. Therefore, our objective was to investigate the effects of a HFD (metabolic stressor) on genetic mechanisms that regulate metabolism during aging. We show that Ames dwarf mice fed HFD for 12 weeks had an increase in subcutaneous and visceral adiposity as a result of diet-induced obesity, yet are more insulin sensitive and have higher levels of adiponectin compared to control mice fed HFD. Furthermore, energy expenditure was higher in Ames dwarf mice fed HFD than in control mice fed HFD. Additionally, we show that transplant of epididymal white adipose tissue (eWAT) from Ames dwarf mice fed HFD into control mice fed HFD improves their insulin sensitivity. We conclude that Ames dwarf mice are resistant to the detrimental metabolic effects of HFD and that visceral adipose tissue of Ames dwarf mice improves insulin sensitivity in control mice fed HFD.

Resistance to the Beneficial Metabolic Effects and Hepatic Antioxidant Defense Actions of Fibroblast Growth Factor 21 Treatment in Growth Hormone-Overexpressing Transgenic Mice.

Fibroblast growth factor 21 (FGF21) modulates a diverse range of biological functions, including glucose and lipid metabolism, adaptive starvation response, and energy homeostasis, but with limited mechanistic insight. FGF21 treatment has been shown to inhibit hepatic growth hormone (GH) intracellular signaling. To evaluate GH axis involvement in FGF21 actions, transgenic mice overexpressing bovine GH were used. Expectedly, in response to FGF21 treatment control littermates showed metabolic improvements whereas GH transgenic mice resisted most of the beneficial effects of FGF21, except an attenuation of the innate hyperinsulinemia. Since FGF21 is believed to exert its effects mostly at the transcriptional level, we analyzed and observed significant upregulation in expression of various genes involved in carbohydrate and lipid metabolism, energy homeostasis, and antioxidant defense in FGF21-treated controls, but not in GH transgenics. The resistance of GH transgenic mice to FGF21-induced changes underlines the necessity of normal GH signaling for the beneficial effects of FGF21.

GH/STAT5 signaling during the growth period in livers of mice overexpressing GH.

GH/STAT5 signaling is desensitized in the liver in adult transgenic mice overexpressing GH; however, these animals present greater body size. To assess whether the STAT5 pathway is active during the growth period in the liver in these animals, and how signaling modulators participate in this process, growing transgenic mice and normal siblings were evaluated. STAT5 does not respond to an acute GH-stimulus, but displays higher basal phosphorylation in the livers of growing GH-overexpressing mice. GH receptor and the positive modulators glucocorticoid receptor and HNF1 display greater abundance in transgenic animals, supporting the activity of STAT5. The negative modulators cytokine-induced suppressor and PTP1B are increased in GH-overexpressing mice. The suppressors SOCS2 and SOCS3 exhibit higher mRNA levels in transgenic mice but lower protein content, indicating that they are being actively degraded. Therefore, STAT5 signaling is increased in the liver in GH-transgenic mice during the growth period, with a balance between positive and negative effectors resulting in accelerated but controlled growth.

Downregulation of the ACE2/Ang-(1-7)/Mas axis in transgenic mice overexpressing GH.

The renin-angiotensin system (RAS) plays a crucial role in the regulation of physiological homeostasis and diseases such as hypertension, coronary artery disease, and chronic renal failure. In this cascade, the angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/AT1 receptor axis induces pathological effects, such as vasoconstriction, cell proliferation, and fibrosis, while the ACE2/Ang-(1-7)/Mas receptor axis is protective for end-organ damage. The altered function of the RAS could be a contributing factor to the cardiac and renal alterations induced by GH excess. To further explore this issue, we evaluated the consequences of chronic GH exposure on the in vivo levels of Ang II, Ang-(1-7), ACE, ACE2, and Mas receptor in the heart and the kidney of GH-transgenic mice (bovine GH (bGH) mice). At the age of 7-8 months, female bGH mice displayed increased systolic blood pressure (SBP), a high degree of both cardiac and renal fibrosis, as well as increased levels of markers of tubular and glomerular damage. Angiotensinogen abundance was increased in the liver and the heart of bGH mice, along with a concomitant increase in cardiac Ang II levels. Importantly, the levels of ACE2, Ang-(1-7), and Mas receptor were markedly decreased in both tissues. In addition, Ang-(1-7) administration reduced SBP to control values in GH-transgenic mice, indicating that the ACE2/Ang-(1-7)/Mas axis is involved in GH-mediated hypertension. The data indicate that the altered expression profile of the ACE2/Ang-(1-7)/Mas axis in the heart and the kidney of bGH mice could contribute to the increased incidence of hypertension, cardiovascular, and renal alterations observed in these animals.

Mitogenic effects of phosphatidylcholine nanoparticles on MCF-7 breast cancer cells.

Lecithins, mainly composed of the phospholipids phosphatidylcholines (PC), have many different uses in the pharmaceutical and clinical field. PC are involved in structural and biological functions as membrane trafficking processes and cellular signaling. Considering the increasing applications of lecithin-based nanosystems for the delivery of therapeutic agents, the aim of the present work was to determine the effects of phosphatidylcholine nanoparticles over breast cancer cellular proliferation and signaling. PC dispersions at 0.01 and 0.1% (w/v) prepared in buffer pH 7.0 and 5.0 were studied in the MCF-7 breast cancer cell line. Neutral 0.1% PC-derived nanoparticles induced the activation of the MEK-ERK1/2 pathway, increased cell viability and induced a 1.2 fold raise in proliferation. These biological effects correlated with the increase of epidermal growth factor receptor (EGFR) content and its altered cellular localization. Results suggest that nanoparticles derived from PC dispersion prepared in buffer pH 7.0 may induce physicochemical changes in the plasma membrane of cancer cells which may affect EGFR cellular localization and/or activity, increasing activation of the MEK-ERK1/2 pathway and inducing proliferation. Results from the present study suggest that possible biological effects of delivery systems based on lecithin nanoparticles should be taken into account in pharmaceutical formulation design.

GH administration patterns differently regulate epidermal growth factor signaling.

Current GH administration protocols imply frequent s.c. injections, resulting in suboptimal compliance. Therefore, there is interest in developing delivery systems for sustained release of the hormone. However, GH has different actions depending on its continuous or pulsatile plasma concentration pattern. GH levels and circulating concentration patterns could be involved in the regulation of epidermal growth factor receptor (EGFR) expression in liver. Aberrant expression of this receptor and/or its hyperactivation has been associated with the pathogenesis of different types of carcinoma. Considering that one of the adverse effects associated with GH overexpression and chronic use of GH is the increased incidence of malignancies, the aim of this study was to analyze the effects of GH plasma concentration patterns on EGFR expression and signaling in livers of mice. For this purpose, GH was administered by s.c. daily injections to produce an intermittent plasma pattern or by osmotic pumps to provoke a continuously elevated GH concentration. Intermittent injections of GH induced upregulation of liver EGFR content, augmented the response to EGF, and the induction of proteins involved in promotion of cell proliferation in female mice. In contrast, continuous GH delivery in male mice was associated with diminished EGFR in liver and decreased EGF-induced signaling and expression of early genes. The results indicate that sustained delivery systems that allow continuous GH plasma patterns would be beneficial in terms of treatment safety with regard to the actions of GH on EGFR signaling and its promitogenic activity.

Oral administration of angiotensin-(1-7) ameliorates type 2 diabetes in rats.

UNLABELLED: Diabetes mellitus type 2 (DM2) is a disease with increasing importance in modern societies and insufficient treatment options. Pharmacological stimulation of insulin signaling, which is blunted in DM2, is a promising approach to treat this disease. It has been shown that activation of the angiotensin (Ang)-(1-7)/Mas axis of the renin-angiotensin system leads to an improved glucose uptake. In this study, we intended to evaluate, whether this effect could be exploited therapeutically. We first confirmed that Ang-(1-7) improves insulin signaling and glucose uptake in vitro in cultured cardiomyocytes. We then evaluated the therapeutic effect of a newly developed hydro-xypropyl-ß-cyclodextrin-based Ang-(1-7) nano-formulation in a novel transgenic rat model of inducible insulin resistance and DM2. The chronic administration of this compound prevented the marked elevation in blood glucose levels in these rats at a dose of 30 µg/kg, reversed the established hyperglycemic state at a dose of 100 µg/kg, and resulted in improved insulin sensitivity, reduced plasma insulin and decreased diabetic nephropathy. In conclusion, an oral Ang-(1-7) formulation reverses hyperglycemia and its consequences in an animal model of DM2 and represents a novel therapeutic option for the treatment of DM2 and other cardio-metabolic diseases. KEY MESSAGE: A novel rat model with inducible diabetes can be used to evaluate new therapies. Angiotensin-(1-7) is effective in an oral formulation packaged in cyclodextrine. Angiotensin-(1-7) is a promising antidiabetic drug.